11
Research Article Scropolioside B Inhibits IL-1 and Cytokines Expression through NF-B and Inflammasome NLRP3 Pathways Tiantian Zhu, 1,2 Liuqiang Zhang, 2 Shuang Ling, 1 Ju Duan, 1 Fei Qian, 1,2 Yiming Li, 2 and Jin-Wen Xu 1 1 Murad Research Institute for Modernized Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China 2 School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Correspondence should be addressed to Yiming Li; [email protected] and Jin-Wen Xu; [email protected] Received 9 June 2014; Revised 6 August 2014; Accepted 20 August 2014; Published 16 October 2014 Academic Editor: Antonio Macci` o Copyright © 2014 Tiantian Zhu et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chronic inflammation is associated with various chronic illnesses including immunity disorders, cancer, neurodegeneration, and vascular diseases. Iridoids are compounds with anti-inflammatory properties. However their anti-inflammatory mechanism remains unclear. Here, we report that scropolioside B, isolated from a Tibetan medicine (Scrophularia dentata Royle ex Benth.), blocked expressions of TNF, IL-1, and IL-32 through NF-B pathway. Scropolioside B inhibited NF-B activity in a dose-dependent manner with IC 50 values of 1.02 mol/L. However, catalpol, similar to scropolioside B, was not effective in inhibiting NF-B activity. Interestingly, scropolioside B and catalpol decreased the expression of NLRP3 and cardiolipin synthetase at both the mRNA and protein level. Our results showed that scropolioside B is superior in inhibiting the expression, maturation, and secretion of IL-1 compared to catalpol. ese observations provide further understanding of the anti-inflammatory effects of iridoids and highlight scropolioside B as a potential drug for the treatment of rheumatoid arthritis and atherosclerosis. 1. Introduction Acute inflammatory responses are essential for pathogen control and tissue repair and can also cause severe tissue dam- age. During chronic infections and age-associated immune dysregulation, inflammatory processes may induce a variety of harmful effects on an organism [1]. Chronic inflammation is associated with chronic illnesses including cancer, neu- rodegeneration, and vascular diseases [24]. Infection or cell damage triggers the release of proinflammatory cytokines such as interleukin- (IL-) 1 and tumor necrosis factor- (TNF-) , which are key mediators of the host immune response. Signal transduction of inflammatory cytokines includes ligands, receptors, coreceptors, and cytosolic and nuclear signaling mechanisms. ese mechanisms can acti- vate the NF-B, JNK, p38 MAPK, STAT, and PI3K signaling pathways [5]. On the other hand, inflammasomes play a key role in the regulation of inflammation and immune responses by producing proinflammatory cytokines [6]. Studies have shown that inflammasomes are involved in atherosclerosis [7], metabolic syndrome [8], type 2 diabetes [9], alcoholic steatohepatitis [10], mucosal immune response [11], rheuma- toid arthritis [12], and gout [13]. e nucleotide-binding domain- (NOD-) like receptor protein 3 (NLRP3) inflamma- some is a multiprotein complex that regulates the maturation of proinflammatory cytokines IL-1 and IL-18. It consists of NOD-like receptor, NLRP3, the adaptor protein ASC (apoptosis-associated speck-like protein containing caspase- 1 activator domain, CARD), and caspase-1. Upon exogenous and endogenous stimuli, the NLRP3 inflammasome forms through activation of NLRP3 and recruitment of ASC and pro-caspase-1, resulting in caspase-1 activation and subse- quently processing of pro-IL-1 and pro-IL-18 into their active forms [14]. Iridoid is derived from Scrophulariaceae, Rubiaceae, Labiatae, Gentianaceae, Oleaceae, and so on; it is mainly Hindawi Publishing Corporation Mediators of Inflammation Volume 2014, Article ID 819053, 10 pages http://dx.doi.org/10.1155/2014/819053

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Research ArticleScropolioside B Inhibits IL-1120573 and Cytokines Expressionthrough NF-120581B and Inflammasome NLRP3 Pathways

Tiantian Zhu12 Liuqiang Zhang2 Shuang Ling1 Ju Duan1 Fei Qian12

Yiming Li2 and Jin-Wen Xu1

1 Murad Research Institute for Modernized Chinese Medicine Shanghai University of Traditional Chinese MedicineShanghai 201203 China

2 School of Pharmacy Shanghai University of Traditional Chinese Medicine Shanghai 201203 China

Correspondence should be addressed to Yiming Li ymlius163com and Jin-Wen Xu jinwenxu88gmailcom

Received 9 June 2014 Revised 6 August 2014 Accepted 20 August 2014 Published 16 October 2014

Academic Editor Antonio Maccio

Copyright copy 2014 Tiantian Zhu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chronic inflammation is associated with various chronic illnesses including immunity disorders cancer neurodegenerationand vascular diseases Iridoids are compounds with anti-inflammatory properties However their anti-inflammatory mechanismremains unclear Here we report that scropolioside B isolated from a Tibetan medicine (Scrophularia dentata Royle ex Benth)blocked expressions of TNF IL-1 and IL-32 through NF-120581B pathway Scropolioside B inhibited NF-120581B activity in a dose-dependentmanner with IC

50values of 102 120583molL However catalpol similar to scropolioside B was not effective in inhibiting NF-120581B activity

Interestingly scropolioside B and catalpol decreased the expression of NLRP3 and cardiolipin synthetase at both the mRNA andprotein level Our results showed that scropolioside B is superior in inhibiting the expression maturation and secretion of IL-1120573compared to catalpol These observations provide further understanding of the anti-inflammatory effects of iridoids and highlightscropolioside B as a potential drug for the treatment of rheumatoid arthritis and atherosclerosis

1 Introduction

Acute inflammatory responses are essential for pathogencontrol and tissue repair and can also cause severe tissue dam-age During chronic infections and age-associated immunedysregulation inflammatory processes may induce a varietyof harmful effects on an organism [1] Chronic inflammationis associated with chronic illnesses including cancer neu-rodegeneration and vascular diseases [2ndash4] Infection or celldamage triggers the release of proinflammatory cytokinessuch as interleukin- (IL-) 1120573 and tumor necrosis factor-(TNF-) 120572 which are key mediators of the host immuneresponse Signal transduction of inflammatory cytokinesincludes ligands receptors coreceptors and cytosolic andnuclear signaling mechanisms These mechanisms can acti-vate the NF-120581B JNK p38 MAPK STAT and PI3K signalingpathways [5] On the other hand inflammasomes play a keyrole in the regulation of inflammation and immune responses

by producing proinflammatory cytokines [6] Studies haveshown that inflammasomes are involved in atherosclerosis[7] metabolic syndrome [8] type 2 diabetes [9] alcoholicsteatohepatitis [10] mucosal immune response [11] rheuma-toid arthritis [12] and gout [13] The nucleotide-bindingdomain- (NOD-) like receptor protein 3 (NLRP3) inflamma-some is a multiprotein complex that regulates the maturationof proinflammatory cytokines IL-1120573 and IL-18 It consistsof NOD-like receptor NLRP3 the adaptor protein ASC(apoptosis-associated speck-like protein containing caspase-1 activator domain CARD) and caspase-1 Upon exogenousand endogenous stimuli the NLRP3 inflammasome formsthrough activation of NLRP3 and recruitment of ASC andpro-caspase-1 resulting in caspase-1 activation and subse-quently processing of pro-IL-1120573 and pro-IL-18 into theiractive forms [14]

Iridoid is derived from Scrophulariaceae RubiaceaeLabiatae Gentianaceae Oleaceae and so on it is mainly

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 819053 10 pageshttpdxdoiorg1011552014819053

2 Mediators of Inflammation

derived from Scrophularia L [15] The iridoids are com-prised of a large family of distinctive bicyclic monoter-penes that possess a wide range of pharmacological prop-erties including anticancer anti-inflammatory antifungaland antibacterial activities [16 17] Scropolioside A exhibitedanti-inflammatory properties against different experimentalmodels of delayed-type hypersensitivity Scropolioside A alsoinhibited the production of prostaglandin E2 leukotriene B4nitric oxide and some interleukin but had no effect on theproduction of IL-10 Moreover it modified the expression ofboth nitric oxide synthase-2 and cyclooxygenase-2 as wellas the activation of NF-120581B in RAW 2647 macrophages [18]Scropolioside D also possessed significant antidiabetic andanti-inflammatory activity [19] However although scrop-olioside B exhibited moderate antibacterial activity againststrains of multidrug and methicillin-resistant Staphylococcusaureus (MRSA) and a panel of rapidly growing mycobac-teria with minimum inhibitory concentration (MIC) valuesranging from 32 to 128120583gmL [20] it had no significanteffect on TXB2-release [21] We previously reported differentanti-inflammatory effects of other iridoid components [2223] Scrophularia dentata Royle ex Benth in Tibet is usedfor antiviral and anti-inflammatory treatment Therefore inthis study we examined scropolioside B isolated from Sdentata Royle ex BenthWe determine whether scropoliosideB exhibits anti-inflammatory effect and further analyze itsunderlying mechanism in human monocytes

2 Materials and Methods

21 Cell Cultures and Reagents Human Embryonic Kidney293 cells (HEK293 cells) were purchased from YongzhengGrubber Products Corporation (Nanjing China) and THP-1cells were from the Chinese Academy of Sciences (ShanghaiChina) Cells were cultured on 100mm tissue culture dishesor 100mL flasks in Dulbeccorsquos modified Eaglersquos medium(DMEM) containing 10 fetal bovine serum (Gibco Invitro-gen USA) at 37∘C in a humidified incubator under 5 CO

2

and 95 air During experiments the cells were plated in 24-well plates or 30mm tissue culture dishes for 16 or 24 h

22 Extraction and Isolation of Scropolioside B NMR spectrawere acquired using a Bruker AM-400 spectrometer ESI-MS and HR-ESI-MS were obtained using an Esquire 3000plus and a Q-TOF-Ultima mass spectrometer respectivelySilica gel (200mesh to 300mesh QingdaoHaiyang ChemicalCo Ltd Qingdao China) C

18reversed-phase silica gel

(150 to 200mesh Fuji Silysia Chemical Ltd Aichi Japan)MCI gel (CHP20P 75 120583m to 150 120583m Mitsubishi ChemicalIndustries Ltd Tokyo Japan) and Sephadex LH-20 gel(Pharmacia Biotech AB Uppsala Sweden) were used forcolumn chromatography (CC)

Plant Material Whole plants of S dentata Royle ex Benthwere collected from Lhasa Tibet China in October 2010The plant was identified by Professor Zhili Zhao (Schoolof Pharmacy Shanghai University of Traditional ChineseMedicine) The voucher specimen (number CX2010) was

deposited at the Herbarium of the Department of TCMChemistry School of Pharmacy of Shanghai University ofTraditional Chinese Medicine (Shanghai China)

23 Luciferase Assay To assay NF-120581B promoter activityTHP-1 cells were transiently transfected with a luciferasereporter gene pNF-120581B-TA-Luc was purchased from Strata-gene (USA) Cells were plated one day prior to transfectionso that cells will be approximately 80 confluent on theday of transfection On the day of transfection DNA wasdiluted to 2 120583g per 100 120583L of serum-free medium and anappropriate amount of FUGENE HD Transfection Reagent(Promega USA) was added to achieve the proper ratio ofreagent toDNAThemixturewas incubated for 0ndash15minutesand 100 120583L was added to each well to be transfected Cellswere transfected for 5 hours before changing to fresh mediaOne hour after transfection TNF-120572 was added to the cellsfor 16ndash20 hours Luciferase activity was measured in the celllysates using the Promega Luciferase Assay System accordingto the manufacturerrsquos instructions (Promega USA)

24 Quantitative Real-Time PCR (qRT-PCR) Total RNA wasextracted using TRIzol (Life Technology Carlsbad CAUSA)according to the manufacturerrsquos instructions Real-time PCRamplification and detection were performed using the SYBRGreen qPCR SuperMix-UDG with ROX (Life Technologies)in a fluorescence thermal cycler (StepOne Real-Time PCRsystem Life Technologies) according to the manufacturerrsquosprotocol Gene expression was normalized using GAPDHas a reference gene Relative mRNA expression levels werecalculated following the ΔΔCt method with the followingprimers GAPDH IL-1120573 TNF-120572 IL-32120573 IL-32120574 CLS1 andNLRP3 in Table 1 All amplifications were conducted withinthe linear range of the assay and normalized to respectiveGAPDH levels using SPSS Version 180 (SPSS Institute IncChicago IL USA)

25 Western Blot After treatment cells were centrifuged andlysed in TritonNP-40 lysis buffer containing 05 Triton X-100 05 Nonidet P-40 10mmolL Tris pH 75 25mmolLKCL 150mmolL NaCl 20mmolL 120573-glycerophosphate50mmolL NaF and 1mmolL Na

3VO4 sonicated by JY92-

2D ultrasonic homogenizer (NingBo Scientz Biotechnol-ogy Co Ltd Zhejiang China) and then centrifuged for10min at 10000 g The supernatant was analyzed for proteinconcentration using a protein assay kit (Bio-Rad HerculesCA USA) and equal amounts of protein (30 120583gsample)were separated by SDS-PAGE and blotted onto nitrocellulosemembranes (Pall China Shanghai China) The blots wereblocked overnight with 5 nonfat dried milk in a buffercontaining 140mmolL NaCl 20mmolL Tris-HCl at pH 75and 01Tween-20 and incubatedwith the following primaryantibodies NLRP3 Rabbit mAb (Cell Signaling Technol-ogy Beverly MA USA) IL-1120573 Mouse Monoclonal IgG

20

(Santa Cruz Biotechnology Dallas TX USA) and HRP-conjugated Monoclonal Mouse Anti-GAPDH (KangChenBio-tech Shanghai China) incubated at 4∘Cwith gentle shak-ing overnight The secondary antibody was conjugated with

Mediators of Inflammation 3

Table 1 Primer sequences of the genes tested in this study

Gene Direction Primer sequences

IL-1120573 Forward 5-AAACAGATGAAGTGCTCCTTCCAGGReverse 5-TGGAGAACACCACTTGTTGCTCCA

TNF-120572 Forward 5-CAGAGGGAAGAGTTCCCCAGReverse 51015840-CCTTGGTCTGGTAGGAGACG

IL-32120573 Forward 5-GAGTTTCTGCTGCTCTCTGTCAReverse 5-ATTTTGAGGATTGGGGTTCAG

IL-32120574 Forward 5-GAGTTTCTGCTGCTCTCTGTCAReverse 5-ATTTTGAGGATTGGGGTTCAG

NLRP3 Forward 5-CTACACACGACTGCGTCTCATCAAReverse 5-GGGTCAAACAGCAACTCCATCTTA

CLS1 Forward 5-GAGTATGCCACAGTATGAAAACCCAReverse 5-CGAGCAATAAATCCATCCAACAA

GAPDH Forward 5-AGAAGGCTGGGGCTCATTTGReverse 5-AGGGGCCATCCACAGTCTTC

horseradish peroxidase The membrane was exposed to highperformance autoradiography film (Fuji Film Corp TokyoJapan) and visualized using the ECL Immobilon Westernchemiluminescent HRP substrate (WBKLS0500) (MilliporeUSA) Quantitative analysis was performed by QuantityOne software Western blot experiments were performed intriplicate

26 ELISA Theculturemedium from the control and treatedcells was collected centrifuged and stored at minus80∘C untiltested IL-1120573 was measured using Abcam Human ELISAKit (Abcam Cambridge England) according to the man-ufacturerrsquos instructions Standard or sample was added toeach well and incubated for 25 h at room temperature Theprepared biotin antibody was then added to each well fol-lowed by incubation for 1 h at room temperature Streptavidinsolution was added and incubated for 45 minutes at roomtemperature Finally TMB One-Step Development Solutionwas added to each well and incubated for 30 minutes at roomtemperature A stop solution was then added to each well andread at 450 nm immediately

27 Date Analysis Each experiment was performed at least 3times The results were presented as means plusmn standard errorof mean (SD) All data was analyzed using SPSS softwareand one-way ANOVA was used to determine the statisticalsignificance of differences between the means Differenceswere statistically significant when 119875 lt 005

3 Results

31 Blocking IL-1120573 and TNF-120572 Expression by Scropolioside BSince scropolioside B contains structures of catalpol and twophenylpropanoids (Figure 1(a)) we compared and tested theanti-inflammatory capabilities of both scropolioside B and

catalpol in THP-1 cells The expression of IL-1120573 and TNF-120572 was significantly induced by lipopolysaccharide (LPS) orpalmitic acid (PA) a free fatty acid with potential proin-flammatory mediators compared to control-treated THP-1 cells (Figure 1) This indicated that cellular exposure toLPS or PA induced the secretion of various cytokines thatlead to the initiation and amplification of inflammation Toinvestigate the anti-inflammatory effect of scropolioside Bwepreincubated THP-1 cells with the compound for 1 h and sub-sequently stimulated the cells with LPS or PA We found thatscropolioside B significantly blocked the increase in IL-1120573 andTNF-120572 levels induced by LPS or PA (Figure 1) However atthe concentration of 50120583molL catalpol did not effectivelyblock expression of IL-1120573 and TNF-120572 although these anti-inflammatory effects had been reported [24 25] Theseobservations suggested that scropolioside B has stronger anti-inflammatory activity compared to catalpol

32 Inhibition of Nuclear Factor 120581BActivation by ScropoliosideB NF-120581B is an essential transcription factor involved in theproduction of several cytokines that mediate the inflam-matory response To investigate overall anti-inflammatoryactivity of scropolioside B we used a luciferase reporterassay to determine nuclear factor kappa B (NF-120581B) activityAfter HEK293 cells were transferred with either the NF-120581Bor the control plasmid cells were treated with or withoutscropolioside B for 1 h and then stimulated with 100 ngmLof TNF-120572 An increase in luciferase activity was observedafter stimulation with TNF-120572 suggesting that NF-120581B wasactivated by TNF-120572 (Figure 2(a)) Pretreatment with scrop-olioside B (008ndash50120583molL) inhibited TNF-120572-induced NF-120581B activation in a concentration-dependentmanner Further-more scropolioside B exhibited an IC

50value of 102 120583molL

(Figure 2(b)) These results showed that scropolioside B-mediated inhibition of inflammatory cytokine induction wasdue to the suppression of NF-120581B

4 Mediators of Inflammation

Scropolioside B

OO

OHO

O

OHO

OH

OHOH

OMe

OO

O

O

H3COCO

Catalpol

OO

OHO

HO

OHO

OH

OH

OH

(a)

lowastlowast

3000

4000

2000

1000

000

IL-1120573

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(b)

lowastlowast

1000

1200

800

600

400

200

000

TNF-120572

relat

ive m

RNA

leve

ls

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

LPS (1120583gmL)

(c)

lowastlowast

1500

1000

2000

500

000

IL-1120573

relat

ive m

RNA

leve

ls

PA (50120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

lowast

300

200

500

600

400

100

000

PA (50120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

TNF-120572

relat

ive m

RNA

leve

ls

(e)

Figure 1 Different chemical structures of scropolioside B and catalpol (a) The effects of scropolioside B and catalpol on LPS-inducedexpression of IL-1120573 and TNF-120572 in THP-1 cells THP-1 cells were pretreated with 50120583molL catalpol or scropolioside B for 1 h and thenstimulated with LPS (1 120583gmL) for another 24 h ((b)-(c)) The effects of scropolioside B and catalpol on PA-induced expression of IL-1120573 andTNF-120572 in THP-1 cells THP-1 cells were pretreated with 50120583molL catalpol or scropolioside B for 1 h and then stimulated with PA (50120583gmL)for another 24 h ((d)-(e)) The expression of IL-1120573 and TNF-120572mRNA was measured by RT-PCRThe data represent the mean values of overthree experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS or PA alone 119875 lt 005 compared to vehiclecontrol lowast119875 lt 005 compared to LPS or PA alone

Mediators of Inflammation 5

lowast

lowast

lowastlowastlowast

600

400

200

000Relat

ive l

ucife

rase

uni

ts (fo

ld ch

ange

ove

r con

trol)

TNF-120572 (100ngmL) minus

minus

+

minus

+

50

+

10

+

04

+

008

+

2Scropolioside B (120583M)

(a)

6000

4000

2000

000

Inhi

bitio

n (

)

minus8 minus7 minus6 minus5 minus4

Concentration (log M)

IC50 = 102 120583M

(b)

Figure 2 Scropolioside B inhibited TNF-120572-inducedNF-120581B activation Cells were preincubated for 1 hourwith different doses of scropoliosideB and then stimulated with 1120583gmL TNF-120572 for 16 hours The results shown are representative of 3 separate experiments Data are expressedas means plusmn SD 119875 lt 005 versus the control lowast119875 lt 005 versus the TNF-120572 (a) IC50 values of 102 120583molL (b)

lowastlowast

3000

2000

1000

000

IL-3

2120573 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowast

4000

3000

2000

1000

000

IL-3

2120574 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(b)

Figure 3 The effects of scropolioside B and catalpol on LPS-induced expression of IL-32120573 and IL-32120574 in THP-1 cells THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The expression of IL-32120573 and IL-32120574mRNA was measured by RT-PCRThe data represent the mean values of over three experiments plusmn SD 119875 lt 001 comparedto vehicle control 119875 lt 005 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone and lowast119875 lt 005 compared to LPS alone

33 Scropolioside B Reduced the IL-32 Expression IL-32 isa proinflammatory cytokine involved in several diseasesincluding infections chronic inflammation and cancerTNF-120572 or LPS are known inducers of IL-32 IL-32-dependenteffects of IL-1120573 on endothelial cell functions [26] We nextdetermined the inhibitory effects of scropolioside B on IL-32 expression Pretreatmentwith scropolioside B significantlydiminished the increase in mRNA expression levels of IL-32120573 and IL-32120574 induced by LPS stimulation (Figures 3(a) and

3(b)) similar to IL-1120573 and TNF-120572 expression pattern in LPS-induced THP-1 cells

34 Scropolioside B Decreases Expression of NLRP3 Inflam-masomes regulate maturation of IL-1120573 and IL-18 and pyrop-tosisNLRP3 is amember of inflammasomeswhich constitutethe compound with ASC and caspase-1 to catalyze thematuration of IL-1120573 [27] To observe whether inflammatory

6 Mediators of Inflammation

lowastlowast

100

150

050

000

NLR

P3 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowastlowastlowast

100

150

050

000

CLS1

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus + minus

(b)

LPS (1120583gmL)

NLRP3

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

100

200

150

050

000

Band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

lowastlowast lowastlowast

(d)

Figure 4 Scropolioside B and catalpol inhibited the expression of CLS1 and NLRP3 in LPS-induced THP-1 cells ((a)-(b)) The effects ofscropolioside B and catalpol on protein expression ofNLRP3 in LPS-inducedTHP-1 cells ((c)-(d)) THP-1 cells were pretreatedwith 50120583molLcatalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The protein expression of NLRP3 was measuredby western blot The expression of CLS1 and NLRP3 mRNA was measured by RT-PCR The data represent the mean values of over threeexperiments plusmn SD 119875 lt 001 compared to vehicle control 119875 lt 005 compared to vehicle control and lowastlowast119875 lt 001 compared to LPS alone

factors induce NLRP3 expression we stimulated THP-1cells with LPS for 24 h We observed that LPS upregulatedNLRP3 mRNA and protein (Figures 4(a) 4(c) and 4(d))Similarly LPS also significantly enhanced mRNA expressionof cardiolipin synthetase 1 (CLS1) (Figure 4(b)) a mitochon-drial enzyme catalysing cardiolipin synthesis necessary forinflammasome NLRP3 activity [28] As shown in Figures4(a)ndash4(d) pretreatment with scropolioside B inhibited the

expressions of NLRP3 mRNA and protein as well as CLS1mRNA We also found that catalpol was as equally effectiveas scropolioside B (Figures 4(a)ndash4(d)) suggesting that thisinhibitory effect may be from the same catalpol structure(Figure 6)

35 Scropolioside B Decreases Expression of Pro-IL-1120573 andIL-1120573 To comprehensively evaluate the inhibitory effect of

Mediators of Inflammation 7

LPS (1120583gmL)

Pro-IL-1120573

IL-1120573

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

500

400

300

200

100

000

Pro-

IL-1120573

band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

lowastlowast

minus minus + minus

minus minus minus +

(b)

lowastlowast

250

200

150

050

100

000

IL-1120573

ban

d de

nsity

(fol

d ba

sal)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

lowastlowast

1500

500

1000

000

IL-1120573

(pg

mL)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

Figure 5 Scropolioside B and catalpol inhibited the expression of pro-IL-1120573 and IL-1120573 in LPS-induced THP-1 cells ((a)ndash(d)) THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 hThe protein expressionof pro-IL-1120573 and IL-1120573 was measured by western blot The levels of cytokines in the medium were measured using an ELISA kit The datarepresent the mean values of over three experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone

scropolioside B and catalpol we performed further studiesabout scropolioside B impact on protein expression of pro-IL-1120573 and IL-1120573 As shown in Figures 5(a)ndash5(d) pretreatmentwith scropolioside B inhibited the expressions of pro-IL-1120573and IL-1120573proteinThe inhibition of pro-IL-1120573 is stronger thanIL-1120573 Catalpol does not inhibit the protein expression of pro-IL-1120573 and IL-1120573

4 Discussion

Scropolioside B which is from S dentata Royle ex Benthhas antipyretic detoxicating effect It is used in Tibetanmedicine such as smallpox measles other infectious feversand inflammatory diseases In this study we demon-strated that scropolioside B an iridoid glycoside containing

8 Mediators of Inflammation

LPSTLR

P50

P50

P65

P65

Scropolioside BScropolioside B

Catalpol

Caspase-1

Pro-IL-1120573 mRNA

Pro-IL-1120573 IL-1120573

Nucleus

NLRP3

Figure 6 A schematic diagram of the regulation of NLRP3 inflam-masome by scropolioside B and catalpol

a catalpol and two phenylpropanoids was more effectivethan catalpol in inhibiting the expressions of IL-1120573 andTNF-120572 in THP-1 cells activated by LPS or palmitic acid(Figure 1) Our results suggested that scropolioside B hashigher anti-inflammatory activity than catalpol althoughsome studies have reported that catalpol does demonstrateanti-inflammatory effects at high dose by inhibiting COX-2activity TNF-120572 formation monocyte chemotactic protein-1 (MCP-1) and nitric oxide production [24 25] As shownin Figure 2 the anti-inflammatory effects of scropolioside Bwere mediated by blocking NF-120581B activity (Figure 2) IL-1120573is involved in the onset of acute local or systemic inflam-mation and contributes to a variety of chronic noninfectiousdiseases including ischemic injury atherosclerosis type 2diabetes and osteoarthritis Endogenousmetabolites such asoxidized fatty acids high glucose uric acid crystals activatedcomplement necrotic cells and cytokines can stimulatethe synthesis of the inactive IL-1120573 precursor which awaitsprocessing by the inflammasome complex to be activated[29]

Inflammasomes regulate maturity of IL-1120573 IL-18 andpyroptosis and recognize microbial products or endogenousmolecules released from damaged cells [27] Inflammasomeshave several member proteins including NLRP1 NLRP3NLRC4 AIM2 and NRP6 [6] Our results showed thatscropolioside B can inhibit mRNA and protein expressionof inflammasome NLRP3 and prevents the secretion of IL-1120573 (Figure 4) Interestingly catalpol has a similar inhibitoryeffect on NLRP3 expression compared to scropolioside Bsuggesting that scropolioside B and catalpol inhibit IL-1120573 andNLRP3 expression by different mechanisms ScropoliosideB and catalpol also block the expression of CLS1 which isan enzyme in the final step of mitochondrial cardiolipinsynthesis by catalysing the transfer of a phosphatidyl residuefrom diacylglycerol to phosphatidylglycerol Iyer et al [28]reported that mitochondrial cardiolipin and reactive oxygenspecies are needed for inflammasome NLRP3 activity Car-diolipin can bind directly to NLRP3 and silencing of cardi-olipin synthesis specifically inhibits inflammasome NLRP3activation [28] Based on these observations we believe

that scropolioside B not only blocks NF-120581B pathway butalso inhibits NLRP3 CLS1 and IL-1120573 expressions Howevercatalpol only prevents the expression of NLRP3 and CLS1(Figure 5)

Our results also showed that scropolioside B but notcatalpol blocked IL-32120573120574 expression (Figure 3) Severalstudies have shown that IL-32 an important proinflamma-tory cytokine in rheumatoid arthritis enhanced IL-6 and IL-8 production in fibroblast-like synoviocytes [30ndash32] Somestudies also have shown that IL-32 is closely associated withliver fibrosis of chronic viral hepatitis [33 34] Furthermorecompared with primary blood monocytes IL-1120573 TNF-120572 orLPS can stimulate high levels of IL-32 expression through theI120581B kinase-120573NF-120581B and ERK pathways in human umbilicalvein endothelial cells aortic macrovascular endothelial cellsand cardiac and pulmonary microvascular endothelial cells[26] Conversely IL-32 also stimulates IL-1120572 IL-1120573 IL-6TNF-120572 and chemokines via NF-120581B p38 MAPK and AP-1activation [26 35] IL-32 promotes angiogenesis propagatingvascular inflammation and exacerbates sepsis in a mousemodel [36 37] Recent studies have shown that atheroscle-rosis maybe associated with IL-32 production [38]

In conclusion scropolioside B significantly diminishedexpression and secretion of IL-1120573 IL-32 and TNF-120572 Weshow that this is mediated by modulating NF-120581B NLRP3and CLS1 levels Additional studies are needed to furtherelucidate other targets by which scropolioside and catalpolregulate inflammation The results of this study strengthenprevious understanding of the anti-inflammatory effects ofiridoids and highlight scropolioside B as a potential drugfor the treatment of rheumatoid arthritis and atheroscleroticdisease

Conflict of Interests

The authors declared no conflict of interests

Authorsrsquo Contribution

Tiantian Zhu and Liuqiang Zhang contributed equally to thiswork

Acknowledgments

This work was supported by grants from the NationalScience and Technology Major Project of the Ministry ofScience and Technology of China (2009ZX09311-003) theNational Natural Science Foundation of China (8117351881274130) the Shanghai 085 Project of Higher EducationConnotation Construction (085ZY1202) and the FundingScheme for Training Young Teachers in Shanghai Colleges(no ZZszy13057)

References

[1] G Pawelec DGoldeck andEDerhovanessian ldquoInflammationageing and chronic diseaserdquo Current Opinion in Immunologyvol 29 no 1 pp 23ndash28 2014

Mediators of Inflammation 9

[2] L M Coussens L Zitvogel and A K Palucka ldquoNeutralizingtumor-promoting chronic inflammation a magic bulletrdquo Sci-ence vol 339 no 6117 pp 286ndash291 2013

[3] G E McKellar D W McCarey N Sattar and I B McInnesldquoRole for TNF in atherosclerosis Lessons from autoimmunediseaserdquo Nature Reviews vol 6 no 6 pp 410ndash417 2009

[4] V H Perry and J Teeling ldquoMicroglia and macrophages of thecentral nervous system the contribution of microglia primingand systemic inflammation to chronic neurodegenerationrdquoSeminars in Immunopathology vol 35 no 5 pp 601ndash612 2013

[5] M L Schmitz A Weber T Roxlau M Gaestel and M KrachtldquoSignal integration crosstalk mechanisms and networks in thefunction of inflammatory cytokinesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1813 no 12 pp 2165ndash21752011

[6] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[7] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 pp 1357ndash1361 2010

[8] K Schroder R Zhou and J Tschopp ldquoThe NLRP3 inflamma-some a sensor formetabolic dangerrdquo Science vol 327 no 5963pp 296ndash300 2010

[9] S L Masters A Dunne S L Subramanian et al ldquoActivationof the NLRP3 inflammasome by islet amyloid polypeptideprovides a mechanism for enhanced IL-1120573 2 in type 2 diabetesrdquoNature Immunology vol 11 no 10 pp 897ndash904 2010

[10] J Petrasek S Bala T Csak et al ldquoIL-1 receptor antagonistameliorates inflammasome-dependent alcoholic steatohepatitisin micerdquo Journal of Clinical Investigation vol 122 no 10 pp3476ndash3489 2012

[11] J Dupaul-Chicoine G Yeretssian K Doiron et al ldquoControl ofintestinal homeostasis colitis and colitis-associated colorectalcancer by the inflammatory caspasesrdquo Immunity vol 32 no 3pp 367ndash378 2010

[12] P I Sidiropoulos G Goulielmos G K Voloudakis E Petrakiand D T Boumpas ldquoInflammasomes and rheumatic diseasesevolving conceptsrdquo Annals of the Rheumatic Diseases vol 67no 10 pp 1382ndash1389 2008

[13] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[14] E Latz T S Xiao andA Stutz ldquoActivation and regulation of theinflammasomesrdquoNature Reviews Immunology vol 13 no 6 pp397ndash411 2013

[15] L-Q Zhang and Y-M Li ldquoAdvances in studies on chemicalconstituents in plants of Scrophularia L and their pharmacolog-ical effects in recent ten yearsrdquo Chinese Traditional and HerbalDrugs vol 42 no 11 pp 2360ndash2368 2011 (Chinese)

[16] A Viljoen N Mncwangi and I Vermaak ldquoAnti-inflammatoryiridoids of botanical originrdquo Current Medicinal Chemistry vol19 no 14 pp 2104ndash2127 2012

[17] R Tundis M R Loizzo F Menichini and G A StattildquoBiological and pharmacological activities of iridoids recentdevelopmentsrdquoMini-Reviews inMedicinal Chemistry vol 8 no4 pp 399ndash420 2008

[18] E Bas M C Recio S Manez et al ldquoNew insight intothe inhibition of the inflammatory response to experimentaldelayed-type hypersensitivity reactions inmice by scropoliosideArdquo European Journal of Pharmacology vol 555 no 2-3 pp 199ndash210 2007

[19] B Ahmed A J Al-Rehaily T A Al-Howiriny K A El-Sayed and M S Ahmad ldquoScropolioside-D2 and harpagoside-B two new iridoid glycosides from Scrophularia deserti andtheir antidiabetic and antiinflammatory activityrdquo Biological andPharmaceutical Bulletin vol 26 no 4 pp 462ndash467 2003

[20] M Stavri K T Mathew and S Gibbons ldquoAntimicrobialconstituents of Scrophularia desertirdquoPhytochemistry vol 67 no14 pp 1530ndash1533 2006

[21] P B Benito A M D Lanza A M S Sen et al ldquoEffects of someiridoids from plant origin on arachidonic acid metabolism incellular systemsrdquo Planta Medica vol 66 no 4 pp 324ndash3282000

[22] P Wang L Sun J Tan J Xu F Guo and Y Li ldquoTwo newglycosidated coumaramides from Clerodendron cyrtophyllumrdquoFitoterapia vol 83 no 8 pp 1494ndash1499 2012

[23] L Zhang L Feng Q Jia et al ldquoEffects of 120573-glucosidasehydrolyzed products of harpagide and harpagoside oncyclooxygenase-2 (COX-2) in vitrordquo Bioorganic and MedicinalChemistry vol 19 no 16 pp 4882ndash4886 2011

[24] K S Park B H Kim and I-M Chang ldquoInhibitory potencies ofseveral iridoids on cyclooxygenase-1 cyclooxygnase-2 enzymesactivities tumor necrosis factor-120572 and nitric oxide produc-tion In Vitrordquo Evidence-based Complementary and AlternativeMedicine vol 7 no 1 pp 41ndash45 2010

[25] H-J Choi H-J Jang T-W Chung et al ldquoCatalpol sup-presses advanced glycation end-products-induced inflamma-tory responses through inhibition of reactive oxygen species inhuman monocytic THP-1 cellsrdquo Fitoterapia vol 86 no 1 pp19ndash28 2013

[26] C A Nold-Petry M F Nold J A Zepp S-H Kim N FVoelkel and C A Dinarello ldquoIL-32-dependent effects of IL-1120573 on endothelial cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no10 pp 3883ndash3888 2009

[27] V A K Rathinam S K Vanaja and K A Fitzgerald ldquoRegu-lation of inflammasome signalingrdquoNature Immunology vol 13no 4 pp 333ndash342 2012

[28] S S Iyer Q He J R Janczy et al ldquoMitochondrial cardiolipinis required for Nlrp3 inflammasome activationrdquo Immunity vol39 no 2 pp 311ndash323 2013

[29] CADinarello ldquoA clinical perspective of IL-1120573 as the gatekeeperof inflammationrdquo European Journal of Immunology vol 41 no5 pp 1203ndash1217 2011

[30] L A B Joosten M G Netea S-H Kim et al ldquoIL-32 aproinflammatory cytokine in rheumatoid arthritisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 9 pp 3298ndash3303 2006

[31] B Heinhuis M I Koenders F A Van De Loo M G NeteaW B Van Den Berg and L A B Joosten ldquoInflammation-dependent secretion and splicing of IL-32120574 in rheumatoidarthritisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 12 pp 4962ndash4967 2011

[32] Y-G Kim C-K Lee S-H Kim W-S Cho S H Mun and BYoo ldquoInterleukin-32120574 enhances the production of IL-6 and IL-8in fibroblast-like synoviocytes Via Erk12 activationrdquo Journal ofClinical Immunology vol 30 no 2 pp 260ndash267 2010

[33] A R Moschen T Fritz A D Clouston et al ldquoInterleukin-32a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosisrdquoHepatology vol 53 no6 pp 1819ndash1829 2011

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 2: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

2 Mediators of Inflammation

derived from Scrophularia L [15] The iridoids are com-prised of a large family of distinctive bicyclic monoter-penes that possess a wide range of pharmacological prop-erties including anticancer anti-inflammatory antifungaland antibacterial activities [16 17] Scropolioside A exhibitedanti-inflammatory properties against different experimentalmodels of delayed-type hypersensitivity Scropolioside A alsoinhibited the production of prostaglandin E2 leukotriene B4nitric oxide and some interleukin but had no effect on theproduction of IL-10 Moreover it modified the expression ofboth nitric oxide synthase-2 and cyclooxygenase-2 as wellas the activation of NF-120581B in RAW 2647 macrophages [18]Scropolioside D also possessed significant antidiabetic andanti-inflammatory activity [19] However although scrop-olioside B exhibited moderate antibacterial activity againststrains of multidrug and methicillin-resistant Staphylococcusaureus (MRSA) and a panel of rapidly growing mycobac-teria with minimum inhibitory concentration (MIC) valuesranging from 32 to 128120583gmL [20] it had no significanteffect on TXB2-release [21] We previously reported differentanti-inflammatory effects of other iridoid components [2223] Scrophularia dentata Royle ex Benth in Tibet is usedfor antiviral and anti-inflammatory treatment Therefore inthis study we examined scropolioside B isolated from Sdentata Royle ex BenthWe determine whether scropoliosideB exhibits anti-inflammatory effect and further analyze itsunderlying mechanism in human monocytes

2 Materials and Methods

21 Cell Cultures and Reagents Human Embryonic Kidney293 cells (HEK293 cells) were purchased from YongzhengGrubber Products Corporation (Nanjing China) and THP-1cells were from the Chinese Academy of Sciences (ShanghaiChina) Cells were cultured on 100mm tissue culture dishesor 100mL flasks in Dulbeccorsquos modified Eaglersquos medium(DMEM) containing 10 fetal bovine serum (Gibco Invitro-gen USA) at 37∘C in a humidified incubator under 5 CO

2

and 95 air During experiments the cells were plated in 24-well plates or 30mm tissue culture dishes for 16 or 24 h

22 Extraction and Isolation of Scropolioside B NMR spectrawere acquired using a Bruker AM-400 spectrometer ESI-MS and HR-ESI-MS were obtained using an Esquire 3000plus and a Q-TOF-Ultima mass spectrometer respectivelySilica gel (200mesh to 300mesh QingdaoHaiyang ChemicalCo Ltd Qingdao China) C

18reversed-phase silica gel

(150 to 200mesh Fuji Silysia Chemical Ltd Aichi Japan)MCI gel (CHP20P 75 120583m to 150 120583m Mitsubishi ChemicalIndustries Ltd Tokyo Japan) and Sephadex LH-20 gel(Pharmacia Biotech AB Uppsala Sweden) were used forcolumn chromatography (CC)

Plant Material Whole plants of S dentata Royle ex Benthwere collected from Lhasa Tibet China in October 2010The plant was identified by Professor Zhili Zhao (Schoolof Pharmacy Shanghai University of Traditional ChineseMedicine) The voucher specimen (number CX2010) was

deposited at the Herbarium of the Department of TCMChemistry School of Pharmacy of Shanghai University ofTraditional Chinese Medicine (Shanghai China)

23 Luciferase Assay To assay NF-120581B promoter activityTHP-1 cells were transiently transfected with a luciferasereporter gene pNF-120581B-TA-Luc was purchased from Strata-gene (USA) Cells were plated one day prior to transfectionso that cells will be approximately 80 confluent on theday of transfection On the day of transfection DNA wasdiluted to 2 120583g per 100 120583L of serum-free medium and anappropriate amount of FUGENE HD Transfection Reagent(Promega USA) was added to achieve the proper ratio ofreagent toDNAThemixturewas incubated for 0ndash15minutesand 100 120583L was added to each well to be transfected Cellswere transfected for 5 hours before changing to fresh mediaOne hour after transfection TNF-120572 was added to the cellsfor 16ndash20 hours Luciferase activity was measured in the celllysates using the Promega Luciferase Assay System accordingto the manufacturerrsquos instructions (Promega USA)

24 Quantitative Real-Time PCR (qRT-PCR) Total RNA wasextracted using TRIzol (Life Technology Carlsbad CAUSA)according to the manufacturerrsquos instructions Real-time PCRamplification and detection were performed using the SYBRGreen qPCR SuperMix-UDG with ROX (Life Technologies)in a fluorescence thermal cycler (StepOne Real-Time PCRsystem Life Technologies) according to the manufacturerrsquosprotocol Gene expression was normalized using GAPDHas a reference gene Relative mRNA expression levels werecalculated following the ΔΔCt method with the followingprimers GAPDH IL-1120573 TNF-120572 IL-32120573 IL-32120574 CLS1 andNLRP3 in Table 1 All amplifications were conducted withinthe linear range of the assay and normalized to respectiveGAPDH levels using SPSS Version 180 (SPSS Institute IncChicago IL USA)

25 Western Blot After treatment cells were centrifuged andlysed in TritonNP-40 lysis buffer containing 05 Triton X-100 05 Nonidet P-40 10mmolL Tris pH 75 25mmolLKCL 150mmolL NaCl 20mmolL 120573-glycerophosphate50mmolL NaF and 1mmolL Na

3VO4 sonicated by JY92-

2D ultrasonic homogenizer (NingBo Scientz Biotechnol-ogy Co Ltd Zhejiang China) and then centrifuged for10min at 10000 g The supernatant was analyzed for proteinconcentration using a protein assay kit (Bio-Rad HerculesCA USA) and equal amounts of protein (30 120583gsample)were separated by SDS-PAGE and blotted onto nitrocellulosemembranes (Pall China Shanghai China) The blots wereblocked overnight with 5 nonfat dried milk in a buffercontaining 140mmolL NaCl 20mmolL Tris-HCl at pH 75and 01Tween-20 and incubatedwith the following primaryantibodies NLRP3 Rabbit mAb (Cell Signaling Technol-ogy Beverly MA USA) IL-1120573 Mouse Monoclonal IgG

20

(Santa Cruz Biotechnology Dallas TX USA) and HRP-conjugated Monoclonal Mouse Anti-GAPDH (KangChenBio-tech Shanghai China) incubated at 4∘Cwith gentle shak-ing overnight The secondary antibody was conjugated with

Mediators of Inflammation 3

Table 1 Primer sequences of the genes tested in this study

Gene Direction Primer sequences

IL-1120573 Forward 5-AAACAGATGAAGTGCTCCTTCCAGGReverse 5-TGGAGAACACCACTTGTTGCTCCA

TNF-120572 Forward 5-CAGAGGGAAGAGTTCCCCAGReverse 51015840-CCTTGGTCTGGTAGGAGACG

IL-32120573 Forward 5-GAGTTTCTGCTGCTCTCTGTCAReverse 5-ATTTTGAGGATTGGGGTTCAG

IL-32120574 Forward 5-GAGTTTCTGCTGCTCTCTGTCAReverse 5-ATTTTGAGGATTGGGGTTCAG

NLRP3 Forward 5-CTACACACGACTGCGTCTCATCAAReverse 5-GGGTCAAACAGCAACTCCATCTTA

CLS1 Forward 5-GAGTATGCCACAGTATGAAAACCCAReverse 5-CGAGCAATAAATCCATCCAACAA

GAPDH Forward 5-AGAAGGCTGGGGCTCATTTGReverse 5-AGGGGCCATCCACAGTCTTC

horseradish peroxidase The membrane was exposed to highperformance autoradiography film (Fuji Film Corp TokyoJapan) and visualized using the ECL Immobilon Westernchemiluminescent HRP substrate (WBKLS0500) (MilliporeUSA) Quantitative analysis was performed by QuantityOne software Western blot experiments were performed intriplicate

26 ELISA Theculturemedium from the control and treatedcells was collected centrifuged and stored at minus80∘C untiltested IL-1120573 was measured using Abcam Human ELISAKit (Abcam Cambridge England) according to the man-ufacturerrsquos instructions Standard or sample was added toeach well and incubated for 25 h at room temperature Theprepared biotin antibody was then added to each well fol-lowed by incubation for 1 h at room temperature Streptavidinsolution was added and incubated for 45 minutes at roomtemperature Finally TMB One-Step Development Solutionwas added to each well and incubated for 30 minutes at roomtemperature A stop solution was then added to each well andread at 450 nm immediately

27 Date Analysis Each experiment was performed at least 3times The results were presented as means plusmn standard errorof mean (SD) All data was analyzed using SPSS softwareand one-way ANOVA was used to determine the statisticalsignificance of differences between the means Differenceswere statistically significant when 119875 lt 005

3 Results

31 Blocking IL-1120573 and TNF-120572 Expression by Scropolioside BSince scropolioside B contains structures of catalpol and twophenylpropanoids (Figure 1(a)) we compared and tested theanti-inflammatory capabilities of both scropolioside B and

catalpol in THP-1 cells The expression of IL-1120573 and TNF-120572 was significantly induced by lipopolysaccharide (LPS) orpalmitic acid (PA) a free fatty acid with potential proin-flammatory mediators compared to control-treated THP-1 cells (Figure 1) This indicated that cellular exposure toLPS or PA induced the secretion of various cytokines thatlead to the initiation and amplification of inflammation Toinvestigate the anti-inflammatory effect of scropolioside Bwepreincubated THP-1 cells with the compound for 1 h and sub-sequently stimulated the cells with LPS or PA We found thatscropolioside B significantly blocked the increase in IL-1120573 andTNF-120572 levels induced by LPS or PA (Figure 1) However atthe concentration of 50120583molL catalpol did not effectivelyblock expression of IL-1120573 and TNF-120572 although these anti-inflammatory effects had been reported [24 25] Theseobservations suggested that scropolioside B has stronger anti-inflammatory activity compared to catalpol

32 Inhibition of Nuclear Factor 120581BActivation by ScropoliosideB NF-120581B is an essential transcription factor involved in theproduction of several cytokines that mediate the inflam-matory response To investigate overall anti-inflammatoryactivity of scropolioside B we used a luciferase reporterassay to determine nuclear factor kappa B (NF-120581B) activityAfter HEK293 cells were transferred with either the NF-120581Bor the control plasmid cells were treated with or withoutscropolioside B for 1 h and then stimulated with 100 ngmLof TNF-120572 An increase in luciferase activity was observedafter stimulation with TNF-120572 suggesting that NF-120581B wasactivated by TNF-120572 (Figure 2(a)) Pretreatment with scrop-olioside B (008ndash50120583molL) inhibited TNF-120572-induced NF-120581B activation in a concentration-dependentmanner Further-more scropolioside B exhibited an IC

50value of 102 120583molL

(Figure 2(b)) These results showed that scropolioside B-mediated inhibition of inflammatory cytokine induction wasdue to the suppression of NF-120581B

4 Mediators of Inflammation

Scropolioside B

OO

OHO

O

OHO

OH

OHOH

OMe

OO

O

O

H3COCO

Catalpol

OO

OHO

HO

OHO

OH

OH

OH

(a)

lowastlowast

3000

4000

2000

1000

000

IL-1120573

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(b)

lowastlowast

1000

1200

800

600

400

200

000

TNF-120572

relat

ive m

RNA

leve

ls

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

LPS (1120583gmL)

(c)

lowastlowast

1500

1000

2000

500

000

IL-1120573

relat

ive m

RNA

leve

ls

PA (50120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

lowast

300

200

500

600

400

100

000

PA (50120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

TNF-120572

relat

ive m

RNA

leve

ls

(e)

Figure 1 Different chemical structures of scropolioside B and catalpol (a) The effects of scropolioside B and catalpol on LPS-inducedexpression of IL-1120573 and TNF-120572 in THP-1 cells THP-1 cells were pretreated with 50120583molL catalpol or scropolioside B for 1 h and thenstimulated with LPS (1 120583gmL) for another 24 h ((b)-(c)) The effects of scropolioside B and catalpol on PA-induced expression of IL-1120573 andTNF-120572 in THP-1 cells THP-1 cells were pretreated with 50120583molL catalpol or scropolioside B for 1 h and then stimulated with PA (50120583gmL)for another 24 h ((d)-(e)) The expression of IL-1120573 and TNF-120572mRNA was measured by RT-PCRThe data represent the mean values of overthree experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS or PA alone 119875 lt 005 compared to vehiclecontrol lowast119875 lt 005 compared to LPS or PA alone

Mediators of Inflammation 5

lowast

lowast

lowastlowastlowast

600

400

200

000Relat

ive l

ucife

rase

uni

ts (fo

ld ch

ange

ove

r con

trol)

TNF-120572 (100ngmL) minus

minus

+

minus

+

50

+

10

+

04

+

008

+

2Scropolioside B (120583M)

(a)

6000

4000

2000

000

Inhi

bitio

n (

)

minus8 minus7 minus6 minus5 minus4

Concentration (log M)

IC50 = 102 120583M

(b)

Figure 2 Scropolioside B inhibited TNF-120572-inducedNF-120581B activation Cells were preincubated for 1 hourwith different doses of scropoliosideB and then stimulated with 1120583gmL TNF-120572 for 16 hours The results shown are representative of 3 separate experiments Data are expressedas means plusmn SD 119875 lt 005 versus the control lowast119875 lt 005 versus the TNF-120572 (a) IC50 values of 102 120583molL (b)

lowastlowast

3000

2000

1000

000

IL-3

2120573 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowast

4000

3000

2000

1000

000

IL-3

2120574 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(b)

Figure 3 The effects of scropolioside B and catalpol on LPS-induced expression of IL-32120573 and IL-32120574 in THP-1 cells THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The expression of IL-32120573 and IL-32120574mRNA was measured by RT-PCRThe data represent the mean values of over three experiments plusmn SD 119875 lt 001 comparedto vehicle control 119875 lt 005 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone and lowast119875 lt 005 compared to LPS alone

33 Scropolioside B Reduced the IL-32 Expression IL-32 isa proinflammatory cytokine involved in several diseasesincluding infections chronic inflammation and cancerTNF-120572 or LPS are known inducers of IL-32 IL-32-dependenteffects of IL-1120573 on endothelial cell functions [26] We nextdetermined the inhibitory effects of scropolioside B on IL-32 expression Pretreatmentwith scropolioside B significantlydiminished the increase in mRNA expression levels of IL-32120573 and IL-32120574 induced by LPS stimulation (Figures 3(a) and

3(b)) similar to IL-1120573 and TNF-120572 expression pattern in LPS-induced THP-1 cells

34 Scropolioside B Decreases Expression of NLRP3 Inflam-masomes regulate maturation of IL-1120573 and IL-18 and pyrop-tosisNLRP3 is amember of inflammasomeswhich constitutethe compound with ASC and caspase-1 to catalyze thematuration of IL-1120573 [27] To observe whether inflammatory

6 Mediators of Inflammation

lowastlowast

100

150

050

000

NLR

P3 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowastlowastlowast

100

150

050

000

CLS1

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus + minus

(b)

LPS (1120583gmL)

NLRP3

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

100

200

150

050

000

Band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

lowastlowast lowastlowast

(d)

Figure 4 Scropolioside B and catalpol inhibited the expression of CLS1 and NLRP3 in LPS-induced THP-1 cells ((a)-(b)) The effects ofscropolioside B and catalpol on protein expression ofNLRP3 in LPS-inducedTHP-1 cells ((c)-(d)) THP-1 cells were pretreatedwith 50120583molLcatalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The protein expression of NLRP3 was measuredby western blot The expression of CLS1 and NLRP3 mRNA was measured by RT-PCR The data represent the mean values of over threeexperiments plusmn SD 119875 lt 001 compared to vehicle control 119875 lt 005 compared to vehicle control and lowastlowast119875 lt 001 compared to LPS alone

factors induce NLRP3 expression we stimulated THP-1cells with LPS for 24 h We observed that LPS upregulatedNLRP3 mRNA and protein (Figures 4(a) 4(c) and 4(d))Similarly LPS also significantly enhanced mRNA expressionof cardiolipin synthetase 1 (CLS1) (Figure 4(b)) a mitochon-drial enzyme catalysing cardiolipin synthesis necessary forinflammasome NLRP3 activity [28] As shown in Figures4(a)ndash4(d) pretreatment with scropolioside B inhibited the

expressions of NLRP3 mRNA and protein as well as CLS1mRNA We also found that catalpol was as equally effectiveas scropolioside B (Figures 4(a)ndash4(d)) suggesting that thisinhibitory effect may be from the same catalpol structure(Figure 6)

35 Scropolioside B Decreases Expression of Pro-IL-1120573 andIL-1120573 To comprehensively evaluate the inhibitory effect of

Mediators of Inflammation 7

LPS (1120583gmL)

Pro-IL-1120573

IL-1120573

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

500

400

300

200

100

000

Pro-

IL-1120573

band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

lowastlowast

minus minus + minus

minus minus minus +

(b)

lowastlowast

250

200

150

050

100

000

IL-1120573

ban

d de

nsity

(fol

d ba

sal)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

lowastlowast

1500

500

1000

000

IL-1120573

(pg

mL)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

Figure 5 Scropolioside B and catalpol inhibited the expression of pro-IL-1120573 and IL-1120573 in LPS-induced THP-1 cells ((a)ndash(d)) THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 hThe protein expressionof pro-IL-1120573 and IL-1120573 was measured by western blot The levels of cytokines in the medium were measured using an ELISA kit The datarepresent the mean values of over three experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone

scropolioside B and catalpol we performed further studiesabout scropolioside B impact on protein expression of pro-IL-1120573 and IL-1120573 As shown in Figures 5(a)ndash5(d) pretreatmentwith scropolioside B inhibited the expressions of pro-IL-1120573and IL-1120573proteinThe inhibition of pro-IL-1120573 is stronger thanIL-1120573 Catalpol does not inhibit the protein expression of pro-IL-1120573 and IL-1120573

4 Discussion

Scropolioside B which is from S dentata Royle ex Benthhas antipyretic detoxicating effect It is used in Tibetanmedicine such as smallpox measles other infectious feversand inflammatory diseases In this study we demon-strated that scropolioside B an iridoid glycoside containing

8 Mediators of Inflammation

LPSTLR

P50

P50

P65

P65

Scropolioside BScropolioside B

Catalpol

Caspase-1

Pro-IL-1120573 mRNA

Pro-IL-1120573 IL-1120573

Nucleus

NLRP3

Figure 6 A schematic diagram of the regulation of NLRP3 inflam-masome by scropolioside B and catalpol

a catalpol and two phenylpropanoids was more effectivethan catalpol in inhibiting the expressions of IL-1120573 andTNF-120572 in THP-1 cells activated by LPS or palmitic acid(Figure 1) Our results suggested that scropolioside B hashigher anti-inflammatory activity than catalpol althoughsome studies have reported that catalpol does demonstrateanti-inflammatory effects at high dose by inhibiting COX-2activity TNF-120572 formation monocyte chemotactic protein-1 (MCP-1) and nitric oxide production [24 25] As shownin Figure 2 the anti-inflammatory effects of scropolioside Bwere mediated by blocking NF-120581B activity (Figure 2) IL-1120573is involved in the onset of acute local or systemic inflam-mation and contributes to a variety of chronic noninfectiousdiseases including ischemic injury atherosclerosis type 2diabetes and osteoarthritis Endogenousmetabolites such asoxidized fatty acids high glucose uric acid crystals activatedcomplement necrotic cells and cytokines can stimulatethe synthesis of the inactive IL-1120573 precursor which awaitsprocessing by the inflammasome complex to be activated[29]

Inflammasomes regulate maturity of IL-1120573 IL-18 andpyroptosis and recognize microbial products or endogenousmolecules released from damaged cells [27] Inflammasomeshave several member proteins including NLRP1 NLRP3NLRC4 AIM2 and NRP6 [6] Our results showed thatscropolioside B can inhibit mRNA and protein expressionof inflammasome NLRP3 and prevents the secretion of IL-1120573 (Figure 4) Interestingly catalpol has a similar inhibitoryeffect on NLRP3 expression compared to scropolioside Bsuggesting that scropolioside B and catalpol inhibit IL-1120573 andNLRP3 expression by different mechanisms ScropoliosideB and catalpol also block the expression of CLS1 which isan enzyme in the final step of mitochondrial cardiolipinsynthesis by catalysing the transfer of a phosphatidyl residuefrom diacylglycerol to phosphatidylglycerol Iyer et al [28]reported that mitochondrial cardiolipin and reactive oxygenspecies are needed for inflammasome NLRP3 activity Car-diolipin can bind directly to NLRP3 and silencing of cardi-olipin synthesis specifically inhibits inflammasome NLRP3activation [28] Based on these observations we believe

that scropolioside B not only blocks NF-120581B pathway butalso inhibits NLRP3 CLS1 and IL-1120573 expressions Howevercatalpol only prevents the expression of NLRP3 and CLS1(Figure 5)

Our results also showed that scropolioside B but notcatalpol blocked IL-32120573120574 expression (Figure 3) Severalstudies have shown that IL-32 an important proinflamma-tory cytokine in rheumatoid arthritis enhanced IL-6 and IL-8 production in fibroblast-like synoviocytes [30ndash32] Somestudies also have shown that IL-32 is closely associated withliver fibrosis of chronic viral hepatitis [33 34] Furthermorecompared with primary blood monocytes IL-1120573 TNF-120572 orLPS can stimulate high levels of IL-32 expression through theI120581B kinase-120573NF-120581B and ERK pathways in human umbilicalvein endothelial cells aortic macrovascular endothelial cellsand cardiac and pulmonary microvascular endothelial cells[26] Conversely IL-32 also stimulates IL-1120572 IL-1120573 IL-6TNF-120572 and chemokines via NF-120581B p38 MAPK and AP-1activation [26 35] IL-32 promotes angiogenesis propagatingvascular inflammation and exacerbates sepsis in a mousemodel [36 37] Recent studies have shown that atheroscle-rosis maybe associated with IL-32 production [38]

In conclusion scropolioside B significantly diminishedexpression and secretion of IL-1120573 IL-32 and TNF-120572 Weshow that this is mediated by modulating NF-120581B NLRP3and CLS1 levels Additional studies are needed to furtherelucidate other targets by which scropolioside and catalpolregulate inflammation The results of this study strengthenprevious understanding of the anti-inflammatory effects ofiridoids and highlight scropolioside B as a potential drugfor the treatment of rheumatoid arthritis and atheroscleroticdisease

Conflict of Interests

The authors declared no conflict of interests

Authorsrsquo Contribution

Tiantian Zhu and Liuqiang Zhang contributed equally to thiswork

Acknowledgments

This work was supported by grants from the NationalScience and Technology Major Project of the Ministry ofScience and Technology of China (2009ZX09311-003) theNational Natural Science Foundation of China (8117351881274130) the Shanghai 085 Project of Higher EducationConnotation Construction (085ZY1202) and the FundingScheme for Training Young Teachers in Shanghai Colleges(no ZZszy13057)

References

[1] G Pawelec DGoldeck andEDerhovanessian ldquoInflammationageing and chronic diseaserdquo Current Opinion in Immunologyvol 29 no 1 pp 23ndash28 2014

Mediators of Inflammation 9

[2] L M Coussens L Zitvogel and A K Palucka ldquoNeutralizingtumor-promoting chronic inflammation a magic bulletrdquo Sci-ence vol 339 no 6117 pp 286ndash291 2013

[3] G E McKellar D W McCarey N Sattar and I B McInnesldquoRole for TNF in atherosclerosis Lessons from autoimmunediseaserdquo Nature Reviews vol 6 no 6 pp 410ndash417 2009

[4] V H Perry and J Teeling ldquoMicroglia and macrophages of thecentral nervous system the contribution of microglia primingand systemic inflammation to chronic neurodegenerationrdquoSeminars in Immunopathology vol 35 no 5 pp 601ndash612 2013

[5] M L Schmitz A Weber T Roxlau M Gaestel and M KrachtldquoSignal integration crosstalk mechanisms and networks in thefunction of inflammatory cytokinesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1813 no 12 pp 2165ndash21752011

[6] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[7] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 pp 1357ndash1361 2010

[8] K Schroder R Zhou and J Tschopp ldquoThe NLRP3 inflamma-some a sensor formetabolic dangerrdquo Science vol 327 no 5963pp 296ndash300 2010

[9] S L Masters A Dunne S L Subramanian et al ldquoActivationof the NLRP3 inflammasome by islet amyloid polypeptideprovides a mechanism for enhanced IL-1120573 2 in type 2 diabetesrdquoNature Immunology vol 11 no 10 pp 897ndash904 2010

[10] J Petrasek S Bala T Csak et al ldquoIL-1 receptor antagonistameliorates inflammasome-dependent alcoholic steatohepatitisin micerdquo Journal of Clinical Investigation vol 122 no 10 pp3476ndash3489 2012

[11] J Dupaul-Chicoine G Yeretssian K Doiron et al ldquoControl ofintestinal homeostasis colitis and colitis-associated colorectalcancer by the inflammatory caspasesrdquo Immunity vol 32 no 3pp 367ndash378 2010

[12] P I Sidiropoulos G Goulielmos G K Voloudakis E Petrakiand D T Boumpas ldquoInflammasomes and rheumatic diseasesevolving conceptsrdquo Annals of the Rheumatic Diseases vol 67no 10 pp 1382ndash1389 2008

[13] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[14] E Latz T S Xiao andA Stutz ldquoActivation and regulation of theinflammasomesrdquoNature Reviews Immunology vol 13 no 6 pp397ndash411 2013

[15] L-Q Zhang and Y-M Li ldquoAdvances in studies on chemicalconstituents in plants of Scrophularia L and their pharmacolog-ical effects in recent ten yearsrdquo Chinese Traditional and HerbalDrugs vol 42 no 11 pp 2360ndash2368 2011 (Chinese)

[16] A Viljoen N Mncwangi and I Vermaak ldquoAnti-inflammatoryiridoids of botanical originrdquo Current Medicinal Chemistry vol19 no 14 pp 2104ndash2127 2012

[17] R Tundis M R Loizzo F Menichini and G A StattildquoBiological and pharmacological activities of iridoids recentdevelopmentsrdquoMini-Reviews inMedicinal Chemistry vol 8 no4 pp 399ndash420 2008

[18] E Bas M C Recio S Manez et al ldquoNew insight intothe inhibition of the inflammatory response to experimentaldelayed-type hypersensitivity reactions inmice by scropoliosideArdquo European Journal of Pharmacology vol 555 no 2-3 pp 199ndash210 2007

[19] B Ahmed A J Al-Rehaily T A Al-Howiriny K A El-Sayed and M S Ahmad ldquoScropolioside-D2 and harpagoside-B two new iridoid glycosides from Scrophularia deserti andtheir antidiabetic and antiinflammatory activityrdquo Biological andPharmaceutical Bulletin vol 26 no 4 pp 462ndash467 2003

[20] M Stavri K T Mathew and S Gibbons ldquoAntimicrobialconstituents of Scrophularia desertirdquoPhytochemistry vol 67 no14 pp 1530ndash1533 2006

[21] P B Benito A M D Lanza A M S Sen et al ldquoEffects of someiridoids from plant origin on arachidonic acid metabolism incellular systemsrdquo Planta Medica vol 66 no 4 pp 324ndash3282000

[22] P Wang L Sun J Tan J Xu F Guo and Y Li ldquoTwo newglycosidated coumaramides from Clerodendron cyrtophyllumrdquoFitoterapia vol 83 no 8 pp 1494ndash1499 2012

[23] L Zhang L Feng Q Jia et al ldquoEffects of 120573-glucosidasehydrolyzed products of harpagide and harpagoside oncyclooxygenase-2 (COX-2) in vitrordquo Bioorganic and MedicinalChemistry vol 19 no 16 pp 4882ndash4886 2011

[24] K S Park B H Kim and I-M Chang ldquoInhibitory potencies ofseveral iridoids on cyclooxygenase-1 cyclooxygnase-2 enzymesactivities tumor necrosis factor-120572 and nitric oxide produc-tion In Vitrordquo Evidence-based Complementary and AlternativeMedicine vol 7 no 1 pp 41ndash45 2010

[25] H-J Choi H-J Jang T-W Chung et al ldquoCatalpol sup-presses advanced glycation end-products-induced inflamma-tory responses through inhibition of reactive oxygen species inhuman monocytic THP-1 cellsrdquo Fitoterapia vol 86 no 1 pp19ndash28 2013

[26] C A Nold-Petry M F Nold J A Zepp S-H Kim N FVoelkel and C A Dinarello ldquoIL-32-dependent effects of IL-1120573 on endothelial cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no10 pp 3883ndash3888 2009

[27] V A K Rathinam S K Vanaja and K A Fitzgerald ldquoRegu-lation of inflammasome signalingrdquoNature Immunology vol 13no 4 pp 333ndash342 2012

[28] S S Iyer Q He J R Janczy et al ldquoMitochondrial cardiolipinis required for Nlrp3 inflammasome activationrdquo Immunity vol39 no 2 pp 311ndash323 2013

[29] CADinarello ldquoA clinical perspective of IL-1120573 as the gatekeeperof inflammationrdquo European Journal of Immunology vol 41 no5 pp 1203ndash1217 2011

[30] L A B Joosten M G Netea S-H Kim et al ldquoIL-32 aproinflammatory cytokine in rheumatoid arthritisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 9 pp 3298ndash3303 2006

[31] B Heinhuis M I Koenders F A Van De Loo M G NeteaW B Van Den Berg and L A B Joosten ldquoInflammation-dependent secretion and splicing of IL-32120574 in rheumatoidarthritisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 12 pp 4962ndash4967 2011

[32] Y-G Kim C-K Lee S-H Kim W-S Cho S H Mun and BYoo ldquoInterleukin-32120574 enhances the production of IL-6 and IL-8in fibroblast-like synoviocytes Via Erk12 activationrdquo Journal ofClinical Immunology vol 30 no 2 pp 260ndash267 2010

[33] A R Moschen T Fritz A D Clouston et al ldquoInterleukin-32a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosisrdquoHepatology vol 53 no6 pp 1819ndash1829 2011

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

Mediators of Inflammation 3

Table 1 Primer sequences of the genes tested in this study

Gene Direction Primer sequences

IL-1120573 Forward 5-AAACAGATGAAGTGCTCCTTCCAGGReverse 5-TGGAGAACACCACTTGTTGCTCCA

TNF-120572 Forward 5-CAGAGGGAAGAGTTCCCCAGReverse 51015840-CCTTGGTCTGGTAGGAGACG

IL-32120573 Forward 5-GAGTTTCTGCTGCTCTCTGTCAReverse 5-ATTTTGAGGATTGGGGTTCAG

IL-32120574 Forward 5-GAGTTTCTGCTGCTCTCTGTCAReverse 5-ATTTTGAGGATTGGGGTTCAG

NLRP3 Forward 5-CTACACACGACTGCGTCTCATCAAReverse 5-GGGTCAAACAGCAACTCCATCTTA

CLS1 Forward 5-GAGTATGCCACAGTATGAAAACCCAReverse 5-CGAGCAATAAATCCATCCAACAA

GAPDH Forward 5-AGAAGGCTGGGGCTCATTTGReverse 5-AGGGGCCATCCACAGTCTTC

horseradish peroxidase The membrane was exposed to highperformance autoradiography film (Fuji Film Corp TokyoJapan) and visualized using the ECL Immobilon Westernchemiluminescent HRP substrate (WBKLS0500) (MilliporeUSA) Quantitative analysis was performed by QuantityOne software Western blot experiments were performed intriplicate

26 ELISA Theculturemedium from the control and treatedcells was collected centrifuged and stored at minus80∘C untiltested IL-1120573 was measured using Abcam Human ELISAKit (Abcam Cambridge England) according to the man-ufacturerrsquos instructions Standard or sample was added toeach well and incubated for 25 h at room temperature Theprepared biotin antibody was then added to each well fol-lowed by incubation for 1 h at room temperature Streptavidinsolution was added and incubated for 45 minutes at roomtemperature Finally TMB One-Step Development Solutionwas added to each well and incubated for 30 minutes at roomtemperature A stop solution was then added to each well andread at 450 nm immediately

27 Date Analysis Each experiment was performed at least 3times The results were presented as means plusmn standard errorof mean (SD) All data was analyzed using SPSS softwareand one-way ANOVA was used to determine the statisticalsignificance of differences between the means Differenceswere statistically significant when 119875 lt 005

3 Results

31 Blocking IL-1120573 and TNF-120572 Expression by Scropolioside BSince scropolioside B contains structures of catalpol and twophenylpropanoids (Figure 1(a)) we compared and tested theanti-inflammatory capabilities of both scropolioside B and

catalpol in THP-1 cells The expression of IL-1120573 and TNF-120572 was significantly induced by lipopolysaccharide (LPS) orpalmitic acid (PA) a free fatty acid with potential proin-flammatory mediators compared to control-treated THP-1 cells (Figure 1) This indicated that cellular exposure toLPS or PA induced the secretion of various cytokines thatlead to the initiation and amplification of inflammation Toinvestigate the anti-inflammatory effect of scropolioside Bwepreincubated THP-1 cells with the compound for 1 h and sub-sequently stimulated the cells with LPS or PA We found thatscropolioside B significantly blocked the increase in IL-1120573 andTNF-120572 levels induced by LPS or PA (Figure 1) However atthe concentration of 50120583molL catalpol did not effectivelyblock expression of IL-1120573 and TNF-120572 although these anti-inflammatory effects had been reported [24 25] Theseobservations suggested that scropolioside B has stronger anti-inflammatory activity compared to catalpol

32 Inhibition of Nuclear Factor 120581BActivation by ScropoliosideB NF-120581B is an essential transcription factor involved in theproduction of several cytokines that mediate the inflam-matory response To investigate overall anti-inflammatoryactivity of scropolioside B we used a luciferase reporterassay to determine nuclear factor kappa B (NF-120581B) activityAfter HEK293 cells were transferred with either the NF-120581Bor the control plasmid cells were treated with or withoutscropolioside B for 1 h and then stimulated with 100 ngmLof TNF-120572 An increase in luciferase activity was observedafter stimulation with TNF-120572 suggesting that NF-120581B wasactivated by TNF-120572 (Figure 2(a)) Pretreatment with scrop-olioside B (008ndash50120583molL) inhibited TNF-120572-induced NF-120581B activation in a concentration-dependentmanner Further-more scropolioside B exhibited an IC

50value of 102 120583molL

(Figure 2(b)) These results showed that scropolioside B-mediated inhibition of inflammatory cytokine induction wasdue to the suppression of NF-120581B

4 Mediators of Inflammation

Scropolioside B

OO

OHO

O

OHO

OH

OHOH

OMe

OO

O

O

H3COCO

Catalpol

OO

OHO

HO

OHO

OH

OH

OH

(a)

lowastlowast

3000

4000

2000

1000

000

IL-1120573

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(b)

lowastlowast

1000

1200

800

600

400

200

000

TNF-120572

relat

ive m

RNA

leve

ls

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

LPS (1120583gmL)

(c)

lowastlowast

1500

1000

2000

500

000

IL-1120573

relat

ive m

RNA

leve

ls

PA (50120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

lowast

300

200

500

600

400

100

000

PA (50120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

TNF-120572

relat

ive m

RNA

leve

ls

(e)

Figure 1 Different chemical structures of scropolioside B and catalpol (a) The effects of scropolioside B and catalpol on LPS-inducedexpression of IL-1120573 and TNF-120572 in THP-1 cells THP-1 cells were pretreated with 50120583molL catalpol or scropolioside B for 1 h and thenstimulated with LPS (1 120583gmL) for another 24 h ((b)-(c)) The effects of scropolioside B and catalpol on PA-induced expression of IL-1120573 andTNF-120572 in THP-1 cells THP-1 cells were pretreated with 50120583molL catalpol or scropolioside B for 1 h and then stimulated with PA (50120583gmL)for another 24 h ((d)-(e)) The expression of IL-1120573 and TNF-120572mRNA was measured by RT-PCRThe data represent the mean values of overthree experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS or PA alone 119875 lt 005 compared to vehiclecontrol lowast119875 lt 005 compared to LPS or PA alone

Mediators of Inflammation 5

lowast

lowast

lowastlowastlowast

600

400

200

000Relat

ive l

ucife

rase

uni

ts (fo

ld ch

ange

ove

r con

trol)

TNF-120572 (100ngmL) minus

minus

+

minus

+

50

+

10

+

04

+

008

+

2Scropolioside B (120583M)

(a)

6000

4000

2000

000

Inhi

bitio

n (

)

minus8 minus7 minus6 minus5 minus4

Concentration (log M)

IC50 = 102 120583M

(b)

Figure 2 Scropolioside B inhibited TNF-120572-inducedNF-120581B activation Cells were preincubated for 1 hourwith different doses of scropoliosideB and then stimulated with 1120583gmL TNF-120572 for 16 hours The results shown are representative of 3 separate experiments Data are expressedas means plusmn SD 119875 lt 005 versus the control lowast119875 lt 005 versus the TNF-120572 (a) IC50 values of 102 120583molL (b)

lowastlowast

3000

2000

1000

000

IL-3

2120573 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowast

4000

3000

2000

1000

000

IL-3

2120574 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(b)

Figure 3 The effects of scropolioside B and catalpol on LPS-induced expression of IL-32120573 and IL-32120574 in THP-1 cells THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The expression of IL-32120573 and IL-32120574mRNA was measured by RT-PCRThe data represent the mean values of over three experiments plusmn SD 119875 lt 001 comparedto vehicle control 119875 lt 005 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone and lowast119875 lt 005 compared to LPS alone

33 Scropolioside B Reduced the IL-32 Expression IL-32 isa proinflammatory cytokine involved in several diseasesincluding infections chronic inflammation and cancerTNF-120572 or LPS are known inducers of IL-32 IL-32-dependenteffects of IL-1120573 on endothelial cell functions [26] We nextdetermined the inhibitory effects of scropolioside B on IL-32 expression Pretreatmentwith scropolioside B significantlydiminished the increase in mRNA expression levels of IL-32120573 and IL-32120574 induced by LPS stimulation (Figures 3(a) and

3(b)) similar to IL-1120573 and TNF-120572 expression pattern in LPS-induced THP-1 cells

34 Scropolioside B Decreases Expression of NLRP3 Inflam-masomes regulate maturation of IL-1120573 and IL-18 and pyrop-tosisNLRP3 is amember of inflammasomeswhich constitutethe compound with ASC and caspase-1 to catalyze thematuration of IL-1120573 [27] To observe whether inflammatory

6 Mediators of Inflammation

lowastlowast

100

150

050

000

NLR

P3 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowastlowastlowast

100

150

050

000

CLS1

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus + minus

(b)

LPS (1120583gmL)

NLRP3

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

100

200

150

050

000

Band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

lowastlowast lowastlowast

(d)

Figure 4 Scropolioside B and catalpol inhibited the expression of CLS1 and NLRP3 in LPS-induced THP-1 cells ((a)-(b)) The effects ofscropolioside B and catalpol on protein expression ofNLRP3 in LPS-inducedTHP-1 cells ((c)-(d)) THP-1 cells were pretreatedwith 50120583molLcatalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The protein expression of NLRP3 was measuredby western blot The expression of CLS1 and NLRP3 mRNA was measured by RT-PCR The data represent the mean values of over threeexperiments plusmn SD 119875 lt 001 compared to vehicle control 119875 lt 005 compared to vehicle control and lowastlowast119875 lt 001 compared to LPS alone

factors induce NLRP3 expression we stimulated THP-1cells with LPS for 24 h We observed that LPS upregulatedNLRP3 mRNA and protein (Figures 4(a) 4(c) and 4(d))Similarly LPS also significantly enhanced mRNA expressionof cardiolipin synthetase 1 (CLS1) (Figure 4(b)) a mitochon-drial enzyme catalysing cardiolipin synthesis necessary forinflammasome NLRP3 activity [28] As shown in Figures4(a)ndash4(d) pretreatment with scropolioside B inhibited the

expressions of NLRP3 mRNA and protein as well as CLS1mRNA We also found that catalpol was as equally effectiveas scropolioside B (Figures 4(a)ndash4(d)) suggesting that thisinhibitory effect may be from the same catalpol structure(Figure 6)

35 Scropolioside B Decreases Expression of Pro-IL-1120573 andIL-1120573 To comprehensively evaluate the inhibitory effect of

Mediators of Inflammation 7

LPS (1120583gmL)

Pro-IL-1120573

IL-1120573

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

500

400

300

200

100

000

Pro-

IL-1120573

band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

lowastlowast

minus minus + minus

minus minus minus +

(b)

lowastlowast

250

200

150

050

100

000

IL-1120573

ban

d de

nsity

(fol

d ba

sal)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

lowastlowast

1500

500

1000

000

IL-1120573

(pg

mL)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

Figure 5 Scropolioside B and catalpol inhibited the expression of pro-IL-1120573 and IL-1120573 in LPS-induced THP-1 cells ((a)ndash(d)) THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 hThe protein expressionof pro-IL-1120573 and IL-1120573 was measured by western blot The levels of cytokines in the medium were measured using an ELISA kit The datarepresent the mean values of over three experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone

scropolioside B and catalpol we performed further studiesabout scropolioside B impact on protein expression of pro-IL-1120573 and IL-1120573 As shown in Figures 5(a)ndash5(d) pretreatmentwith scropolioside B inhibited the expressions of pro-IL-1120573and IL-1120573proteinThe inhibition of pro-IL-1120573 is stronger thanIL-1120573 Catalpol does not inhibit the protein expression of pro-IL-1120573 and IL-1120573

4 Discussion

Scropolioside B which is from S dentata Royle ex Benthhas antipyretic detoxicating effect It is used in Tibetanmedicine such as smallpox measles other infectious feversand inflammatory diseases In this study we demon-strated that scropolioside B an iridoid glycoside containing

8 Mediators of Inflammation

LPSTLR

P50

P50

P65

P65

Scropolioside BScropolioside B

Catalpol

Caspase-1

Pro-IL-1120573 mRNA

Pro-IL-1120573 IL-1120573

Nucleus

NLRP3

Figure 6 A schematic diagram of the regulation of NLRP3 inflam-masome by scropolioside B and catalpol

a catalpol and two phenylpropanoids was more effectivethan catalpol in inhibiting the expressions of IL-1120573 andTNF-120572 in THP-1 cells activated by LPS or palmitic acid(Figure 1) Our results suggested that scropolioside B hashigher anti-inflammatory activity than catalpol althoughsome studies have reported that catalpol does demonstrateanti-inflammatory effects at high dose by inhibiting COX-2activity TNF-120572 formation monocyte chemotactic protein-1 (MCP-1) and nitric oxide production [24 25] As shownin Figure 2 the anti-inflammatory effects of scropolioside Bwere mediated by blocking NF-120581B activity (Figure 2) IL-1120573is involved in the onset of acute local or systemic inflam-mation and contributes to a variety of chronic noninfectiousdiseases including ischemic injury atherosclerosis type 2diabetes and osteoarthritis Endogenousmetabolites such asoxidized fatty acids high glucose uric acid crystals activatedcomplement necrotic cells and cytokines can stimulatethe synthesis of the inactive IL-1120573 precursor which awaitsprocessing by the inflammasome complex to be activated[29]

Inflammasomes regulate maturity of IL-1120573 IL-18 andpyroptosis and recognize microbial products or endogenousmolecules released from damaged cells [27] Inflammasomeshave several member proteins including NLRP1 NLRP3NLRC4 AIM2 and NRP6 [6] Our results showed thatscropolioside B can inhibit mRNA and protein expressionof inflammasome NLRP3 and prevents the secretion of IL-1120573 (Figure 4) Interestingly catalpol has a similar inhibitoryeffect on NLRP3 expression compared to scropolioside Bsuggesting that scropolioside B and catalpol inhibit IL-1120573 andNLRP3 expression by different mechanisms ScropoliosideB and catalpol also block the expression of CLS1 which isan enzyme in the final step of mitochondrial cardiolipinsynthesis by catalysing the transfer of a phosphatidyl residuefrom diacylglycerol to phosphatidylglycerol Iyer et al [28]reported that mitochondrial cardiolipin and reactive oxygenspecies are needed for inflammasome NLRP3 activity Car-diolipin can bind directly to NLRP3 and silencing of cardi-olipin synthesis specifically inhibits inflammasome NLRP3activation [28] Based on these observations we believe

that scropolioside B not only blocks NF-120581B pathway butalso inhibits NLRP3 CLS1 and IL-1120573 expressions Howevercatalpol only prevents the expression of NLRP3 and CLS1(Figure 5)

Our results also showed that scropolioside B but notcatalpol blocked IL-32120573120574 expression (Figure 3) Severalstudies have shown that IL-32 an important proinflamma-tory cytokine in rheumatoid arthritis enhanced IL-6 and IL-8 production in fibroblast-like synoviocytes [30ndash32] Somestudies also have shown that IL-32 is closely associated withliver fibrosis of chronic viral hepatitis [33 34] Furthermorecompared with primary blood monocytes IL-1120573 TNF-120572 orLPS can stimulate high levels of IL-32 expression through theI120581B kinase-120573NF-120581B and ERK pathways in human umbilicalvein endothelial cells aortic macrovascular endothelial cellsand cardiac and pulmonary microvascular endothelial cells[26] Conversely IL-32 also stimulates IL-1120572 IL-1120573 IL-6TNF-120572 and chemokines via NF-120581B p38 MAPK and AP-1activation [26 35] IL-32 promotes angiogenesis propagatingvascular inflammation and exacerbates sepsis in a mousemodel [36 37] Recent studies have shown that atheroscle-rosis maybe associated with IL-32 production [38]

In conclusion scropolioside B significantly diminishedexpression and secretion of IL-1120573 IL-32 and TNF-120572 Weshow that this is mediated by modulating NF-120581B NLRP3and CLS1 levels Additional studies are needed to furtherelucidate other targets by which scropolioside and catalpolregulate inflammation The results of this study strengthenprevious understanding of the anti-inflammatory effects ofiridoids and highlight scropolioside B as a potential drugfor the treatment of rheumatoid arthritis and atheroscleroticdisease

Conflict of Interests

The authors declared no conflict of interests

Authorsrsquo Contribution

Tiantian Zhu and Liuqiang Zhang contributed equally to thiswork

Acknowledgments

This work was supported by grants from the NationalScience and Technology Major Project of the Ministry ofScience and Technology of China (2009ZX09311-003) theNational Natural Science Foundation of China (8117351881274130) the Shanghai 085 Project of Higher EducationConnotation Construction (085ZY1202) and the FundingScheme for Training Young Teachers in Shanghai Colleges(no ZZszy13057)

References

[1] G Pawelec DGoldeck andEDerhovanessian ldquoInflammationageing and chronic diseaserdquo Current Opinion in Immunologyvol 29 no 1 pp 23ndash28 2014

Mediators of Inflammation 9

[2] L M Coussens L Zitvogel and A K Palucka ldquoNeutralizingtumor-promoting chronic inflammation a magic bulletrdquo Sci-ence vol 339 no 6117 pp 286ndash291 2013

[3] G E McKellar D W McCarey N Sattar and I B McInnesldquoRole for TNF in atherosclerosis Lessons from autoimmunediseaserdquo Nature Reviews vol 6 no 6 pp 410ndash417 2009

[4] V H Perry and J Teeling ldquoMicroglia and macrophages of thecentral nervous system the contribution of microglia primingand systemic inflammation to chronic neurodegenerationrdquoSeminars in Immunopathology vol 35 no 5 pp 601ndash612 2013

[5] M L Schmitz A Weber T Roxlau M Gaestel and M KrachtldquoSignal integration crosstalk mechanisms and networks in thefunction of inflammatory cytokinesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1813 no 12 pp 2165ndash21752011

[6] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[7] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 pp 1357ndash1361 2010

[8] K Schroder R Zhou and J Tschopp ldquoThe NLRP3 inflamma-some a sensor formetabolic dangerrdquo Science vol 327 no 5963pp 296ndash300 2010

[9] S L Masters A Dunne S L Subramanian et al ldquoActivationof the NLRP3 inflammasome by islet amyloid polypeptideprovides a mechanism for enhanced IL-1120573 2 in type 2 diabetesrdquoNature Immunology vol 11 no 10 pp 897ndash904 2010

[10] J Petrasek S Bala T Csak et al ldquoIL-1 receptor antagonistameliorates inflammasome-dependent alcoholic steatohepatitisin micerdquo Journal of Clinical Investigation vol 122 no 10 pp3476ndash3489 2012

[11] J Dupaul-Chicoine G Yeretssian K Doiron et al ldquoControl ofintestinal homeostasis colitis and colitis-associated colorectalcancer by the inflammatory caspasesrdquo Immunity vol 32 no 3pp 367ndash378 2010

[12] P I Sidiropoulos G Goulielmos G K Voloudakis E Petrakiand D T Boumpas ldquoInflammasomes and rheumatic diseasesevolving conceptsrdquo Annals of the Rheumatic Diseases vol 67no 10 pp 1382ndash1389 2008

[13] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[14] E Latz T S Xiao andA Stutz ldquoActivation and regulation of theinflammasomesrdquoNature Reviews Immunology vol 13 no 6 pp397ndash411 2013

[15] L-Q Zhang and Y-M Li ldquoAdvances in studies on chemicalconstituents in plants of Scrophularia L and their pharmacolog-ical effects in recent ten yearsrdquo Chinese Traditional and HerbalDrugs vol 42 no 11 pp 2360ndash2368 2011 (Chinese)

[16] A Viljoen N Mncwangi and I Vermaak ldquoAnti-inflammatoryiridoids of botanical originrdquo Current Medicinal Chemistry vol19 no 14 pp 2104ndash2127 2012

[17] R Tundis M R Loizzo F Menichini and G A StattildquoBiological and pharmacological activities of iridoids recentdevelopmentsrdquoMini-Reviews inMedicinal Chemistry vol 8 no4 pp 399ndash420 2008

[18] E Bas M C Recio S Manez et al ldquoNew insight intothe inhibition of the inflammatory response to experimentaldelayed-type hypersensitivity reactions inmice by scropoliosideArdquo European Journal of Pharmacology vol 555 no 2-3 pp 199ndash210 2007

[19] B Ahmed A J Al-Rehaily T A Al-Howiriny K A El-Sayed and M S Ahmad ldquoScropolioside-D2 and harpagoside-B two new iridoid glycosides from Scrophularia deserti andtheir antidiabetic and antiinflammatory activityrdquo Biological andPharmaceutical Bulletin vol 26 no 4 pp 462ndash467 2003

[20] M Stavri K T Mathew and S Gibbons ldquoAntimicrobialconstituents of Scrophularia desertirdquoPhytochemistry vol 67 no14 pp 1530ndash1533 2006

[21] P B Benito A M D Lanza A M S Sen et al ldquoEffects of someiridoids from plant origin on arachidonic acid metabolism incellular systemsrdquo Planta Medica vol 66 no 4 pp 324ndash3282000

[22] P Wang L Sun J Tan J Xu F Guo and Y Li ldquoTwo newglycosidated coumaramides from Clerodendron cyrtophyllumrdquoFitoterapia vol 83 no 8 pp 1494ndash1499 2012

[23] L Zhang L Feng Q Jia et al ldquoEffects of 120573-glucosidasehydrolyzed products of harpagide and harpagoside oncyclooxygenase-2 (COX-2) in vitrordquo Bioorganic and MedicinalChemistry vol 19 no 16 pp 4882ndash4886 2011

[24] K S Park B H Kim and I-M Chang ldquoInhibitory potencies ofseveral iridoids on cyclooxygenase-1 cyclooxygnase-2 enzymesactivities tumor necrosis factor-120572 and nitric oxide produc-tion In Vitrordquo Evidence-based Complementary and AlternativeMedicine vol 7 no 1 pp 41ndash45 2010

[25] H-J Choi H-J Jang T-W Chung et al ldquoCatalpol sup-presses advanced glycation end-products-induced inflamma-tory responses through inhibition of reactive oxygen species inhuman monocytic THP-1 cellsrdquo Fitoterapia vol 86 no 1 pp19ndash28 2013

[26] C A Nold-Petry M F Nold J A Zepp S-H Kim N FVoelkel and C A Dinarello ldquoIL-32-dependent effects of IL-1120573 on endothelial cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no10 pp 3883ndash3888 2009

[27] V A K Rathinam S K Vanaja and K A Fitzgerald ldquoRegu-lation of inflammasome signalingrdquoNature Immunology vol 13no 4 pp 333ndash342 2012

[28] S S Iyer Q He J R Janczy et al ldquoMitochondrial cardiolipinis required for Nlrp3 inflammasome activationrdquo Immunity vol39 no 2 pp 311ndash323 2013

[29] CADinarello ldquoA clinical perspective of IL-1120573 as the gatekeeperof inflammationrdquo European Journal of Immunology vol 41 no5 pp 1203ndash1217 2011

[30] L A B Joosten M G Netea S-H Kim et al ldquoIL-32 aproinflammatory cytokine in rheumatoid arthritisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 9 pp 3298ndash3303 2006

[31] B Heinhuis M I Koenders F A Van De Loo M G NeteaW B Van Den Berg and L A B Joosten ldquoInflammation-dependent secretion and splicing of IL-32120574 in rheumatoidarthritisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 12 pp 4962ndash4967 2011

[32] Y-G Kim C-K Lee S-H Kim W-S Cho S H Mun and BYoo ldquoInterleukin-32120574 enhances the production of IL-6 and IL-8in fibroblast-like synoviocytes Via Erk12 activationrdquo Journal ofClinical Immunology vol 30 no 2 pp 260ndash267 2010

[33] A R Moschen T Fritz A D Clouston et al ldquoInterleukin-32a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosisrdquoHepatology vol 53 no6 pp 1819ndash1829 2011

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

4 Mediators of Inflammation

Scropolioside B

OO

OHO

O

OHO

OH

OHOH

OMe

OO

O

O

H3COCO

Catalpol

OO

OHO

HO

OHO

OH

OH

OH

(a)

lowastlowast

3000

4000

2000

1000

000

IL-1120573

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(b)

lowastlowast

1000

1200

800

600

400

200

000

TNF-120572

relat

ive m

RNA

leve

ls

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

LPS (1120583gmL)

(c)

lowastlowast

1500

1000

2000

500

000

IL-1120573

relat

ive m

RNA

leve

ls

PA (50120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

lowast

300

200

500

600

400

100

000

PA (50120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

TNF-120572

relat

ive m

RNA

leve

ls

(e)

Figure 1 Different chemical structures of scropolioside B and catalpol (a) The effects of scropolioside B and catalpol on LPS-inducedexpression of IL-1120573 and TNF-120572 in THP-1 cells THP-1 cells were pretreated with 50120583molL catalpol or scropolioside B for 1 h and thenstimulated with LPS (1 120583gmL) for another 24 h ((b)-(c)) The effects of scropolioside B and catalpol on PA-induced expression of IL-1120573 andTNF-120572 in THP-1 cells THP-1 cells were pretreated with 50120583molL catalpol or scropolioside B for 1 h and then stimulated with PA (50120583gmL)for another 24 h ((d)-(e)) The expression of IL-1120573 and TNF-120572mRNA was measured by RT-PCRThe data represent the mean values of overthree experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS or PA alone 119875 lt 005 compared to vehiclecontrol lowast119875 lt 005 compared to LPS or PA alone

Mediators of Inflammation 5

lowast

lowast

lowastlowastlowast

600

400

200

000Relat

ive l

ucife

rase

uni

ts (fo

ld ch

ange

ove

r con

trol)

TNF-120572 (100ngmL) minus

minus

+

minus

+

50

+

10

+

04

+

008

+

2Scropolioside B (120583M)

(a)

6000

4000

2000

000

Inhi

bitio

n (

)

minus8 minus7 minus6 minus5 minus4

Concentration (log M)

IC50 = 102 120583M

(b)

Figure 2 Scropolioside B inhibited TNF-120572-inducedNF-120581B activation Cells were preincubated for 1 hourwith different doses of scropoliosideB and then stimulated with 1120583gmL TNF-120572 for 16 hours The results shown are representative of 3 separate experiments Data are expressedas means plusmn SD 119875 lt 005 versus the control lowast119875 lt 005 versus the TNF-120572 (a) IC50 values of 102 120583molL (b)

lowastlowast

3000

2000

1000

000

IL-3

2120573 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowast

4000

3000

2000

1000

000

IL-3

2120574 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(b)

Figure 3 The effects of scropolioside B and catalpol on LPS-induced expression of IL-32120573 and IL-32120574 in THP-1 cells THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The expression of IL-32120573 and IL-32120574mRNA was measured by RT-PCRThe data represent the mean values of over three experiments plusmn SD 119875 lt 001 comparedto vehicle control 119875 lt 005 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone and lowast119875 lt 005 compared to LPS alone

33 Scropolioside B Reduced the IL-32 Expression IL-32 isa proinflammatory cytokine involved in several diseasesincluding infections chronic inflammation and cancerTNF-120572 or LPS are known inducers of IL-32 IL-32-dependenteffects of IL-1120573 on endothelial cell functions [26] We nextdetermined the inhibitory effects of scropolioside B on IL-32 expression Pretreatmentwith scropolioside B significantlydiminished the increase in mRNA expression levels of IL-32120573 and IL-32120574 induced by LPS stimulation (Figures 3(a) and

3(b)) similar to IL-1120573 and TNF-120572 expression pattern in LPS-induced THP-1 cells

34 Scropolioside B Decreases Expression of NLRP3 Inflam-masomes regulate maturation of IL-1120573 and IL-18 and pyrop-tosisNLRP3 is amember of inflammasomeswhich constitutethe compound with ASC and caspase-1 to catalyze thematuration of IL-1120573 [27] To observe whether inflammatory

6 Mediators of Inflammation

lowastlowast

100

150

050

000

NLR

P3 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowastlowastlowast

100

150

050

000

CLS1

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus + minus

(b)

LPS (1120583gmL)

NLRP3

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

100

200

150

050

000

Band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

lowastlowast lowastlowast

(d)

Figure 4 Scropolioside B and catalpol inhibited the expression of CLS1 and NLRP3 in LPS-induced THP-1 cells ((a)-(b)) The effects ofscropolioside B and catalpol on protein expression ofNLRP3 in LPS-inducedTHP-1 cells ((c)-(d)) THP-1 cells were pretreatedwith 50120583molLcatalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The protein expression of NLRP3 was measuredby western blot The expression of CLS1 and NLRP3 mRNA was measured by RT-PCR The data represent the mean values of over threeexperiments plusmn SD 119875 lt 001 compared to vehicle control 119875 lt 005 compared to vehicle control and lowastlowast119875 lt 001 compared to LPS alone

factors induce NLRP3 expression we stimulated THP-1cells with LPS for 24 h We observed that LPS upregulatedNLRP3 mRNA and protein (Figures 4(a) 4(c) and 4(d))Similarly LPS also significantly enhanced mRNA expressionof cardiolipin synthetase 1 (CLS1) (Figure 4(b)) a mitochon-drial enzyme catalysing cardiolipin synthesis necessary forinflammasome NLRP3 activity [28] As shown in Figures4(a)ndash4(d) pretreatment with scropolioside B inhibited the

expressions of NLRP3 mRNA and protein as well as CLS1mRNA We also found that catalpol was as equally effectiveas scropolioside B (Figures 4(a)ndash4(d)) suggesting that thisinhibitory effect may be from the same catalpol structure(Figure 6)

35 Scropolioside B Decreases Expression of Pro-IL-1120573 andIL-1120573 To comprehensively evaluate the inhibitory effect of

Mediators of Inflammation 7

LPS (1120583gmL)

Pro-IL-1120573

IL-1120573

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

500

400

300

200

100

000

Pro-

IL-1120573

band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

lowastlowast

minus minus + minus

minus minus minus +

(b)

lowastlowast

250

200

150

050

100

000

IL-1120573

ban

d de

nsity

(fol

d ba

sal)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

lowastlowast

1500

500

1000

000

IL-1120573

(pg

mL)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

Figure 5 Scropolioside B and catalpol inhibited the expression of pro-IL-1120573 and IL-1120573 in LPS-induced THP-1 cells ((a)ndash(d)) THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 hThe protein expressionof pro-IL-1120573 and IL-1120573 was measured by western blot The levels of cytokines in the medium were measured using an ELISA kit The datarepresent the mean values of over three experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone

scropolioside B and catalpol we performed further studiesabout scropolioside B impact on protein expression of pro-IL-1120573 and IL-1120573 As shown in Figures 5(a)ndash5(d) pretreatmentwith scropolioside B inhibited the expressions of pro-IL-1120573and IL-1120573proteinThe inhibition of pro-IL-1120573 is stronger thanIL-1120573 Catalpol does not inhibit the protein expression of pro-IL-1120573 and IL-1120573

4 Discussion

Scropolioside B which is from S dentata Royle ex Benthhas antipyretic detoxicating effect It is used in Tibetanmedicine such as smallpox measles other infectious feversand inflammatory diseases In this study we demon-strated that scropolioside B an iridoid glycoside containing

8 Mediators of Inflammation

LPSTLR

P50

P50

P65

P65

Scropolioside BScropolioside B

Catalpol

Caspase-1

Pro-IL-1120573 mRNA

Pro-IL-1120573 IL-1120573

Nucleus

NLRP3

Figure 6 A schematic diagram of the regulation of NLRP3 inflam-masome by scropolioside B and catalpol

a catalpol and two phenylpropanoids was more effectivethan catalpol in inhibiting the expressions of IL-1120573 andTNF-120572 in THP-1 cells activated by LPS or palmitic acid(Figure 1) Our results suggested that scropolioside B hashigher anti-inflammatory activity than catalpol althoughsome studies have reported that catalpol does demonstrateanti-inflammatory effects at high dose by inhibiting COX-2activity TNF-120572 formation monocyte chemotactic protein-1 (MCP-1) and nitric oxide production [24 25] As shownin Figure 2 the anti-inflammatory effects of scropolioside Bwere mediated by blocking NF-120581B activity (Figure 2) IL-1120573is involved in the onset of acute local or systemic inflam-mation and contributes to a variety of chronic noninfectiousdiseases including ischemic injury atherosclerosis type 2diabetes and osteoarthritis Endogenousmetabolites such asoxidized fatty acids high glucose uric acid crystals activatedcomplement necrotic cells and cytokines can stimulatethe synthesis of the inactive IL-1120573 precursor which awaitsprocessing by the inflammasome complex to be activated[29]

Inflammasomes regulate maturity of IL-1120573 IL-18 andpyroptosis and recognize microbial products or endogenousmolecules released from damaged cells [27] Inflammasomeshave several member proteins including NLRP1 NLRP3NLRC4 AIM2 and NRP6 [6] Our results showed thatscropolioside B can inhibit mRNA and protein expressionof inflammasome NLRP3 and prevents the secretion of IL-1120573 (Figure 4) Interestingly catalpol has a similar inhibitoryeffect on NLRP3 expression compared to scropolioside Bsuggesting that scropolioside B and catalpol inhibit IL-1120573 andNLRP3 expression by different mechanisms ScropoliosideB and catalpol also block the expression of CLS1 which isan enzyme in the final step of mitochondrial cardiolipinsynthesis by catalysing the transfer of a phosphatidyl residuefrom diacylglycerol to phosphatidylglycerol Iyer et al [28]reported that mitochondrial cardiolipin and reactive oxygenspecies are needed for inflammasome NLRP3 activity Car-diolipin can bind directly to NLRP3 and silencing of cardi-olipin synthesis specifically inhibits inflammasome NLRP3activation [28] Based on these observations we believe

that scropolioside B not only blocks NF-120581B pathway butalso inhibits NLRP3 CLS1 and IL-1120573 expressions Howevercatalpol only prevents the expression of NLRP3 and CLS1(Figure 5)

Our results also showed that scropolioside B but notcatalpol blocked IL-32120573120574 expression (Figure 3) Severalstudies have shown that IL-32 an important proinflamma-tory cytokine in rheumatoid arthritis enhanced IL-6 and IL-8 production in fibroblast-like synoviocytes [30ndash32] Somestudies also have shown that IL-32 is closely associated withliver fibrosis of chronic viral hepatitis [33 34] Furthermorecompared with primary blood monocytes IL-1120573 TNF-120572 orLPS can stimulate high levels of IL-32 expression through theI120581B kinase-120573NF-120581B and ERK pathways in human umbilicalvein endothelial cells aortic macrovascular endothelial cellsand cardiac and pulmonary microvascular endothelial cells[26] Conversely IL-32 also stimulates IL-1120572 IL-1120573 IL-6TNF-120572 and chemokines via NF-120581B p38 MAPK and AP-1activation [26 35] IL-32 promotes angiogenesis propagatingvascular inflammation and exacerbates sepsis in a mousemodel [36 37] Recent studies have shown that atheroscle-rosis maybe associated with IL-32 production [38]

In conclusion scropolioside B significantly diminishedexpression and secretion of IL-1120573 IL-32 and TNF-120572 Weshow that this is mediated by modulating NF-120581B NLRP3and CLS1 levels Additional studies are needed to furtherelucidate other targets by which scropolioside and catalpolregulate inflammation The results of this study strengthenprevious understanding of the anti-inflammatory effects ofiridoids and highlight scropolioside B as a potential drugfor the treatment of rheumatoid arthritis and atheroscleroticdisease

Conflict of Interests

The authors declared no conflict of interests

Authorsrsquo Contribution

Tiantian Zhu and Liuqiang Zhang contributed equally to thiswork

Acknowledgments

This work was supported by grants from the NationalScience and Technology Major Project of the Ministry ofScience and Technology of China (2009ZX09311-003) theNational Natural Science Foundation of China (8117351881274130) the Shanghai 085 Project of Higher EducationConnotation Construction (085ZY1202) and the FundingScheme for Training Young Teachers in Shanghai Colleges(no ZZszy13057)

References

[1] G Pawelec DGoldeck andEDerhovanessian ldquoInflammationageing and chronic diseaserdquo Current Opinion in Immunologyvol 29 no 1 pp 23ndash28 2014

Mediators of Inflammation 9

[2] L M Coussens L Zitvogel and A K Palucka ldquoNeutralizingtumor-promoting chronic inflammation a magic bulletrdquo Sci-ence vol 339 no 6117 pp 286ndash291 2013

[3] G E McKellar D W McCarey N Sattar and I B McInnesldquoRole for TNF in atherosclerosis Lessons from autoimmunediseaserdquo Nature Reviews vol 6 no 6 pp 410ndash417 2009

[4] V H Perry and J Teeling ldquoMicroglia and macrophages of thecentral nervous system the contribution of microglia primingand systemic inflammation to chronic neurodegenerationrdquoSeminars in Immunopathology vol 35 no 5 pp 601ndash612 2013

[5] M L Schmitz A Weber T Roxlau M Gaestel and M KrachtldquoSignal integration crosstalk mechanisms and networks in thefunction of inflammatory cytokinesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1813 no 12 pp 2165ndash21752011

[6] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[7] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 pp 1357ndash1361 2010

[8] K Schroder R Zhou and J Tschopp ldquoThe NLRP3 inflamma-some a sensor formetabolic dangerrdquo Science vol 327 no 5963pp 296ndash300 2010

[9] S L Masters A Dunne S L Subramanian et al ldquoActivationof the NLRP3 inflammasome by islet amyloid polypeptideprovides a mechanism for enhanced IL-1120573 2 in type 2 diabetesrdquoNature Immunology vol 11 no 10 pp 897ndash904 2010

[10] J Petrasek S Bala T Csak et al ldquoIL-1 receptor antagonistameliorates inflammasome-dependent alcoholic steatohepatitisin micerdquo Journal of Clinical Investigation vol 122 no 10 pp3476ndash3489 2012

[11] J Dupaul-Chicoine G Yeretssian K Doiron et al ldquoControl ofintestinal homeostasis colitis and colitis-associated colorectalcancer by the inflammatory caspasesrdquo Immunity vol 32 no 3pp 367ndash378 2010

[12] P I Sidiropoulos G Goulielmos G K Voloudakis E Petrakiand D T Boumpas ldquoInflammasomes and rheumatic diseasesevolving conceptsrdquo Annals of the Rheumatic Diseases vol 67no 10 pp 1382ndash1389 2008

[13] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[14] E Latz T S Xiao andA Stutz ldquoActivation and regulation of theinflammasomesrdquoNature Reviews Immunology vol 13 no 6 pp397ndash411 2013

[15] L-Q Zhang and Y-M Li ldquoAdvances in studies on chemicalconstituents in plants of Scrophularia L and their pharmacolog-ical effects in recent ten yearsrdquo Chinese Traditional and HerbalDrugs vol 42 no 11 pp 2360ndash2368 2011 (Chinese)

[16] A Viljoen N Mncwangi and I Vermaak ldquoAnti-inflammatoryiridoids of botanical originrdquo Current Medicinal Chemistry vol19 no 14 pp 2104ndash2127 2012

[17] R Tundis M R Loizzo F Menichini and G A StattildquoBiological and pharmacological activities of iridoids recentdevelopmentsrdquoMini-Reviews inMedicinal Chemistry vol 8 no4 pp 399ndash420 2008

[18] E Bas M C Recio S Manez et al ldquoNew insight intothe inhibition of the inflammatory response to experimentaldelayed-type hypersensitivity reactions inmice by scropoliosideArdquo European Journal of Pharmacology vol 555 no 2-3 pp 199ndash210 2007

[19] B Ahmed A J Al-Rehaily T A Al-Howiriny K A El-Sayed and M S Ahmad ldquoScropolioside-D2 and harpagoside-B two new iridoid glycosides from Scrophularia deserti andtheir antidiabetic and antiinflammatory activityrdquo Biological andPharmaceutical Bulletin vol 26 no 4 pp 462ndash467 2003

[20] M Stavri K T Mathew and S Gibbons ldquoAntimicrobialconstituents of Scrophularia desertirdquoPhytochemistry vol 67 no14 pp 1530ndash1533 2006

[21] P B Benito A M D Lanza A M S Sen et al ldquoEffects of someiridoids from plant origin on arachidonic acid metabolism incellular systemsrdquo Planta Medica vol 66 no 4 pp 324ndash3282000

[22] P Wang L Sun J Tan J Xu F Guo and Y Li ldquoTwo newglycosidated coumaramides from Clerodendron cyrtophyllumrdquoFitoterapia vol 83 no 8 pp 1494ndash1499 2012

[23] L Zhang L Feng Q Jia et al ldquoEffects of 120573-glucosidasehydrolyzed products of harpagide and harpagoside oncyclooxygenase-2 (COX-2) in vitrordquo Bioorganic and MedicinalChemistry vol 19 no 16 pp 4882ndash4886 2011

[24] K S Park B H Kim and I-M Chang ldquoInhibitory potencies ofseveral iridoids on cyclooxygenase-1 cyclooxygnase-2 enzymesactivities tumor necrosis factor-120572 and nitric oxide produc-tion In Vitrordquo Evidence-based Complementary and AlternativeMedicine vol 7 no 1 pp 41ndash45 2010

[25] H-J Choi H-J Jang T-W Chung et al ldquoCatalpol sup-presses advanced glycation end-products-induced inflamma-tory responses through inhibition of reactive oxygen species inhuman monocytic THP-1 cellsrdquo Fitoterapia vol 86 no 1 pp19ndash28 2013

[26] C A Nold-Petry M F Nold J A Zepp S-H Kim N FVoelkel and C A Dinarello ldquoIL-32-dependent effects of IL-1120573 on endothelial cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no10 pp 3883ndash3888 2009

[27] V A K Rathinam S K Vanaja and K A Fitzgerald ldquoRegu-lation of inflammasome signalingrdquoNature Immunology vol 13no 4 pp 333ndash342 2012

[28] S S Iyer Q He J R Janczy et al ldquoMitochondrial cardiolipinis required for Nlrp3 inflammasome activationrdquo Immunity vol39 no 2 pp 311ndash323 2013

[29] CADinarello ldquoA clinical perspective of IL-1120573 as the gatekeeperof inflammationrdquo European Journal of Immunology vol 41 no5 pp 1203ndash1217 2011

[30] L A B Joosten M G Netea S-H Kim et al ldquoIL-32 aproinflammatory cytokine in rheumatoid arthritisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 9 pp 3298ndash3303 2006

[31] B Heinhuis M I Koenders F A Van De Loo M G NeteaW B Van Den Berg and L A B Joosten ldquoInflammation-dependent secretion and splicing of IL-32120574 in rheumatoidarthritisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 12 pp 4962ndash4967 2011

[32] Y-G Kim C-K Lee S-H Kim W-S Cho S H Mun and BYoo ldquoInterleukin-32120574 enhances the production of IL-6 and IL-8in fibroblast-like synoviocytes Via Erk12 activationrdquo Journal ofClinical Immunology vol 30 no 2 pp 260ndash267 2010

[33] A R Moschen T Fritz A D Clouston et al ldquoInterleukin-32a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosisrdquoHepatology vol 53 no6 pp 1819ndash1829 2011

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

Mediators of Inflammation 5

lowast

lowast

lowastlowastlowast

600

400

200

000Relat

ive l

ucife

rase

uni

ts (fo

ld ch

ange

ove

r con

trol)

TNF-120572 (100ngmL) minus

minus

+

minus

+

50

+

10

+

04

+

008

+

2Scropolioside B (120583M)

(a)

6000

4000

2000

000

Inhi

bitio

n (

)

minus8 minus7 minus6 minus5 minus4

Concentration (log M)

IC50 = 102 120583M

(b)

Figure 2 Scropolioside B inhibited TNF-120572-inducedNF-120581B activation Cells were preincubated for 1 hourwith different doses of scropoliosideB and then stimulated with 1120583gmL TNF-120572 for 16 hours The results shown are representative of 3 separate experiments Data are expressedas means plusmn SD 119875 lt 005 versus the control lowast119875 lt 005 versus the TNF-120572 (a) IC50 values of 102 120583molL (b)

lowastlowast

3000

2000

1000

000

IL-3

2120573 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowast

4000

3000

2000

1000

000

IL-3

2120574 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(b)

Figure 3 The effects of scropolioside B and catalpol on LPS-induced expression of IL-32120573 and IL-32120574 in THP-1 cells THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The expression of IL-32120573 and IL-32120574mRNA was measured by RT-PCRThe data represent the mean values of over three experiments plusmn SD 119875 lt 001 comparedto vehicle control 119875 lt 005 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone and lowast119875 lt 005 compared to LPS alone

33 Scropolioside B Reduced the IL-32 Expression IL-32 isa proinflammatory cytokine involved in several diseasesincluding infections chronic inflammation and cancerTNF-120572 or LPS are known inducers of IL-32 IL-32-dependenteffects of IL-1120573 on endothelial cell functions [26] We nextdetermined the inhibitory effects of scropolioside B on IL-32 expression Pretreatmentwith scropolioside B significantlydiminished the increase in mRNA expression levels of IL-32120573 and IL-32120574 induced by LPS stimulation (Figures 3(a) and

3(b)) similar to IL-1120573 and TNF-120572 expression pattern in LPS-induced THP-1 cells

34 Scropolioside B Decreases Expression of NLRP3 Inflam-masomes regulate maturation of IL-1120573 and IL-18 and pyrop-tosisNLRP3 is amember of inflammasomeswhich constitutethe compound with ASC and caspase-1 to catalyze thematuration of IL-1120573 [27] To observe whether inflammatory

6 Mediators of Inflammation

lowastlowast

100

150

050

000

NLR

P3 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowastlowastlowast

100

150

050

000

CLS1

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus + minus

(b)

LPS (1120583gmL)

NLRP3

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

100

200

150

050

000

Band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

lowastlowast lowastlowast

(d)

Figure 4 Scropolioside B and catalpol inhibited the expression of CLS1 and NLRP3 in LPS-induced THP-1 cells ((a)-(b)) The effects ofscropolioside B and catalpol on protein expression ofNLRP3 in LPS-inducedTHP-1 cells ((c)-(d)) THP-1 cells were pretreatedwith 50120583molLcatalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The protein expression of NLRP3 was measuredby western blot The expression of CLS1 and NLRP3 mRNA was measured by RT-PCR The data represent the mean values of over threeexperiments plusmn SD 119875 lt 001 compared to vehicle control 119875 lt 005 compared to vehicle control and lowastlowast119875 lt 001 compared to LPS alone

factors induce NLRP3 expression we stimulated THP-1cells with LPS for 24 h We observed that LPS upregulatedNLRP3 mRNA and protein (Figures 4(a) 4(c) and 4(d))Similarly LPS also significantly enhanced mRNA expressionof cardiolipin synthetase 1 (CLS1) (Figure 4(b)) a mitochon-drial enzyme catalysing cardiolipin synthesis necessary forinflammasome NLRP3 activity [28] As shown in Figures4(a)ndash4(d) pretreatment with scropolioside B inhibited the

expressions of NLRP3 mRNA and protein as well as CLS1mRNA We also found that catalpol was as equally effectiveas scropolioside B (Figures 4(a)ndash4(d)) suggesting that thisinhibitory effect may be from the same catalpol structure(Figure 6)

35 Scropolioside B Decreases Expression of Pro-IL-1120573 andIL-1120573 To comprehensively evaluate the inhibitory effect of

Mediators of Inflammation 7

LPS (1120583gmL)

Pro-IL-1120573

IL-1120573

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

500

400

300

200

100

000

Pro-

IL-1120573

band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

lowastlowast

minus minus + minus

minus minus minus +

(b)

lowastlowast

250

200

150

050

100

000

IL-1120573

ban

d de

nsity

(fol

d ba

sal)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

lowastlowast

1500

500

1000

000

IL-1120573

(pg

mL)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

Figure 5 Scropolioside B and catalpol inhibited the expression of pro-IL-1120573 and IL-1120573 in LPS-induced THP-1 cells ((a)ndash(d)) THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 hThe protein expressionof pro-IL-1120573 and IL-1120573 was measured by western blot The levels of cytokines in the medium were measured using an ELISA kit The datarepresent the mean values of over three experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone

scropolioside B and catalpol we performed further studiesabout scropolioside B impact on protein expression of pro-IL-1120573 and IL-1120573 As shown in Figures 5(a)ndash5(d) pretreatmentwith scropolioside B inhibited the expressions of pro-IL-1120573and IL-1120573proteinThe inhibition of pro-IL-1120573 is stronger thanIL-1120573 Catalpol does not inhibit the protein expression of pro-IL-1120573 and IL-1120573

4 Discussion

Scropolioside B which is from S dentata Royle ex Benthhas antipyretic detoxicating effect It is used in Tibetanmedicine such as smallpox measles other infectious feversand inflammatory diseases In this study we demon-strated that scropolioside B an iridoid glycoside containing

8 Mediators of Inflammation

LPSTLR

P50

P50

P65

P65

Scropolioside BScropolioside B

Catalpol

Caspase-1

Pro-IL-1120573 mRNA

Pro-IL-1120573 IL-1120573

Nucleus

NLRP3

Figure 6 A schematic diagram of the regulation of NLRP3 inflam-masome by scropolioside B and catalpol

a catalpol and two phenylpropanoids was more effectivethan catalpol in inhibiting the expressions of IL-1120573 andTNF-120572 in THP-1 cells activated by LPS or palmitic acid(Figure 1) Our results suggested that scropolioside B hashigher anti-inflammatory activity than catalpol althoughsome studies have reported that catalpol does demonstrateanti-inflammatory effects at high dose by inhibiting COX-2activity TNF-120572 formation monocyte chemotactic protein-1 (MCP-1) and nitric oxide production [24 25] As shownin Figure 2 the anti-inflammatory effects of scropolioside Bwere mediated by blocking NF-120581B activity (Figure 2) IL-1120573is involved in the onset of acute local or systemic inflam-mation and contributes to a variety of chronic noninfectiousdiseases including ischemic injury atherosclerosis type 2diabetes and osteoarthritis Endogenousmetabolites such asoxidized fatty acids high glucose uric acid crystals activatedcomplement necrotic cells and cytokines can stimulatethe synthesis of the inactive IL-1120573 precursor which awaitsprocessing by the inflammasome complex to be activated[29]

Inflammasomes regulate maturity of IL-1120573 IL-18 andpyroptosis and recognize microbial products or endogenousmolecules released from damaged cells [27] Inflammasomeshave several member proteins including NLRP1 NLRP3NLRC4 AIM2 and NRP6 [6] Our results showed thatscropolioside B can inhibit mRNA and protein expressionof inflammasome NLRP3 and prevents the secretion of IL-1120573 (Figure 4) Interestingly catalpol has a similar inhibitoryeffect on NLRP3 expression compared to scropolioside Bsuggesting that scropolioside B and catalpol inhibit IL-1120573 andNLRP3 expression by different mechanisms ScropoliosideB and catalpol also block the expression of CLS1 which isan enzyme in the final step of mitochondrial cardiolipinsynthesis by catalysing the transfer of a phosphatidyl residuefrom diacylglycerol to phosphatidylglycerol Iyer et al [28]reported that mitochondrial cardiolipin and reactive oxygenspecies are needed for inflammasome NLRP3 activity Car-diolipin can bind directly to NLRP3 and silencing of cardi-olipin synthesis specifically inhibits inflammasome NLRP3activation [28] Based on these observations we believe

that scropolioside B not only blocks NF-120581B pathway butalso inhibits NLRP3 CLS1 and IL-1120573 expressions Howevercatalpol only prevents the expression of NLRP3 and CLS1(Figure 5)

Our results also showed that scropolioside B but notcatalpol blocked IL-32120573120574 expression (Figure 3) Severalstudies have shown that IL-32 an important proinflamma-tory cytokine in rheumatoid arthritis enhanced IL-6 and IL-8 production in fibroblast-like synoviocytes [30ndash32] Somestudies also have shown that IL-32 is closely associated withliver fibrosis of chronic viral hepatitis [33 34] Furthermorecompared with primary blood monocytes IL-1120573 TNF-120572 orLPS can stimulate high levels of IL-32 expression through theI120581B kinase-120573NF-120581B and ERK pathways in human umbilicalvein endothelial cells aortic macrovascular endothelial cellsand cardiac and pulmonary microvascular endothelial cells[26] Conversely IL-32 also stimulates IL-1120572 IL-1120573 IL-6TNF-120572 and chemokines via NF-120581B p38 MAPK and AP-1activation [26 35] IL-32 promotes angiogenesis propagatingvascular inflammation and exacerbates sepsis in a mousemodel [36 37] Recent studies have shown that atheroscle-rosis maybe associated with IL-32 production [38]

In conclusion scropolioside B significantly diminishedexpression and secretion of IL-1120573 IL-32 and TNF-120572 Weshow that this is mediated by modulating NF-120581B NLRP3and CLS1 levels Additional studies are needed to furtherelucidate other targets by which scropolioside and catalpolregulate inflammation The results of this study strengthenprevious understanding of the anti-inflammatory effects ofiridoids and highlight scropolioside B as a potential drugfor the treatment of rheumatoid arthritis and atheroscleroticdisease

Conflict of Interests

The authors declared no conflict of interests

Authorsrsquo Contribution

Tiantian Zhu and Liuqiang Zhang contributed equally to thiswork

Acknowledgments

This work was supported by grants from the NationalScience and Technology Major Project of the Ministry ofScience and Technology of China (2009ZX09311-003) theNational Natural Science Foundation of China (8117351881274130) the Shanghai 085 Project of Higher EducationConnotation Construction (085ZY1202) and the FundingScheme for Training Young Teachers in Shanghai Colleges(no ZZszy13057)

References

[1] G Pawelec DGoldeck andEDerhovanessian ldquoInflammationageing and chronic diseaserdquo Current Opinion in Immunologyvol 29 no 1 pp 23ndash28 2014

Mediators of Inflammation 9

[2] L M Coussens L Zitvogel and A K Palucka ldquoNeutralizingtumor-promoting chronic inflammation a magic bulletrdquo Sci-ence vol 339 no 6117 pp 286ndash291 2013

[3] G E McKellar D W McCarey N Sattar and I B McInnesldquoRole for TNF in atherosclerosis Lessons from autoimmunediseaserdquo Nature Reviews vol 6 no 6 pp 410ndash417 2009

[4] V H Perry and J Teeling ldquoMicroglia and macrophages of thecentral nervous system the contribution of microglia primingand systemic inflammation to chronic neurodegenerationrdquoSeminars in Immunopathology vol 35 no 5 pp 601ndash612 2013

[5] M L Schmitz A Weber T Roxlau M Gaestel and M KrachtldquoSignal integration crosstalk mechanisms and networks in thefunction of inflammatory cytokinesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1813 no 12 pp 2165ndash21752011

[6] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[7] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 pp 1357ndash1361 2010

[8] K Schroder R Zhou and J Tschopp ldquoThe NLRP3 inflamma-some a sensor formetabolic dangerrdquo Science vol 327 no 5963pp 296ndash300 2010

[9] S L Masters A Dunne S L Subramanian et al ldquoActivationof the NLRP3 inflammasome by islet amyloid polypeptideprovides a mechanism for enhanced IL-1120573 2 in type 2 diabetesrdquoNature Immunology vol 11 no 10 pp 897ndash904 2010

[10] J Petrasek S Bala T Csak et al ldquoIL-1 receptor antagonistameliorates inflammasome-dependent alcoholic steatohepatitisin micerdquo Journal of Clinical Investigation vol 122 no 10 pp3476ndash3489 2012

[11] J Dupaul-Chicoine G Yeretssian K Doiron et al ldquoControl ofintestinal homeostasis colitis and colitis-associated colorectalcancer by the inflammatory caspasesrdquo Immunity vol 32 no 3pp 367ndash378 2010

[12] P I Sidiropoulos G Goulielmos G K Voloudakis E Petrakiand D T Boumpas ldquoInflammasomes and rheumatic diseasesevolving conceptsrdquo Annals of the Rheumatic Diseases vol 67no 10 pp 1382ndash1389 2008

[13] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[14] E Latz T S Xiao andA Stutz ldquoActivation and regulation of theinflammasomesrdquoNature Reviews Immunology vol 13 no 6 pp397ndash411 2013

[15] L-Q Zhang and Y-M Li ldquoAdvances in studies on chemicalconstituents in plants of Scrophularia L and their pharmacolog-ical effects in recent ten yearsrdquo Chinese Traditional and HerbalDrugs vol 42 no 11 pp 2360ndash2368 2011 (Chinese)

[16] A Viljoen N Mncwangi and I Vermaak ldquoAnti-inflammatoryiridoids of botanical originrdquo Current Medicinal Chemistry vol19 no 14 pp 2104ndash2127 2012

[17] R Tundis M R Loizzo F Menichini and G A StattildquoBiological and pharmacological activities of iridoids recentdevelopmentsrdquoMini-Reviews inMedicinal Chemistry vol 8 no4 pp 399ndash420 2008

[18] E Bas M C Recio S Manez et al ldquoNew insight intothe inhibition of the inflammatory response to experimentaldelayed-type hypersensitivity reactions inmice by scropoliosideArdquo European Journal of Pharmacology vol 555 no 2-3 pp 199ndash210 2007

[19] B Ahmed A J Al-Rehaily T A Al-Howiriny K A El-Sayed and M S Ahmad ldquoScropolioside-D2 and harpagoside-B two new iridoid glycosides from Scrophularia deserti andtheir antidiabetic and antiinflammatory activityrdquo Biological andPharmaceutical Bulletin vol 26 no 4 pp 462ndash467 2003

[20] M Stavri K T Mathew and S Gibbons ldquoAntimicrobialconstituents of Scrophularia desertirdquoPhytochemistry vol 67 no14 pp 1530ndash1533 2006

[21] P B Benito A M D Lanza A M S Sen et al ldquoEffects of someiridoids from plant origin on arachidonic acid metabolism incellular systemsrdquo Planta Medica vol 66 no 4 pp 324ndash3282000

[22] P Wang L Sun J Tan J Xu F Guo and Y Li ldquoTwo newglycosidated coumaramides from Clerodendron cyrtophyllumrdquoFitoterapia vol 83 no 8 pp 1494ndash1499 2012

[23] L Zhang L Feng Q Jia et al ldquoEffects of 120573-glucosidasehydrolyzed products of harpagide and harpagoside oncyclooxygenase-2 (COX-2) in vitrordquo Bioorganic and MedicinalChemistry vol 19 no 16 pp 4882ndash4886 2011

[24] K S Park B H Kim and I-M Chang ldquoInhibitory potencies ofseveral iridoids on cyclooxygenase-1 cyclooxygnase-2 enzymesactivities tumor necrosis factor-120572 and nitric oxide produc-tion In Vitrordquo Evidence-based Complementary and AlternativeMedicine vol 7 no 1 pp 41ndash45 2010

[25] H-J Choi H-J Jang T-W Chung et al ldquoCatalpol sup-presses advanced glycation end-products-induced inflamma-tory responses through inhibition of reactive oxygen species inhuman monocytic THP-1 cellsrdquo Fitoterapia vol 86 no 1 pp19ndash28 2013

[26] C A Nold-Petry M F Nold J A Zepp S-H Kim N FVoelkel and C A Dinarello ldquoIL-32-dependent effects of IL-1120573 on endothelial cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no10 pp 3883ndash3888 2009

[27] V A K Rathinam S K Vanaja and K A Fitzgerald ldquoRegu-lation of inflammasome signalingrdquoNature Immunology vol 13no 4 pp 333ndash342 2012

[28] S S Iyer Q He J R Janczy et al ldquoMitochondrial cardiolipinis required for Nlrp3 inflammasome activationrdquo Immunity vol39 no 2 pp 311ndash323 2013

[29] CADinarello ldquoA clinical perspective of IL-1120573 as the gatekeeperof inflammationrdquo European Journal of Immunology vol 41 no5 pp 1203ndash1217 2011

[30] L A B Joosten M G Netea S-H Kim et al ldquoIL-32 aproinflammatory cytokine in rheumatoid arthritisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 9 pp 3298ndash3303 2006

[31] B Heinhuis M I Koenders F A Van De Loo M G NeteaW B Van Den Berg and L A B Joosten ldquoInflammation-dependent secretion and splicing of IL-32120574 in rheumatoidarthritisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 12 pp 4962ndash4967 2011

[32] Y-G Kim C-K Lee S-H Kim W-S Cho S H Mun and BYoo ldquoInterleukin-32120574 enhances the production of IL-6 and IL-8in fibroblast-like synoviocytes Via Erk12 activationrdquo Journal ofClinical Immunology vol 30 no 2 pp 260ndash267 2010

[33] A R Moschen T Fritz A D Clouston et al ldquoInterleukin-32a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosisrdquoHepatology vol 53 no6 pp 1819ndash1829 2011

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

6 Mediators of Inflammation

lowastlowast

100

150

050

000

NLR

P3 re

lativ

e mRN

A le

vels

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

lowastlowastlowastlowast

100

150

050

000

CLS1

relat

ive m

RNA

leve

ls

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus + minus

(b)

LPS (1120583gmL)

NLRP3

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

100

200

150

050

000

Band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

lowastlowast lowastlowast

(d)

Figure 4 Scropolioside B and catalpol inhibited the expression of CLS1 and NLRP3 in LPS-induced THP-1 cells ((a)-(b)) The effects ofscropolioside B and catalpol on protein expression ofNLRP3 in LPS-inducedTHP-1 cells ((c)-(d)) THP-1 cells were pretreatedwith 50120583molLcatalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 h The protein expression of NLRP3 was measuredby western blot The expression of CLS1 and NLRP3 mRNA was measured by RT-PCR The data represent the mean values of over threeexperiments plusmn SD 119875 lt 001 compared to vehicle control 119875 lt 005 compared to vehicle control and lowastlowast119875 lt 001 compared to LPS alone

factors induce NLRP3 expression we stimulated THP-1cells with LPS for 24 h We observed that LPS upregulatedNLRP3 mRNA and protein (Figures 4(a) 4(c) and 4(d))Similarly LPS also significantly enhanced mRNA expressionof cardiolipin synthetase 1 (CLS1) (Figure 4(b)) a mitochon-drial enzyme catalysing cardiolipin synthesis necessary forinflammasome NLRP3 activity [28] As shown in Figures4(a)ndash4(d) pretreatment with scropolioside B inhibited the

expressions of NLRP3 mRNA and protein as well as CLS1mRNA We also found that catalpol was as equally effectiveas scropolioside B (Figures 4(a)ndash4(d)) suggesting that thisinhibitory effect may be from the same catalpol structure(Figure 6)

35 Scropolioside B Decreases Expression of Pro-IL-1120573 andIL-1120573 To comprehensively evaluate the inhibitory effect of

Mediators of Inflammation 7

LPS (1120583gmL)

Pro-IL-1120573

IL-1120573

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

500

400

300

200

100

000

Pro-

IL-1120573

band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

lowastlowast

minus minus + minus

minus minus minus +

(b)

lowastlowast

250

200

150

050

100

000

IL-1120573

ban

d de

nsity

(fol

d ba

sal)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

lowastlowast

1500

500

1000

000

IL-1120573

(pg

mL)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

Figure 5 Scropolioside B and catalpol inhibited the expression of pro-IL-1120573 and IL-1120573 in LPS-induced THP-1 cells ((a)ndash(d)) THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 hThe protein expressionof pro-IL-1120573 and IL-1120573 was measured by western blot The levels of cytokines in the medium were measured using an ELISA kit The datarepresent the mean values of over three experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone

scropolioside B and catalpol we performed further studiesabout scropolioside B impact on protein expression of pro-IL-1120573 and IL-1120573 As shown in Figures 5(a)ndash5(d) pretreatmentwith scropolioside B inhibited the expressions of pro-IL-1120573and IL-1120573proteinThe inhibition of pro-IL-1120573 is stronger thanIL-1120573 Catalpol does not inhibit the protein expression of pro-IL-1120573 and IL-1120573

4 Discussion

Scropolioside B which is from S dentata Royle ex Benthhas antipyretic detoxicating effect It is used in Tibetanmedicine such as smallpox measles other infectious feversand inflammatory diseases In this study we demon-strated that scropolioside B an iridoid glycoside containing

8 Mediators of Inflammation

LPSTLR

P50

P50

P65

P65

Scropolioside BScropolioside B

Catalpol

Caspase-1

Pro-IL-1120573 mRNA

Pro-IL-1120573 IL-1120573

Nucleus

NLRP3

Figure 6 A schematic diagram of the regulation of NLRP3 inflam-masome by scropolioside B and catalpol

a catalpol and two phenylpropanoids was more effectivethan catalpol in inhibiting the expressions of IL-1120573 andTNF-120572 in THP-1 cells activated by LPS or palmitic acid(Figure 1) Our results suggested that scropolioside B hashigher anti-inflammatory activity than catalpol althoughsome studies have reported that catalpol does demonstrateanti-inflammatory effects at high dose by inhibiting COX-2activity TNF-120572 formation monocyte chemotactic protein-1 (MCP-1) and nitric oxide production [24 25] As shownin Figure 2 the anti-inflammatory effects of scropolioside Bwere mediated by blocking NF-120581B activity (Figure 2) IL-1120573is involved in the onset of acute local or systemic inflam-mation and contributes to a variety of chronic noninfectiousdiseases including ischemic injury atherosclerosis type 2diabetes and osteoarthritis Endogenousmetabolites such asoxidized fatty acids high glucose uric acid crystals activatedcomplement necrotic cells and cytokines can stimulatethe synthesis of the inactive IL-1120573 precursor which awaitsprocessing by the inflammasome complex to be activated[29]

Inflammasomes regulate maturity of IL-1120573 IL-18 andpyroptosis and recognize microbial products or endogenousmolecules released from damaged cells [27] Inflammasomeshave several member proteins including NLRP1 NLRP3NLRC4 AIM2 and NRP6 [6] Our results showed thatscropolioside B can inhibit mRNA and protein expressionof inflammasome NLRP3 and prevents the secretion of IL-1120573 (Figure 4) Interestingly catalpol has a similar inhibitoryeffect on NLRP3 expression compared to scropolioside Bsuggesting that scropolioside B and catalpol inhibit IL-1120573 andNLRP3 expression by different mechanisms ScropoliosideB and catalpol also block the expression of CLS1 which isan enzyme in the final step of mitochondrial cardiolipinsynthesis by catalysing the transfer of a phosphatidyl residuefrom diacylglycerol to phosphatidylglycerol Iyer et al [28]reported that mitochondrial cardiolipin and reactive oxygenspecies are needed for inflammasome NLRP3 activity Car-diolipin can bind directly to NLRP3 and silencing of cardi-olipin synthesis specifically inhibits inflammasome NLRP3activation [28] Based on these observations we believe

that scropolioside B not only blocks NF-120581B pathway butalso inhibits NLRP3 CLS1 and IL-1120573 expressions Howevercatalpol only prevents the expression of NLRP3 and CLS1(Figure 5)

Our results also showed that scropolioside B but notcatalpol blocked IL-32120573120574 expression (Figure 3) Severalstudies have shown that IL-32 an important proinflamma-tory cytokine in rheumatoid arthritis enhanced IL-6 and IL-8 production in fibroblast-like synoviocytes [30ndash32] Somestudies also have shown that IL-32 is closely associated withliver fibrosis of chronic viral hepatitis [33 34] Furthermorecompared with primary blood monocytes IL-1120573 TNF-120572 orLPS can stimulate high levels of IL-32 expression through theI120581B kinase-120573NF-120581B and ERK pathways in human umbilicalvein endothelial cells aortic macrovascular endothelial cellsand cardiac and pulmonary microvascular endothelial cells[26] Conversely IL-32 also stimulates IL-1120572 IL-1120573 IL-6TNF-120572 and chemokines via NF-120581B p38 MAPK and AP-1activation [26 35] IL-32 promotes angiogenesis propagatingvascular inflammation and exacerbates sepsis in a mousemodel [36 37] Recent studies have shown that atheroscle-rosis maybe associated with IL-32 production [38]

In conclusion scropolioside B significantly diminishedexpression and secretion of IL-1120573 IL-32 and TNF-120572 Weshow that this is mediated by modulating NF-120581B NLRP3and CLS1 levels Additional studies are needed to furtherelucidate other targets by which scropolioside and catalpolregulate inflammation The results of this study strengthenprevious understanding of the anti-inflammatory effects ofiridoids and highlight scropolioside B as a potential drugfor the treatment of rheumatoid arthritis and atheroscleroticdisease

Conflict of Interests

The authors declared no conflict of interests

Authorsrsquo Contribution

Tiantian Zhu and Liuqiang Zhang contributed equally to thiswork

Acknowledgments

This work was supported by grants from the NationalScience and Technology Major Project of the Ministry ofScience and Technology of China (2009ZX09311-003) theNational Natural Science Foundation of China (8117351881274130) the Shanghai 085 Project of Higher EducationConnotation Construction (085ZY1202) and the FundingScheme for Training Young Teachers in Shanghai Colleges(no ZZszy13057)

References

[1] G Pawelec DGoldeck andEDerhovanessian ldquoInflammationageing and chronic diseaserdquo Current Opinion in Immunologyvol 29 no 1 pp 23ndash28 2014

Mediators of Inflammation 9

[2] L M Coussens L Zitvogel and A K Palucka ldquoNeutralizingtumor-promoting chronic inflammation a magic bulletrdquo Sci-ence vol 339 no 6117 pp 286ndash291 2013

[3] G E McKellar D W McCarey N Sattar and I B McInnesldquoRole for TNF in atherosclerosis Lessons from autoimmunediseaserdquo Nature Reviews vol 6 no 6 pp 410ndash417 2009

[4] V H Perry and J Teeling ldquoMicroglia and macrophages of thecentral nervous system the contribution of microglia primingand systemic inflammation to chronic neurodegenerationrdquoSeminars in Immunopathology vol 35 no 5 pp 601ndash612 2013

[5] M L Schmitz A Weber T Roxlau M Gaestel and M KrachtldquoSignal integration crosstalk mechanisms and networks in thefunction of inflammatory cytokinesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1813 no 12 pp 2165ndash21752011

[6] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[7] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 pp 1357ndash1361 2010

[8] K Schroder R Zhou and J Tschopp ldquoThe NLRP3 inflamma-some a sensor formetabolic dangerrdquo Science vol 327 no 5963pp 296ndash300 2010

[9] S L Masters A Dunne S L Subramanian et al ldquoActivationof the NLRP3 inflammasome by islet amyloid polypeptideprovides a mechanism for enhanced IL-1120573 2 in type 2 diabetesrdquoNature Immunology vol 11 no 10 pp 897ndash904 2010

[10] J Petrasek S Bala T Csak et al ldquoIL-1 receptor antagonistameliorates inflammasome-dependent alcoholic steatohepatitisin micerdquo Journal of Clinical Investigation vol 122 no 10 pp3476ndash3489 2012

[11] J Dupaul-Chicoine G Yeretssian K Doiron et al ldquoControl ofintestinal homeostasis colitis and colitis-associated colorectalcancer by the inflammatory caspasesrdquo Immunity vol 32 no 3pp 367ndash378 2010

[12] P I Sidiropoulos G Goulielmos G K Voloudakis E Petrakiand D T Boumpas ldquoInflammasomes and rheumatic diseasesevolving conceptsrdquo Annals of the Rheumatic Diseases vol 67no 10 pp 1382ndash1389 2008

[13] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[14] E Latz T S Xiao andA Stutz ldquoActivation and regulation of theinflammasomesrdquoNature Reviews Immunology vol 13 no 6 pp397ndash411 2013

[15] L-Q Zhang and Y-M Li ldquoAdvances in studies on chemicalconstituents in plants of Scrophularia L and their pharmacolog-ical effects in recent ten yearsrdquo Chinese Traditional and HerbalDrugs vol 42 no 11 pp 2360ndash2368 2011 (Chinese)

[16] A Viljoen N Mncwangi and I Vermaak ldquoAnti-inflammatoryiridoids of botanical originrdquo Current Medicinal Chemistry vol19 no 14 pp 2104ndash2127 2012

[17] R Tundis M R Loizzo F Menichini and G A StattildquoBiological and pharmacological activities of iridoids recentdevelopmentsrdquoMini-Reviews inMedicinal Chemistry vol 8 no4 pp 399ndash420 2008

[18] E Bas M C Recio S Manez et al ldquoNew insight intothe inhibition of the inflammatory response to experimentaldelayed-type hypersensitivity reactions inmice by scropoliosideArdquo European Journal of Pharmacology vol 555 no 2-3 pp 199ndash210 2007

[19] B Ahmed A J Al-Rehaily T A Al-Howiriny K A El-Sayed and M S Ahmad ldquoScropolioside-D2 and harpagoside-B two new iridoid glycosides from Scrophularia deserti andtheir antidiabetic and antiinflammatory activityrdquo Biological andPharmaceutical Bulletin vol 26 no 4 pp 462ndash467 2003

[20] M Stavri K T Mathew and S Gibbons ldquoAntimicrobialconstituents of Scrophularia desertirdquoPhytochemistry vol 67 no14 pp 1530ndash1533 2006

[21] P B Benito A M D Lanza A M S Sen et al ldquoEffects of someiridoids from plant origin on arachidonic acid metabolism incellular systemsrdquo Planta Medica vol 66 no 4 pp 324ndash3282000

[22] P Wang L Sun J Tan J Xu F Guo and Y Li ldquoTwo newglycosidated coumaramides from Clerodendron cyrtophyllumrdquoFitoterapia vol 83 no 8 pp 1494ndash1499 2012

[23] L Zhang L Feng Q Jia et al ldquoEffects of 120573-glucosidasehydrolyzed products of harpagide and harpagoside oncyclooxygenase-2 (COX-2) in vitrordquo Bioorganic and MedicinalChemistry vol 19 no 16 pp 4882ndash4886 2011

[24] K S Park B H Kim and I-M Chang ldquoInhibitory potencies ofseveral iridoids on cyclooxygenase-1 cyclooxygnase-2 enzymesactivities tumor necrosis factor-120572 and nitric oxide produc-tion In Vitrordquo Evidence-based Complementary and AlternativeMedicine vol 7 no 1 pp 41ndash45 2010

[25] H-J Choi H-J Jang T-W Chung et al ldquoCatalpol sup-presses advanced glycation end-products-induced inflamma-tory responses through inhibition of reactive oxygen species inhuman monocytic THP-1 cellsrdquo Fitoterapia vol 86 no 1 pp19ndash28 2013

[26] C A Nold-Petry M F Nold J A Zepp S-H Kim N FVoelkel and C A Dinarello ldquoIL-32-dependent effects of IL-1120573 on endothelial cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no10 pp 3883ndash3888 2009

[27] V A K Rathinam S K Vanaja and K A Fitzgerald ldquoRegu-lation of inflammasome signalingrdquoNature Immunology vol 13no 4 pp 333ndash342 2012

[28] S S Iyer Q He J R Janczy et al ldquoMitochondrial cardiolipinis required for Nlrp3 inflammasome activationrdquo Immunity vol39 no 2 pp 311ndash323 2013

[29] CADinarello ldquoA clinical perspective of IL-1120573 as the gatekeeperof inflammationrdquo European Journal of Immunology vol 41 no5 pp 1203ndash1217 2011

[30] L A B Joosten M G Netea S-H Kim et al ldquoIL-32 aproinflammatory cytokine in rheumatoid arthritisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 9 pp 3298ndash3303 2006

[31] B Heinhuis M I Koenders F A Van De Loo M G NeteaW B Van Den Berg and L A B Joosten ldquoInflammation-dependent secretion and splicing of IL-32120574 in rheumatoidarthritisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 12 pp 4962ndash4967 2011

[32] Y-G Kim C-K Lee S-H Kim W-S Cho S H Mun and BYoo ldquoInterleukin-32120574 enhances the production of IL-6 and IL-8in fibroblast-like synoviocytes Via Erk12 activationrdquo Journal ofClinical Immunology vol 30 no 2 pp 260ndash267 2010

[33] A R Moschen T Fritz A D Clouston et al ldquoInterleukin-32a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosisrdquoHepatology vol 53 no6 pp 1819ndash1829 2011

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

Mediators of Inflammation 7

LPS (1120583gmL)

Pro-IL-1120573

IL-1120573

GAPDH

Catalpol (50120583M)Scropolioside B

(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(a)

500

400

300

200

100

000

Pro-

IL-1120573

band

den

sity

(fold

bas

al)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

lowastlowast

minus minus + minus

minus minus minus +

(b)

lowastlowast

250

200

150

050

100

000

IL-1120573

ban

d de

nsity

(fol

d ba

sal)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(c)

lowastlowast

1500

500

1000

000

IL-1120573

(pg

mL)

LPS (1120583gmL)Catalpol (50120583M)

Scropolioside B(50120583M)

minus + + +

minus minus + minus

minus minus minus +

(d)

Figure 5 Scropolioside B and catalpol inhibited the expression of pro-IL-1120573 and IL-1120573 in LPS-induced THP-1 cells ((a)ndash(d)) THP-1 cells werepretreated with 50 120583molL catalpol or scropolioside B for 1 h and then stimulated with LPS (1120583gmL) for another 24 hThe protein expressionof pro-IL-1120573 and IL-1120573 was measured by western blot The levels of cytokines in the medium were measured using an ELISA kit The datarepresent the mean values of over three experiments plusmn SD 119875 lt 001 compared to vehicle control lowastlowast119875 lt 001 compared to LPS alone

scropolioside B and catalpol we performed further studiesabout scropolioside B impact on protein expression of pro-IL-1120573 and IL-1120573 As shown in Figures 5(a)ndash5(d) pretreatmentwith scropolioside B inhibited the expressions of pro-IL-1120573and IL-1120573proteinThe inhibition of pro-IL-1120573 is stronger thanIL-1120573 Catalpol does not inhibit the protein expression of pro-IL-1120573 and IL-1120573

4 Discussion

Scropolioside B which is from S dentata Royle ex Benthhas antipyretic detoxicating effect It is used in Tibetanmedicine such as smallpox measles other infectious feversand inflammatory diseases In this study we demon-strated that scropolioside B an iridoid glycoside containing

8 Mediators of Inflammation

LPSTLR

P50

P50

P65

P65

Scropolioside BScropolioside B

Catalpol

Caspase-1

Pro-IL-1120573 mRNA

Pro-IL-1120573 IL-1120573

Nucleus

NLRP3

Figure 6 A schematic diagram of the regulation of NLRP3 inflam-masome by scropolioside B and catalpol

a catalpol and two phenylpropanoids was more effectivethan catalpol in inhibiting the expressions of IL-1120573 andTNF-120572 in THP-1 cells activated by LPS or palmitic acid(Figure 1) Our results suggested that scropolioside B hashigher anti-inflammatory activity than catalpol althoughsome studies have reported that catalpol does demonstrateanti-inflammatory effects at high dose by inhibiting COX-2activity TNF-120572 formation monocyte chemotactic protein-1 (MCP-1) and nitric oxide production [24 25] As shownin Figure 2 the anti-inflammatory effects of scropolioside Bwere mediated by blocking NF-120581B activity (Figure 2) IL-1120573is involved in the onset of acute local or systemic inflam-mation and contributes to a variety of chronic noninfectiousdiseases including ischemic injury atherosclerosis type 2diabetes and osteoarthritis Endogenousmetabolites such asoxidized fatty acids high glucose uric acid crystals activatedcomplement necrotic cells and cytokines can stimulatethe synthesis of the inactive IL-1120573 precursor which awaitsprocessing by the inflammasome complex to be activated[29]

Inflammasomes regulate maturity of IL-1120573 IL-18 andpyroptosis and recognize microbial products or endogenousmolecules released from damaged cells [27] Inflammasomeshave several member proteins including NLRP1 NLRP3NLRC4 AIM2 and NRP6 [6] Our results showed thatscropolioside B can inhibit mRNA and protein expressionof inflammasome NLRP3 and prevents the secretion of IL-1120573 (Figure 4) Interestingly catalpol has a similar inhibitoryeffect on NLRP3 expression compared to scropolioside Bsuggesting that scropolioside B and catalpol inhibit IL-1120573 andNLRP3 expression by different mechanisms ScropoliosideB and catalpol also block the expression of CLS1 which isan enzyme in the final step of mitochondrial cardiolipinsynthesis by catalysing the transfer of a phosphatidyl residuefrom diacylglycerol to phosphatidylglycerol Iyer et al [28]reported that mitochondrial cardiolipin and reactive oxygenspecies are needed for inflammasome NLRP3 activity Car-diolipin can bind directly to NLRP3 and silencing of cardi-olipin synthesis specifically inhibits inflammasome NLRP3activation [28] Based on these observations we believe

that scropolioside B not only blocks NF-120581B pathway butalso inhibits NLRP3 CLS1 and IL-1120573 expressions Howevercatalpol only prevents the expression of NLRP3 and CLS1(Figure 5)

Our results also showed that scropolioside B but notcatalpol blocked IL-32120573120574 expression (Figure 3) Severalstudies have shown that IL-32 an important proinflamma-tory cytokine in rheumatoid arthritis enhanced IL-6 and IL-8 production in fibroblast-like synoviocytes [30ndash32] Somestudies also have shown that IL-32 is closely associated withliver fibrosis of chronic viral hepatitis [33 34] Furthermorecompared with primary blood monocytes IL-1120573 TNF-120572 orLPS can stimulate high levels of IL-32 expression through theI120581B kinase-120573NF-120581B and ERK pathways in human umbilicalvein endothelial cells aortic macrovascular endothelial cellsand cardiac and pulmonary microvascular endothelial cells[26] Conversely IL-32 also stimulates IL-1120572 IL-1120573 IL-6TNF-120572 and chemokines via NF-120581B p38 MAPK and AP-1activation [26 35] IL-32 promotes angiogenesis propagatingvascular inflammation and exacerbates sepsis in a mousemodel [36 37] Recent studies have shown that atheroscle-rosis maybe associated with IL-32 production [38]

In conclusion scropolioside B significantly diminishedexpression and secretion of IL-1120573 IL-32 and TNF-120572 Weshow that this is mediated by modulating NF-120581B NLRP3and CLS1 levels Additional studies are needed to furtherelucidate other targets by which scropolioside and catalpolregulate inflammation The results of this study strengthenprevious understanding of the anti-inflammatory effects ofiridoids and highlight scropolioside B as a potential drugfor the treatment of rheumatoid arthritis and atheroscleroticdisease

Conflict of Interests

The authors declared no conflict of interests

Authorsrsquo Contribution

Tiantian Zhu and Liuqiang Zhang contributed equally to thiswork

Acknowledgments

This work was supported by grants from the NationalScience and Technology Major Project of the Ministry ofScience and Technology of China (2009ZX09311-003) theNational Natural Science Foundation of China (8117351881274130) the Shanghai 085 Project of Higher EducationConnotation Construction (085ZY1202) and the FundingScheme for Training Young Teachers in Shanghai Colleges(no ZZszy13057)

References

[1] G Pawelec DGoldeck andEDerhovanessian ldquoInflammationageing and chronic diseaserdquo Current Opinion in Immunologyvol 29 no 1 pp 23ndash28 2014

Mediators of Inflammation 9

[2] L M Coussens L Zitvogel and A K Palucka ldquoNeutralizingtumor-promoting chronic inflammation a magic bulletrdquo Sci-ence vol 339 no 6117 pp 286ndash291 2013

[3] G E McKellar D W McCarey N Sattar and I B McInnesldquoRole for TNF in atherosclerosis Lessons from autoimmunediseaserdquo Nature Reviews vol 6 no 6 pp 410ndash417 2009

[4] V H Perry and J Teeling ldquoMicroglia and macrophages of thecentral nervous system the contribution of microglia primingand systemic inflammation to chronic neurodegenerationrdquoSeminars in Immunopathology vol 35 no 5 pp 601ndash612 2013

[5] M L Schmitz A Weber T Roxlau M Gaestel and M KrachtldquoSignal integration crosstalk mechanisms and networks in thefunction of inflammatory cytokinesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1813 no 12 pp 2165ndash21752011

[6] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[7] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 pp 1357ndash1361 2010

[8] K Schroder R Zhou and J Tschopp ldquoThe NLRP3 inflamma-some a sensor formetabolic dangerrdquo Science vol 327 no 5963pp 296ndash300 2010

[9] S L Masters A Dunne S L Subramanian et al ldquoActivationof the NLRP3 inflammasome by islet amyloid polypeptideprovides a mechanism for enhanced IL-1120573 2 in type 2 diabetesrdquoNature Immunology vol 11 no 10 pp 897ndash904 2010

[10] J Petrasek S Bala T Csak et al ldquoIL-1 receptor antagonistameliorates inflammasome-dependent alcoholic steatohepatitisin micerdquo Journal of Clinical Investigation vol 122 no 10 pp3476ndash3489 2012

[11] J Dupaul-Chicoine G Yeretssian K Doiron et al ldquoControl ofintestinal homeostasis colitis and colitis-associated colorectalcancer by the inflammatory caspasesrdquo Immunity vol 32 no 3pp 367ndash378 2010

[12] P I Sidiropoulos G Goulielmos G K Voloudakis E Petrakiand D T Boumpas ldquoInflammasomes and rheumatic diseasesevolving conceptsrdquo Annals of the Rheumatic Diseases vol 67no 10 pp 1382ndash1389 2008

[13] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[14] E Latz T S Xiao andA Stutz ldquoActivation and regulation of theinflammasomesrdquoNature Reviews Immunology vol 13 no 6 pp397ndash411 2013

[15] L-Q Zhang and Y-M Li ldquoAdvances in studies on chemicalconstituents in plants of Scrophularia L and their pharmacolog-ical effects in recent ten yearsrdquo Chinese Traditional and HerbalDrugs vol 42 no 11 pp 2360ndash2368 2011 (Chinese)

[16] A Viljoen N Mncwangi and I Vermaak ldquoAnti-inflammatoryiridoids of botanical originrdquo Current Medicinal Chemistry vol19 no 14 pp 2104ndash2127 2012

[17] R Tundis M R Loizzo F Menichini and G A StattildquoBiological and pharmacological activities of iridoids recentdevelopmentsrdquoMini-Reviews inMedicinal Chemistry vol 8 no4 pp 399ndash420 2008

[18] E Bas M C Recio S Manez et al ldquoNew insight intothe inhibition of the inflammatory response to experimentaldelayed-type hypersensitivity reactions inmice by scropoliosideArdquo European Journal of Pharmacology vol 555 no 2-3 pp 199ndash210 2007

[19] B Ahmed A J Al-Rehaily T A Al-Howiriny K A El-Sayed and M S Ahmad ldquoScropolioside-D2 and harpagoside-B two new iridoid glycosides from Scrophularia deserti andtheir antidiabetic and antiinflammatory activityrdquo Biological andPharmaceutical Bulletin vol 26 no 4 pp 462ndash467 2003

[20] M Stavri K T Mathew and S Gibbons ldquoAntimicrobialconstituents of Scrophularia desertirdquoPhytochemistry vol 67 no14 pp 1530ndash1533 2006

[21] P B Benito A M D Lanza A M S Sen et al ldquoEffects of someiridoids from plant origin on arachidonic acid metabolism incellular systemsrdquo Planta Medica vol 66 no 4 pp 324ndash3282000

[22] P Wang L Sun J Tan J Xu F Guo and Y Li ldquoTwo newglycosidated coumaramides from Clerodendron cyrtophyllumrdquoFitoterapia vol 83 no 8 pp 1494ndash1499 2012

[23] L Zhang L Feng Q Jia et al ldquoEffects of 120573-glucosidasehydrolyzed products of harpagide and harpagoside oncyclooxygenase-2 (COX-2) in vitrordquo Bioorganic and MedicinalChemistry vol 19 no 16 pp 4882ndash4886 2011

[24] K S Park B H Kim and I-M Chang ldquoInhibitory potencies ofseveral iridoids on cyclooxygenase-1 cyclooxygnase-2 enzymesactivities tumor necrosis factor-120572 and nitric oxide produc-tion In Vitrordquo Evidence-based Complementary and AlternativeMedicine vol 7 no 1 pp 41ndash45 2010

[25] H-J Choi H-J Jang T-W Chung et al ldquoCatalpol sup-presses advanced glycation end-products-induced inflamma-tory responses through inhibition of reactive oxygen species inhuman monocytic THP-1 cellsrdquo Fitoterapia vol 86 no 1 pp19ndash28 2013

[26] C A Nold-Petry M F Nold J A Zepp S-H Kim N FVoelkel and C A Dinarello ldquoIL-32-dependent effects of IL-1120573 on endothelial cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no10 pp 3883ndash3888 2009

[27] V A K Rathinam S K Vanaja and K A Fitzgerald ldquoRegu-lation of inflammasome signalingrdquoNature Immunology vol 13no 4 pp 333ndash342 2012

[28] S S Iyer Q He J R Janczy et al ldquoMitochondrial cardiolipinis required for Nlrp3 inflammasome activationrdquo Immunity vol39 no 2 pp 311ndash323 2013

[29] CADinarello ldquoA clinical perspective of IL-1120573 as the gatekeeperof inflammationrdquo European Journal of Immunology vol 41 no5 pp 1203ndash1217 2011

[30] L A B Joosten M G Netea S-H Kim et al ldquoIL-32 aproinflammatory cytokine in rheumatoid arthritisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 9 pp 3298ndash3303 2006

[31] B Heinhuis M I Koenders F A Van De Loo M G NeteaW B Van Den Berg and L A B Joosten ldquoInflammation-dependent secretion and splicing of IL-32120574 in rheumatoidarthritisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 12 pp 4962ndash4967 2011

[32] Y-G Kim C-K Lee S-H Kim W-S Cho S H Mun and BYoo ldquoInterleukin-32120574 enhances the production of IL-6 and IL-8in fibroblast-like synoviocytes Via Erk12 activationrdquo Journal ofClinical Immunology vol 30 no 2 pp 260ndash267 2010

[33] A R Moschen T Fritz A D Clouston et al ldquoInterleukin-32a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosisrdquoHepatology vol 53 no6 pp 1819ndash1829 2011

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

8 Mediators of Inflammation

LPSTLR

P50

P50

P65

P65

Scropolioside BScropolioside B

Catalpol

Caspase-1

Pro-IL-1120573 mRNA

Pro-IL-1120573 IL-1120573

Nucleus

NLRP3

Figure 6 A schematic diagram of the regulation of NLRP3 inflam-masome by scropolioside B and catalpol

a catalpol and two phenylpropanoids was more effectivethan catalpol in inhibiting the expressions of IL-1120573 andTNF-120572 in THP-1 cells activated by LPS or palmitic acid(Figure 1) Our results suggested that scropolioside B hashigher anti-inflammatory activity than catalpol althoughsome studies have reported that catalpol does demonstrateanti-inflammatory effects at high dose by inhibiting COX-2activity TNF-120572 formation monocyte chemotactic protein-1 (MCP-1) and nitric oxide production [24 25] As shownin Figure 2 the anti-inflammatory effects of scropolioside Bwere mediated by blocking NF-120581B activity (Figure 2) IL-1120573is involved in the onset of acute local or systemic inflam-mation and contributes to a variety of chronic noninfectiousdiseases including ischemic injury atherosclerosis type 2diabetes and osteoarthritis Endogenousmetabolites such asoxidized fatty acids high glucose uric acid crystals activatedcomplement necrotic cells and cytokines can stimulatethe synthesis of the inactive IL-1120573 precursor which awaitsprocessing by the inflammasome complex to be activated[29]

Inflammasomes regulate maturity of IL-1120573 IL-18 andpyroptosis and recognize microbial products or endogenousmolecules released from damaged cells [27] Inflammasomeshave several member proteins including NLRP1 NLRP3NLRC4 AIM2 and NRP6 [6] Our results showed thatscropolioside B can inhibit mRNA and protein expressionof inflammasome NLRP3 and prevents the secretion of IL-1120573 (Figure 4) Interestingly catalpol has a similar inhibitoryeffect on NLRP3 expression compared to scropolioside Bsuggesting that scropolioside B and catalpol inhibit IL-1120573 andNLRP3 expression by different mechanisms ScropoliosideB and catalpol also block the expression of CLS1 which isan enzyme in the final step of mitochondrial cardiolipinsynthesis by catalysing the transfer of a phosphatidyl residuefrom diacylglycerol to phosphatidylglycerol Iyer et al [28]reported that mitochondrial cardiolipin and reactive oxygenspecies are needed for inflammasome NLRP3 activity Car-diolipin can bind directly to NLRP3 and silencing of cardi-olipin synthesis specifically inhibits inflammasome NLRP3activation [28] Based on these observations we believe

that scropolioside B not only blocks NF-120581B pathway butalso inhibits NLRP3 CLS1 and IL-1120573 expressions Howevercatalpol only prevents the expression of NLRP3 and CLS1(Figure 5)

Our results also showed that scropolioside B but notcatalpol blocked IL-32120573120574 expression (Figure 3) Severalstudies have shown that IL-32 an important proinflamma-tory cytokine in rheumatoid arthritis enhanced IL-6 and IL-8 production in fibroblast-like synoviocytes [30ndash32] Somestudies also have shown that IL-32 is closely associated withliver fibrosis of chronic viral hepatitis [33 34] Furthermorecompared with primary blood monocytes IL-1120573 TNF-120572 orLPS can stimulate high levels of IL-32 expression through theI120581B kinase-120573NF-120581B and ERK pathways in human umbilicalvein endothelial cells aortic macrovascular endothelial cellsand cardiac and pulmonary microvascular endothelial cells[26] Conversely IL-32 also stimulates IL-1120572 IL-1120573 IL-6TNF-120572 and chemokines via NF-120581B p38 MAPK and AP-1activation [26 35] IL-32 promotes angiogenesis propagatingvascular inflammation and exacerbates sepsis in a mousemodel [36 37] Recent studies have shown that atheroscle-rosis maybe associated with IL-32 production [38]

In conclusion scropolioside B significantly diminishedexpression and secretion of IL-1120573 IL-32 and TNF-120572 Weshow that this is mediated by modulating NF-120581B NLRP3and CLS1 levels Additional studies are needed to furtherelucidate other targets by which scropolioside and catalpolregulate inflammation The results of this study strengthenprevious understanding of the anti-inflammatory effects ofiridoids and highlight scropolioside B as a potential drugfor the treatment of rheumatoid arthritis and atheroscleroticdisease

Conflict of Interests

The authors declared no conflict of interests

Authorsrsquo Contribution

Tiantian Zhu and Liuqiang Zhang contributed equally to thiswork

Acknowledgments

This work was supported by grants from the NationalScience and Technology Major Project of the Ministry ofScience and Technology of China (2009ZX09311-003) theNational Natural Science Foundation of China (8117351881274130) the Shanghai 085 Project of Higher EducationConnotation Construction (085ZY1202) and the FundingScheme for Training Young Teachers in Shanghai Colleges(no ZZszy13057)

References

[1] G Pawelec DGoldeck andEDerhovanessian ldquoInflammationageing and chronic diseaserdquo Current Opinion in Immunologyvol 29 no 1 pp 23ndash28 2014

Mediators of Inflammation 9

[2] L M Coussens L Zitvogel and A K Palucka ldquoNeutralizingtumor-promoting chronic inflammation a magic bulletrdquo Sci-ence vol 339 no 6117 pp 286ndash291 2013

[3] G E McKellar D W McCarey N Sattar and I B McInnesldquoRole for TNF in atherosclerosis Lessons from autoimmunediseaserdquo Nature Reviews vol 6 no 6 pp 410ndash417 2009

[4] V H Perry and J Teeling ldquoMicroglia and macrophages of thecentral nervous system the contribution of microglia primingand systemic inflammation to chronic neurodegenerationrdquoSeminars in Immunopathology vol 35 no 5 pp 601ndash612 2013

[5] M L Schmitz A Weber T Roxlau M Gaestel and M KrachtldquoSignal integration crosstalk mechanisms and networks in thefunction of inflammatory cytokinesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1813 no 12 pp 2165ndash21752011

[6] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[7] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 pp 1357ndash1361 2010

[8] K Schroder R Zhou and J Tschopp ldquoThe NLRP3 inflamma-some a sensor formetabolic dangerrdquo Science vol 327 no 5963pp 296ndash300 2010

[9] S L Masters A Dunne S L Subramanian et al ldquoActivationof the NLRP3 inflammasome by islet amyloid polypeptideprovides a mechanism for enhanced IL-1120573 2 in type 2 diabetesrdquoNature Immunology vol 11 no 10 pp 897ndash904 2010

[10] J Petrasek S Bala T Csak et al ldquoIL-1 receptor antagonistameliorates inflammasome-dependent alcoholic steatohepatitisin micerdquo Journal of Clinical Investigation vol 122 no 10 pp3476ndash3489 2012

[11] J Dupaul-Chicoine G Yeretssian K Doiron et al ldquoControl ofintestinal homeostasis colitis and colitis-associated colorectalcancer by the inflammatory caspasesrdquo Immunity vol 32 no 3pp 367ndash378 2010

[12] P I Sidiropoulos G Goulielmos G K Voloudakis E Petrakiand D T Boumpas ldquoInflammasomes and rheumatic diseasesevolving conceptsrdquo Annals of the Rheumatic Diseases vol 67no 10 pp 1382ndash1389 2008

[13] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[14] E Latz T S Xiao andA Stutz ldquoActivation and regulation of theinflammasomesrdquoNature Reviews Immunology vol 13 no 6 pp397ndash411 2013

[15] L-Q Zhang and Y-M Li ldquoAdvances in studies on chemicalconstituents in plants of Scrophularia L and their pharmacolog-ical effects in recent ten yearsrdquo Chinese Traditional and HerbalDrugs vol 42 no 11 pp 2360ndash2368 2011 (Chinese)

[16] A Viljoen N Mncwangi and I Vermaak ldquoAnti-inflammatoryiridoids of botanical originrdquo Current Medicinal Chemistry vol19 no 14 pp 2104ndash2127 2012

[17] R Tundis M R Loizzo F Menichini and G A StattildquoBiological and pharmacological activities of iridoids recentdevelopmentsrdquoMini-Reviews inMedicinal Chemistry vol 8 no4 pp 399ndash420 2008

[18] E Bas M C Recio S Manez et al ldquoNew insight intothe inhibition of the inflammatory response to experimentaldelayed-type hypersensitivity reactions inmice by scropoliosideArdquo European Journal of Pharmacology vol 555 no 2-3 pp 199ndash210 2007

[19] B Ahmed A J Al-Rehaily T A Al-Howiriny K A El-Sayed and M S Ahmad ldquoScropolioside-D2 and harpagoside-B two new iridoid glycosides from Scrophularia deserti andtheir antidiabetic and antiinflammatory activityrdquo Biological andPharmaceutical Bulletin vol 26 no 4 pp 462ndash467 2003

[20] M Stavri K T Mathew and S Gibbons ldquoAntimicrobialconstituents of Scrophularia desertirdquoPhytochemistry vol 67 no14 pp 1530ndash1533 2006

[21] P B Benito A M D Lanza A M S Sen et al ldquoEffects of someiridoids from plant origin on arachidonic acid metabolism incellular systemsrdquo Planta Medica vol 66 no 4 pp 324ndash3282000

[22] P Wang L Sun J Tan J Xu F Guo and Y Li ldquoTwo newglycosidated coumaramides from Clerodendron cyrtophyllumrdquoFitoterapia vol 83 no 8 pp 1494ndash1499 2012

[23] L Zhang L Feng Q Jia et al ldquoEffects of 120573-glucosidasehydrolyzed products of harpagide and harpagoside oncyclooxygenase-2 (COX-2) in vitrordquo Bioorganic and MedicinalChemistry vol 19 no 16 pp 4882ndash4886 2011

[24] K S Park B H Kim and I-M Chang ldquoInhibitory potencies ofseveral iridoids on cyclooxygenase-1 cyclooxygnase-2 enzymesactivities tumor necrosis factor-120572 and nitric oxide produc-tion In Vitrordquo Evidence-based Complementary and AlternativeMedicine vol 7 no 1 pp 41ndash45 2010

[25] H-J Choi H-J Jang T-W Chung et al ldquoCatalpol sup-presses advanced glycation end-products-induced inflamma-tory responses through inhibition of reactive oxygen species inhuman monocytic THP-1 cellsrdquo Fitoterapia vol 86 no 1 pp19ndash28 2013

[26] C A Nold-Petry M F Nold J A Zepp S-H Kim N FVoelkel and C A Dinarello ldquoIL-32-dependent effects of IL-1120573 on endothelial cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no10 pp 3883ndash3888 2009

[27] V A K Rathinam S K Vanaja and K A Fitzgerald ldquoRegu-lation of inflammasome signalingrdquoNature Immunology vol 13no 4 pp 333ndash342 2012

[28] S S Iyer Q He J R Janczy et al ldquoMitochondrial cardiolipinis required for Nlrp3 inflammasome activationrdquo Immunity vol39 no 2 pp 311ndash323 2013

[29] CADinarello ldquoA clinical perspective of IL-1120573 as the gatekeeperof inflammationrdquo European Journal of Immunology vol 41 no5 pp 1203ndash1217 2011

[30] L A B Joosten M G Netea S-H Kim et al ldquoIL-32 aproinflammatory cytokine in rheumatoid arthritisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 9 pp 3298ndash3303 2006

[31] B Heinhuis M I Koenders F A Van De Loo M G NeteaW B Van Den Berg and L A B Joosten ldquoInflammation-dependent secretion and splicing of IL-32120574 in rheumatoidarthritisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 12 pp 4962ndash4967 2011

[32] Y-G Kim C-K Lee S-H Kim W-S Cho S H Mun and BYoo ldquoInterleukin-32120574 enhances the production of IL-6 and IL-8in fibroblast-like synoviocytes Via Erk12 activationrdquo Journal ofClinical Immunology vol 30 no 2 pp 260ndash267 2010

[33] A R Moschen T Fritz A D Clouston et al ldquoInterleukin-32a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosisrdquoHepatology vol 53 no6 pp 1819ndash1829 2011

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 9: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

Mediators of Inflammation 9

[2] L M Coussens L Zitvogel and A K Palucka ldquoNeutralizingtumor-promoting chronic inflammation a magic bulletrdquo Sci-ence vol 339 no 6117 pp 286ndash291 2013

[3] G E McKellar D W McCarey N Sattar and I B McInnesldquoRole for TNF in atherosclerosis Lessons from autoimmunediseaserdquo Nature Reviews vol 6 no 6 pp 410ndash417 2009

[4] V H Perry and J Teeling ldquoMicroglia and macrophages of thecentral nervous system the contribution of microglia primingand systemic inflammation to chronic neurodegenerationrdquoSeminars in Immunopathology vol 35 no 5 pp 601ndash612 2013

[5] M L Schmitz A Weber T Roxlau M Gaestel and M KrachtldquoSignal integration crosstalk mechanisms and networks in thefunction of inflammatory cytokinesrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1813 no 12 pp 2165ndash21752011

[6] T Strowig J Henao-Mejia E Elinav and R Flavell ldquoInflam-masomes in health and diseaserdquo Nature vol 481 no 7381 pp278ndash286 2012

[7] P Duewell H Kono K J Rayner et al ldquoNLRP3 inflammasomesare required for atherogenesis and activated by cholesterolcrystalsrdquo Nature vol 464 pp 1357ndash1361 2010

[8] K Schroder R Zhou and J Tschopp ldquoThe NLRP3 inflamma-some a sensor formetabolic dangerrdquo Science vol 327 no 5963pp 296ndash300 2010

[9] S L Masters A Dunne S L Subramanian et al ldquoActivationof the NLRP3 inflammasome by islet amyloid polypeptideprovides a mechanism for enhanced IL-1120573 2 in type 2 diabetesrdquoNature Immunology vol 11 no 10 pp 897ndash904 2010

[10] J Petrasek S Bala T Csak et al ldquoIL-1 receptor antagonistameliorates inflammasome-dependent alcoholic steatohepatitisin micerdquo Journal of Clinical Investigation vol 122 no 10 pp3476ndash3489 2012

[11] J Dupaul-Chicoine G Yeretssian K Doiron et al ldquoControl ofintestinal homeostasis colitis and colitis-associated colorectalcancer by the inflammatory caspasesrdquo Immunity vol 32 no 3pp 367ndash378 2010

[12] P I Sidiropoulos G Goulielmos G K Voloudakis E Petrakiand D T Boumpas ldquoInflammasomes and rheumatic diseasesevolving conceptsrdquo Annals of the Rheumatic Diseases vol 67no 10 pp 1382ndash1389 2008

[13] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[14] E Latz T S Xiao andA Stutz ldquoActivation and regulation of theinflammasomesrdquoNature Reviews Immunology vol 13 no 6 pp397ndash411 2013

[15] L-Q Zhang and Y-M Li ldquoAdvances in studies on chemicalconstituents in plants of Scrophularia L and their pharmacolog-ical effects in recent ten yearsrdquo Chinese Traditional and HerbalDrugs vol 42 no 11 pp 2360ndash2368 2011 (Chinese)

[16] A Viljoen N Mncwangi and I Vermaak ldquoAnti-inflammatoryiridoids of botanical originrdquo Current Medicinal Chemistry vol19 no 14 pp 2104ndash2127 2012

[17] R Tundis M R Loizzo F Menichini and G A StattildquoBiological and pharmacological activities of iridoids recentdevelopmentsrdquoMini-Reviews inMedicinal Chemistry vol 8 no4 pp 399ndash420 2008

[18] E Bas M C Recio S Manez et al ldquoNew insight intothe inhibition of the inflammatory response to experimentaldelayed-type hypersensitivity reactions inmice by scropoliosideArdquo European Journal of Pharmacology vol 555 no 2-3 pp 199ndash210 2007

[19] B Ahmed A J Al-Rehaily T A Al-Howiriny K A El-Sayed and M S Ahmad ldquoScropolioside-D2 and harpagoside-B two new iridoid glycosides from Scrophularia deserti andtheir antidiabetic and antiinflammatory activityrdquo Biological andPharmaceutical Bulletin vol 26 no 4 pp 462ndash467 2003

[20] M Stavri K T Mathew and S Gibbons ldquoAntimicrobialconstituents of Scrophularia desertirdquoPhytochemistry vol 67 no14 pp 1530ndash1533 2006

[21] P B Benito A M D Lanza A M S Sen et al ldquoEffects of someiridoids from plant origin on arachidonic acid metabolism incellular systemsrdquo Planta Medica vol 66 no 4 pp 324ndash3282000

[22] P Wang L Sun J Tan J Xu F Guo and Y Li ldquoTwo newglycosidated coumaramides from Clerodendron cyrtophyllumrdquoFitoterapia vol 83 no 8 pp 1494ndash1499 2012

[23] L Zhang L Feng Q Jia et al ldquoEffects of 120573-glucosidasehydrolyzed products of harpagide and harpagoside oncyclooxygenase-2 (COX-2) in vitrordquo Bioorganic and MedicinalChemistry vol 19 no 16 pp 4882ndash4886 2011

[24] K S Park B H Kim and I-M Chang ldquoInhibitory potencies ofseveral iridoids on cyclooxygenase-1 cyclooxygnase-2 enzymesactivities tumor necrosis factor-120572 and nitric oxide produc-tion In Vitrordquo Evidence-based Complementary and AlternativeMedicine vol 7 no 1 pp 41ndash45 2010

[25] H-J Choi H-J Jang T-W Chung et al ldquoCatalpol sup-presses advanced glycation end-products-induced inflamma-tory responses through inhibition of reactive oxygen species inhuman monocytic THP-1 cellsrdquo Fitoterapia vol 86 no 1 pp19ndash28 2013

[26] C A Nold-Petry M F Nold J A Zepp S-H Kim N FVoelkel and C A Dinarello ldquoIL-32-dependent effects of IL-1120573 on endothelial cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no10 pp 3883ndash3888 2009

[27] V A K Rathinam S K Vanaja and K A Fitzgerald ldquoRegu-lation of inflammasome signalingrdquoNature Immunology vol 13no 4 pp 333ndash342 2012

[28] S S Iyer Q He J R Janczy et al ldquoMitochondrial cardiolipinis required for Nlrp3 inflammasome activationrdquo Immunity vol39 no 2 pp 311ndash323 2013

[29] CADinarello ldquoA clinical perspective of IL-1120573 as the gatekeeperof inflammationrdquo European Journal of Immunology vol 41 no5 pp 1203ndash1217 2011

[30] L A B Joosten M G Netea S-H Kim et al ldquoIL-32 aproinflammatory cytokine in rheumatoid arthritisrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 103 no 9 pp 3298ndash3303 2006

[31] B Heinhuis M I Koenders F A Van De Loo M G NeteaW B Van Den Berg and L A B Joosten ldquoInflammation-dependent secretion and splicing of IL-32120574 in rheumatoidarthritisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 12 pp 4962ndash4967 2011

[32] Y-G Kim C-K Lee S-H Kim W-S Cho S H Mun and BYoo ldquoInterleukin-32120574 enhances the production of IL-6 and IL-8in fibroblast-like synoviocytes Via Erk12 activationrdquo Journal ofClinical Immunology vol 30 no 2 pp 260ndash267 2010

[33] A R Moschen T Fritz A D Clouston et al ldquoInterleukin-32a new proinflammatory cytokine involved in hepatitis C virus-related liver inflammation and fibrosisrdquoHepatology vol 53 no6 pp 1819ndash1829 2011

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 10: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

10 Mediators of Inflammation

[34] Q Xu X Pan X Shu et al ldquoIncreased interleukin-32 expressionin chronic hepatitis B virus-infected liverrdquo Journal of Infectionvol 65 no 4 pp 336ndash342 2012

[35] S-H Kim S-Y Han T Azam D-Y Yoon and C A DinarelloldquoInterleukin-32 a cytokine and inducer of TNF120572rdquo Immunityvol 22 no 1 pp 131ndash142 2005

[36] C A Nold-Petry I Rudloff Y Baumer et al ldquoIL-32 promotesangiogenesisrdquo Journal of Immunology vol 192 no 2 pp 589ndash602 2014

[37] H Kobayashi J Huang F Ye Y Shyr T S Blackwell and PC Lin ldquoInterleukin-32120573 propagates vascular inflammation andexacerbates sepsis in a mouse modelrdquo PLoS ONE vol 5 no 3Article ID e9458 2010

[38] B Heinhuis C D Popa B L J H van Tits et al ldquoTowards arole of interleukin-32 in atherosclerosisrdquo Cytokine vol 64 no1 pp 433ndash440 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 11: Research Article Scropolioside B Inhibits IL-1 and Cytokines ...downloads.hindawi.com/journals/mi/2014/819053.pdfnuclear signaling mechanisms. ese mechanisms can acti-vate the NF-

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom