Research Article Potential Mechanisms of an ...downloads.hindawi.com/journals/ecam/2014/982913.pdf · Research Article Potential Mechanisms of an Antiadenomyosis Chinese Herbal Formula

Research ArticlePotential Mechanisms of an AntiadenomyosisChinese Herbal Formula Shaoyao-Gancao Decoction inPrimary Cell Culture Model

Yong-Ge Guan1 Jin-Bin Liao23 Kun-Yin Li1 Yu-Cui Li2 Yang Song4

Jing Ling1 and Zi-Ren Su2

1 First Affiliated Hospital Guangzhou University of Chinese Medicine Guangzhou 510405 China2 School of Chinese Materia Medica Guangzhou University of Chinese Medicine Guangzhou 510006 China3 Guangdong Second Province Hospital of Traditional Chinese Medicine Guangzhou 510095 China4 School of Nursing Guangzhou University of Chinese Medicine Guangzhou 510405 China

Correspondence should be addressed to Kun-Yin Li lky0303gzucmeducn and Zi-Ren Su suzirengzucmeducn

Received 5 August 2014 Revised 11 October 2014 Accepted 15 October 2014 Published 10 November 2014

Academic Editor Min Ye

Copyright copy 2014 Yong-Ge Guan et alThis 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

Background Shaoyao-Gancao Decoction (SGD) a well-known traditional Chinese medicine prescription has been widely usedto treat adenomyosis dysmenorrhea abdominal pain and inflammation in Asia However the mechanism underlying theeffectiveness of SGD in the treatment of adenomyosis still remains elusiveThe present study aimed to investigate the bioactivity ofSGD and its underlying molecular mechanisms using cultured human adenomyosis-derived cellsMethods Human adenomyosis-derived cells were treated with SGD and its major constituents (paeoniflorin and liquiritin) in vitro Effects of SGD paeoniflorinand liquiritin on cell proliferation and apoptosis were examined by MTT assay and flow cytometry analyses The effects of SGDpaeoniflorin and liquiritin on the production of PGE

2and PGF

2120572were assayed using ELISA ER-120572 and OTR mRNA expression

levels were also evaluated by real-time qRT-PCR Results SGD paeoniflorin and liquiritin inhibited proliferation and inducedapoptosis of human adenomyosis-derived cells in a dose-dependent manner SGD and paeoniflorin significantly reduced the PGE

2

and PGF2120572production Furthermore they remarkably decreased themRNA levels of ER-120572 andOTRConclusionsThe results of this

study provide possiblemechanisms for the bioactivity of SGD for treating adenomyosis and contribute to the ethnopharmacologicalknowledge about this prescription

1 Introduction

Adenomyosis also known as endometriosis interna is a com-mon gynecological disorder defined as the benign invasionof endometrial glands and stroma into the myometrium [1]The reported prevalence of adenomyosis varies from 5 to70 [2] Adenomyosis is not a fatal disease but it does have atremendous impact on the quality of a womanrsquos life causingmany health problems such as dysmenorrhea menorrha-gia metrorrhagia early pregnancy-stage miscarriages oreven subfertility [3] Treatment of adenomyosis has been achallenge with hysterectomy being the treatment of choicefor severe and symptomatic adenomyosis yet hysterectomycan be quite traumatic for women who still wish to have

a family [4] Even though adenomyosis is recognized ashormone sensitive progestogenic agents are ineffective [5]Other therapeutic options include gonadotrophin-releasinghormone (GnRH) agonists that could induce suppressionof adenomyosis However their use is confined by shortduration and easy recurrence after discontinuation of therapy[6 7] Moreover uterine artery embolization (UAE) has beeninvestigated as a possible therapy for adenomyosis but theeffectiveness of UAE in the treatment of adenomyosis is stilllimited [8 9] Thus novel therapeutic strategies are urgentlyneeded to improve the clinical management of patients withadenomyosis

Shaoyao-Gancao Decoction (SGD Shakuyaku-Kanzo-toin Japanese) a well-known traditional Chinese medicine

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 982913 11 pageshttpdxdoiorg1011552014982913

2 Evidence-Based Complementary and Alternative Medicine

prescription was sourced from the Chinese Medical ClassicstextmdashShanghan lun in 210 CEThe herbal prescription whichis made up of two herbs (Paeoniae Radix and GlycyrrhizaeRadix ldquoShaoyaordquo and ldquoGancaordquo in Chinese resp) is com-monly used in the treatment of gynecological disorders inChina including dysmenorrheal [10 11] menorrhagia [12]and infertility [10] Clinically SGD has been used veryefficaciously and widely to treat adenomyosis in women intraditional Chinese medical practice since it has the advan-tage of noninvasive and lessno side effects [13 14] Similarherbal prescription has also been used in Japan and otherOriental countries [15] In additionmodern pharmacologicalstudies have demonstrated that SGD possesses analgesic [16]and anti-inflammatory [17] effects Moreover SGD treatmentresulted in a significantly low incidence of adenomyosis in anexperimental animal model [18]

However little is known about the potential mechanismsof the antiadenomyosis effect of SGD In this study we ex-amined the effects of SGD and itsmajor constituents (paeoni-florin and liquiritin) on the proliferation and apoptosis ofhuman adenomyosis-derived cells In addition their effectson prostaglandin E

2(PGE2) and prostaglandin F

2120572(PGF2120572)

production and estrogen receptor-120572 (ER-120572) mRNA andoxytocin receptor (OTR) mRNA expression were also inves-tigated

2 Materials and Methods

21 Materials Acetonitrile (Merk Darmstadt Germany)Paeoniflorin and liquiritin were obtained fromThe NationalInstitute for the Control of Pharmaceutical and BiologicalProducts (Beijing China) 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide (MTT) Apoptosis DetectionKit (Catalog no V13241) sodium dodecyl (lauryl) sulphate-hydrochloride (SDS-HCl) and Trizol were purchased fromInvitrogen Dulbeccorsquos modified Eaglersquos medium (DMEM)Collagenase I 025 trypsin fetal bovine serum (FBS) andphosphate buffered saline (PBS) were obtained from GibcoM-MLV RTase cDNA Synthesis Kit and SYBR Premix Ex Taqwere obtained from Takara (Dalian China) PGE

2enzyme-

linked immunosorbent assay (ELISA) kit was obtained fromRampD Systems (Minneapolis USA) PGF

2120572ELISA kit was

purchased from ENZO Life Sciences

22 Single Herb and SGD Preparation Paeoniae Radix andGlycyrrhizae Radix were purchased from Caizhilin MaterialsCompany of Guangzhou and authenticated by professor Zi-Ren Su in the School of Pharmacy of Guangzhou Universityof Chinese Medicine

According to the preparation method of SGD by Shenet al with slight modifications [19] extract solutions of twosingle herbs and SGD were prepared in the following proce-dure Two single herbs and SGD were macerated for 30minwith eight times (vw) distilled water The medical solutionswere heated to boiling and then decocted for 40min and thefiltrates were collected The residues were decocted again for30min with eight times (vw) distilled water then collected

filtrates again and were mixed with previous collected fil-trates condensed under reduced pressure at 80∘C to obtain aconcentration equivalent to 1 gmL crude herb materials andstored at minus20∘C

23 Ultra-Fast Liquid Chromatography (UFLC) AnalysisUltra-fast liquid chromatography (UFLC) profiles of twosingle herbs and SGD were performed as the followingprocedures extract solutions of Paeoniae Radix (02mL)Glycyrrhizae Radix (008mL) and SGD (028mL) with aconcentration equivalent to 1 gmL crude herbmaterials weredried and then extractedwithmethanol-water (10mL 50 50vv) under ultrasonication for 30min The solutions werefiltered and then determined for UFLC analysis

UFLC equipment was controlled with LC-20AD pump(Shimadzu Co Kyoto Japan) using a YMC-UltraHT ProC18

column (50mm times 20mm ID 2 120583m YMC Inc) Theelution gradient was carried out with binary solvent systemconsisting of 05 acetic acid in H

2O (solvent A) and MeCN

(solvent B) A linear gradient profile with the followingproportions (vv) of solvent B was applied gradient profile0 to 10min and 5 of B 10 to 20min and 5 to 15 of B 20to 30min and 15 to 30 of B 30 to 40min and 30 to 50of B and 40 to 45min and 50 to 70 of BThe flow rate wascontrolled with LC 20AD at 04mLmin injection volume5 120583L and the column temperature was maintained at 28∘CThe effluents from the column were detected at absorbenciesranging from 200 to 400 nm using a photodiode-array detec-torThe three-dimensional data collection and processing forpeak analysis were accomplished with CLASS-LC10 SystemAnalysis Software (Shimadzu Co Kyoto Japan)

24 Tissue Preparation Adenomyosis specimens were ob-tained at the time of laparoscopy or laparotomy performedfor adenomyosis treatment from eight Chinese women aged20ndash45 years All patients had regular menstrual cycles Nopatients were undergoing hormone therapy prior to surgeryAll biopsy specimens were collected at the First AffiliatedHospital of Guangzhou University of Traditional ChineseMedicine between November 2009 and August 2010 withthe permission of the local ethics committee and writteninformed consent was obtained from each patient

25 Cell Isolation and Culture The biopsy specimens wereimmediately placed in ice-cold DMEM for transport to thelaboratory The tissues were then dissociated as previouslydescribed with some modifications [20] The biopsy speci-mens were washed once with PBS and twice with DMEMThe biopsy specimens were then minced into small piecesand incubated at 37∘C for 5 to 6 hours in Hankrsquos balanced saltsolution supplemented with 01 type I collagenase DMEMcontaining 10 FBS was added The cell suspensions werefiltered through a 200mesh stainless steel screen and the fil-trates were centrifuged (1000 rpm 8min 3 times) Cells weresuspended at a concentration of 5 times 105 cellmL in DMEMsupplemented with 10FBS then transferred to a tissueculture dish and incubated at 37∘C In order to accumulateenough cells for further study cultured cells of individual

Evidence-Based Complementary and Alternative Medicine 3

patients were pooled and cryopreserved The lower passagenumber (three to six) of cells was used for experiments toavoid changes in phenotype and gene expression

26 Cell Survival Assay Isolated cells were first placed in96-well microtiter plates at a density of 5 times 104 cells per welland cultivated in 100 120583L of the DMEM containing 10FBSAfter a 3-day preincubation period culture media werereplaced with 100120583L DMEM and then incubated at 37∘C for24 h The supernatants were removed and 100 120583L of freshDMEM containing various concentrations of SGD (01ndash500mgmL) paeoniflorin (00001ndash10mgmL) liquiritin(00001ndash10mgmL) and mifepristone (0005ndash100 120583M aspositive control) was added Cells were incubated at 37∘Cfor 24 h then 10120583L MTT (5mgmL) was added and thecells were incubated at 37∘C for 4 h After the medium andMTT were removed 100120583L of SDS-HCL was added to eachwell and then placed on a plate shaker for 10min at roomtemperature For each well absorbance at 570 nm was meas-ured using a microtiter plate ELISA reader The cell survivalrate was calculated as follows percent survival = (meanexperimental absorbancemean control absorbance) times 100

27 FlowCytometric Analysis of Cell Apoptosis Theflow cyto-metric analysis of annexinV-FITC and propidium iodide (PI)stained cells was performed using the Apoptosis DetectionKit [21] AnnexinV binds to extracellular phosphatidylserinea marker for both apoptotic cells and necrotic cells PI canenter only cells in which the integrity of the membrane hasbeen compromised which can constitute necrotic or lateapoptotic cellsTherefore healthy cells are doubly negative toannexin V and PI Cells stained positively only for annexin Vare early apoptotic Cells that are doubly positive for annexinV and PI are considered to be in late apoptosis [22] Accord-ing to the manufacturerrsquos protocol after treatments withSGD (10 and 20mgmL) paeoniflorin (025 and 05mgmL)liquiritin (025 and 05mgmL) and mifepristone (10 120583M aspositive control) for 24 hours cells were collected andwashedwith PBS Cells were harvested by incubating with 025trypsin for 3ndash6min and then DMEM containing 10FBSwas added Cells were centrifuged at 1000 rpm for 5min andthenwashed 3 timeswith PBS followed by being resuspendedin 500120583L of binding buffer containing 5 120583L annexin V-FITCand 5 120583L PI and then incubated for 5min in the dark at roomtemperature Following this the cells were analyzed by flowcytometry The percentage of cells undergoing apoptosis wasdetermined by three independent experiments

28 Measurement of PGE2and PGF

2120572Production Human

adenomyosis-derived cells were pretreated with SGD (10 and20mgmL) paeoniflorin (025 and 05mgmL) and liquiritin(025 and 05mgmL) for 24 hours The concentrations ofPGE2and PGF

2120572in the culture supernatant were determined

by ELISA kits according to the manufacturerrsquos protocol

29 Real-Time Quantitative Reverse Transcription-PolymeraseChain Reaction (qRT-PCR) Analysis The mRNA levels ofER-120572 and OTR were determined by qRT-PCR analysis Cells

preincubated with or without SGD (10 and 20mgmL)paeoniflorin (025 and 05mgmL) and liquiritin (025and 05mgmL) for 24 hours and then total RNA wasisolated using Trizol Reagent according to the manufacturerrsquosprotocol Total RNA was reverse-transcribed using a cDNAsynthesis kit qRT-PCRwas performed on theThermal CyclerDice Real Time System using SYBR Premix Ex Taq Theforward and reverse primers were 51015840-GTCTCGTCTGGC-GCTCCAT-31015840 and 51015840-CCCTGGTTCCTGTCCAAGAG-31015840for ER120572 mRNA 51015840-AGGAAGCCTCGGCCTTCAT-31015840 and51015840-GAGGTGGCCCGTGAACAG-31015840 for OTR mRNA 51015840-GCATGGGTCAGAAGGATTCCT-31015840 and 51015840-TCGTCC-CAGTTGGTGACGAT-31015840 for 120573-actin mRNA The relativeexpression of ER-120572 and OTR mRNA was normalized to theamount of 120573-actin mRNA levels

210 Statistical Analysis of Data For all groups data arepresented as themean plus orminus standard deviation (SD)Statistical significance within a parameter was evaluated byone-way analysis of variance (ANOVA) using the SPSS Valueof 119875 lt 005 was considered statistically significant

3 Results

31 Chemical Profiles of SGD UFLC profiles of two singleherbs and SGDwere shown in Figure 1 In order to control thequality of SGD we also detected the two active compositionsof five batches of SGD by UFLC with standard compositionsThe results indicated all the five batches contained two activecompoundsThe RSD of the relative peak areas were between032 and 257

32 Cell Morphology Inverted microscope was selectedfor the identification of human adenomyosis-derived cellsExamination of these cells in culture showed monolayersof cells displaying a fibroblast-like stellate or spindle-likemorphology as reported by Suzuki-Kakisaka et al [23]

33 Cytotoxic Effects of SGD and Its Constituents onHuman Adenomyosis-Derived Cells The inhibitory effectof SGD paeoniflorin and liquiritin on the growth ofhuman adenomyosis-derived cells was evaluated using MTTassay The effects on human adenomyosis-derived cells areshown in Figure 2 where the cells were treated with dif-ferent concentrations of SGD (01ndash500mgmL) paeoni-florin (00001ndash10mgmL) liquiritin (00001ndash10mgmL) andmifepristone (0005ndash100 120583M) for 24 h The results showedthat SGD paeoniflorin liquiritin and mifepristone inhibitedthe survival of human adenomyosis-derived cells in a dose-dependent manner The IC

50value of SGD paeoniflorin

liquiritin and mifepristone was 2506mgmL 103mgmL162mgmL and 1061 120583M respectively

34 SGD and Its Constituents Induce Apoptosis in HumanAdenomyosis-Derived Cells Simultaneous staining withannexin V-FITC and PI distinguished between healthy earlyapoptotic late apoptotic and necrotic cells [24] After 24 h oftreatmentwith orwithout different concentrations of SGD (10


0 10 20 30 40(min)

0

10

20

30

40

50

60

70

(mAU

)

O

O

OHOHHO

OHO

O

HO

OO

OHOHHO

O OH

Paeoniflorin

LiquiritinHOH

O

OHO

254nm 4nm (100)

(a)

0 10 20 30 40(min)

Paeoniflorin

0

10

20

30

40

50

60

70

80

(mAU

)

254nm 4nm (100)

(b)

0 10 20 30 40

Liquiritin

0102030405060708090

100110

(mAU

)

(min)

254nm 4nm (100)

(c)

Figure 1 UFLC pattern of SGD and single herb (a) SGD (b) Bai Shao (Paeonia lactiflora Pall Radix) and (c) Gan Cao (Glycyrrhiza uralensisFisch Radix et Rhizoma) each herb was extracted with water as described in the section of methods The dried powder of each herb wasextracted with methanol-water (10mL 50 50 vv) under ultrasonication for 30min The solutions were filtered and then determined forUFLC analysis

and 20mgmL) paeoniflorin (025 and 05mgmL) liquiritin(025 and 05mgmL) and mifepristone (10 120583M) humanadenomyosis-derived cells were analyzed by flow cytometryFigure 3 shows the percentages of apoptotic cells that were

undergoing early apoptosis and late apoptosis The apoptosisrate in human adenomyosis-derived cells was 68plusmn08whenthe cells were not treated with SGD paeoniflorin liquiritinand mifepristone (control group) However the percentage


100

80

60

40

20

00 100 200 300 400 500

Concentrations of SGD (mgmL)

Cel

l sur

viva

l (

)

(a)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 2 4 6 8 10

Concentrations of PA and LQ (mgmL)

PALQ

(b)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 20 40 60 80 100

Concentrations of MIF (120583M)

(c)

Figure 2 Cytotoxic effects of SGD and its constituents on human adenomyosis-derived cells After treatments with (a) SGD (b) paeoniflorin(PA) and liquiritin (LQ) and (c) mifepristone (MIF) the survival of human adenomyosis-derived cells was measured by the MTT assay Theresults displayed that SGD paeoniflorin liquiritin andmifepristone significantly inhibited the survival of human adenomyosis-derived cellsThe IC

50values were 2506mgmL 103mgmL 162mgmL and 1061 120583M respectively Representative graphs are shown

of apoptotic human adenomyosis-derived cells was increasedto 175 plusmn 36 (119875 lt 005) 235 plusmn 32 (119875 lt 001) 230 plusmn 26(119875 lt 001) 283 plusmn 26 (119875 lt 001) 107 plusmn 18 132 plusmn 22(119875 lt 005) and 185 plusmn 42 (119875 lt 005) in the presence ofSGD (10 and 20mgmL) paeoniflorin (025 and 05mgmL)liquiritin (025 and 05mgmL) and mifepristone (10 120583M)respectively These results demonstrated that SGD (10 and20mgmL) paeoniflorin (025 and 05mgmL) liquiritin(05mgmL) and mifepristone (10 120583M) could significantlyinduce the apoptosis of human adenomyosis-derived cells

35 Detection of PGE2and PGF

2120572in the Supernatants To

investigate the potential mechanism underlying the effec-tiveness of SGD in the treatment of adenomyosis-associated

dysmenorrhea the levels of two well-established pain media-tors namely PGE

2and PGF

2120572 were examined As shown in

Figure 4 SGD (10 and 20mgmL) and paeoniflorin (025 and05mgmL) dose-dependently and significantly decreasedthe production of PGE

2(6900 plusmn 253 pg106 cells 119875 lt 001

3717 plusmn 458 pg106 cells 119875 lt 001 45881 plusmn 4081 pg106 cells119875 lt 001 32216 plusmn 3841 pg106 cells 119875 lt 001) andPGF2120572

(96607 plusmn 4025 pg106 cells 119875 lt 001 85670 plusmn2694 pg106 cells 119875 lt 001 169993 plusmn 6497 pg106 cells 119875 lt001 137204 plusmn 6215 pg106 cells 119875 lt 001) while PGE

2and

PGF2120572

in control group were 159263 plusmn 5135 and 255358 plusmn2569 pg106 cells respectively These results suggested thatthe decrease in production of PGE

2and PGF

2120572might be

a potential mechanism in relieving adenomyosis-associateddysmenorrhea using SGD Meanwhile the activity of SGD


102 103101

100

100

102

103

101

PI-P

MT4

Log

Annexin V-FITC Log Annexin V-FITC Log Annexin V-FITC Log Annexin V-FITC Log

Annexin V-FITC LogAnnexin V-FITC LogAnnexin V-FITC LogAnnexin V-FITC Log

Control

102 103101

100

100

102

103

101

SGD 10

102 103101

100

100

102

103

101

SGD 20

102 103101

100

100

102

103

101

PA 025

100

102

103

101

102 103101100

LQ 025

100

102

103

101

PI-P

MT4

Log

102 103101100

PA 05

100

102

103

101

102 103101100

LQ 05

100

102

103

101

102 103101100

MIF 10

(a)

MIF (120583M)SGD (mgmL) PA (mgmL) LQ (mgmL)

Apop

tosis

inde

x (

)

35

30

25

20

15

10

5

0

Control 10 20 025 05 025 05 10

lowast

lowast

lowast

lowastlowast

lowastlowastlowastlowast

(b)

Figure 3 SGD and its constituents paeoniflorin (PA) and liquiritin (LQ) treatment results in apoptosis of human adenomyosis-derived cellsApoptotic cells were assessed by annexin V-FITC and PI staining as described in Section 2 Mifepristone (MIF) was used as positive controlThe data were expressed as meanplusmn SDThree independent experiments were performed lowast119875 lt 005 and lowastlowast119875 lt 001 indicated the groups withsignificant difference when compared to the control group

in decreasing PGE2and PGF

2120572production might be partly

ascribable to paeoniflorin

36 ER-120572 mRNA and OTR mRNA Expression in HumanAdenomyosis-Derived Cells Theeffects of SGD paeoniflorinand liquiritin on the expression of ER-120572 mRNA and OTRmRNAwere investigated by qRT-PCR As shown in Figure 5treatments with SGD (10 and 20mgmL) paeoniflorin (025and 05mgmL) and liquiritin (025 and 05mgmL) sig-nificantly decreased the mRNA levels of ER-120572 (0000390 plusmn

0000099 119875 lt 001 0000136 plusmn 0000051 119875 lt 0010006342plusmn0000343119875 lt 001 0001910plusmn0000704119875 lt 0010013642plusmn0003817119875 lt 001 0005028plusmn0000456119875 lt 001)in comparison to control group (0353722 plusmn 0060531) Inaddition SGD (10 and 20mgmL) paeoniflorin (025 and05mgmL) and liquiritin (025 and 05mgmL) obviouslydecreased the mRNA levels of OTR (00157 plusmn 00066 119875 lt005 00034 plusmn 00007 119875 lt 001 00141 plusmn 00079 119875 lt 00100033 plusmn 00007 119875 lt 001 00037 plusmn 00003 119875 lt 00100028 plusmn 00009 119875 lt 001) in a dose-dependent manner incomparison to control group (00693 plusmn 00164)

Evidence-Based Complementary and Alternative Medicine 7PG

E 2co

ncen

trat

ion

(pg106

cells

)

(mgmL)

1800

1600

1400

1200

1000

800

600

400

200

0

lowastlowast lowastlowast

lowastlowast

lowastlowast

SGD PA LQControl 10 20 025 05 025 05

(a)

PGF 2

120572co

ncen

trat

ion

(pg106

cells

)

3500

3000

2500

2000

1500

1000

500

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(b)

Figure 4 Effects of SGD and its constituents on the production of PGE2and PGF

2120572in human adenomyosis-derived cells Cells were treated

with SGD paeoniflorin (PA) and liquiritin (LQ) Panels (a) and (b) show results of PGE2and PGF

2120572 respectively Both PGE

2and PGF

2120572

production in the supernatant were significantly decreased by the treatment of SGD and PA Data represented the mean plusmn SD of threeindependent experiments Significant lower PGE

2and PGF

2120572compared to the control group (lowast119875 lt 005 and lowastlowast119875 lt 001)

Relat

ive e

xpre

ssio

n of

ER-120572

mRN

A

045

040

035

002

001

000

lowastlowast


lowastlowast


(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(a)

Relat

ive e

xpre

ssio

n of

OTR

mRN

A

010

008

006

004

002

000


lowastlowast


lowast

(mgmL)

SGD PA LQ

Control 10 20 025 05 025 05

(b)

Figure 5 Effects of SGD and its constituents on the expression of (a) ER-120572mRNA and (b) OTRmRNA in human adenomyosis-derived cellsThe mRNA levels in human adenomyosis-derived cells from control and SGD paeoniflorin (PA) and liquiritin (LQ) treatment groups weredetermined by qRT-PCR Data represented the mean plusmn SD of three independent experiments lowast119875 lt 005 and lowastlowast119875 lt 001 versus controls

4 Discussion

Traditional Chinese medicines have attracted interest andacceptance in many countries with the merits of few sideeffects affordability and local availability [25] Howeverthere are few of the traditional Chinese medicines that havebeen illustrated clearly how it works SGD a famous tradi-tional Chinese prescription is widely used in clinic to relievemany types of pain especially dysmenorrhea in womenwith endometriosis and adenomyosis [11 14] Therefore thepotential mechanism of the antiadenomyosis effects of thistraditional prescription is worth investigation In this studywe provide evidence that SGD and its active ingredients

paeoniflorin and liquiritin have a beneficial effect in thetreatment of adenomyosis

Cell proliferation has been shown to have multiple effectsin embryonic development and pattern formation includ-ing cell growth morphogenesis and gene expression [26]However abnormal cell proliferation may cause cancer andadenomyosis has been found to be associated with abnormalendometrial proliferation [27 28] The present study showedthat SGD had a significant inhibitory effect on the survivalof human adenomyosis-derived cells in a dose-dependentmanner Furthermore increased cellular proliferation hasbeen shown to contribute to the pathogenesis of abnormaluterine bleeding associated with adenomyosis [29] These


results showed that SGD could reduce proliferation of humanadenomyosis-derived cells which indicated the treatmentof SGD could prevent the formation of new adenomyoticlesions

Apoptosis a process of programmed cell death is essen-tial for embryonic development proper development andfunctioning of the immune system and the maintenanceof tissue homeostasis [30] Accumulated evidence suggeststhat apoptosis helps to maintain cellular homeostasis duringthe menstrual cycle by eliminating senescent cells from thefunctional layer of the uterine endometrium during the latesecretory and menstrual phase of the cycle [31] Howeverinappropriate apoptosis (either too little or too much) maycontribute to the pathology of many human diseases includ-ing neurodegenerative diseases ischemic damage autoim-mune disorders and cancer [32] It has been reported that adecrease in apoptosis led to ectopic survival and implantationof endometrial cells and development of adenomyosis [33]These studies indicate that apoptosis-inducing agents can bea potential therapeutic strategy for the treatment of adeno-myosis [34] In the present study quantification of apoptosisvia annexin V and PI staining was performed and the resultsshowed that SGD dose-dependently enhanced apoptosis inhuman adenomyosis-derived cells These results suggestedthat the prevention and treatment of adenomyosis usingSGD might be partially attributed to increased apoptosis inadenomyotic cell

Dysmenorrhea defined as painful menstruation involv-ing low abdominal pains is a major clinical symptom ofadenomyosis [35 36] Prostaglandins (PGs) especially PGE

2

and PGF2120572 are bioactive lipids derived from arachidonic acid

and play an important role in the etiology of dysmenorrhoea[37 38] In previous studies it has been reported that theconcentrations of PGE

2and PGF

2120572were higher in the men-

strual fluid of women with dysmenorrhoea than in womenwith painless periods [37] The increased production ofPGE2and PGF

2120572could upregulate cyclooxygenase-2 (COX-

2) which is an essential enzyme for the synthesis of PGE2

and PGF2120572 causing dysmenorrhea in adenomyosis [39 40]

Therefore suppression of PGE2and PGF

2120572synthesis has

become the main treatment of dysmenorrhoea [41] In thepresent study an assay on the levels of PGE

2and PGF

2120572was

performed Results showed that treatment with SGD dose-dependently and significantly decreased the production ofPGE2and PGF

2120572 A similar result has been reported by Imai

et al that incubation of human endometrial cells with SGDinduced a significant decrease in PG levels [14] In additionShibata et al also reported that concentrations of PGE

2

and PGF2120572

in the culture medium of human myometrialbiopsies were significantly decreased by the addition of SGD[42] The above findings suggested that SGD might alleviateadenomyosis-associated dysmenorrhea by decreasing PGE

2

and PGF2120572

productionAdenomyosis is most frequent in women of reproductive

age and regress after menopause which is well regardedas an estrogen-dependent condition [43] The estrogen andits classical estrogen receptors (ERs) ER-120572 and ER-120573 havebeen shown to be partly responsible for the cell proliferationapoptosis differentiation and development [44] ER-120572 is

the principal ER expressed in the endometrium and it isconsidered to be crucial in the development of endometrioidendometrial carcinoma [45] In addition expression of ERsis elevated in the adenomyotic uteri [46] and ER-120572 isassociated with the onset and growth of adenomyosis [47]Consequently an assay on the mRNA level of ER-120572 can helpus understand the mechanism underlying the effectivenessof SGD in the treatment of adenomyosis By qRT-PCRanalysis we found that SGD treatment remarkably inhibitedthe expression of ER-120572mRNA

Although the cause for OTR overexpression in adeno-myosis is currently unclear it has been shown that OTR isinvolved in adenomyosis-associated dysmenorrhea and maybe potential therapeutic target in treating the symptom andperhaps chronic pelvic pain in womenwith adenomyosis [4849] Meanwhile myometrial OTR expression is also found tobe associated with the severity of dysmenorrhea in womenwith adenomyosis [48] In the present study we found thattreatment of human adenomyosis-derived cells with SGDcould significantly reduce the expression of OTR mRNAleading possibly to pain alleviation This finding supportedthe traditional use of SGD in the treatment of adenomyosis-associated dysmenorrhea

In addition it is widely accepted that the combined actionof multiple constituents in traditional Chinese medicine isconsidered to be crucial for its therapeutic effects [50] Yinet al reported that total 58 compounds from SGD havebeen detected by UPLC-ESI-Q-TOF-MS and 50 compoundsamong them were identified or tentatively characterizedincluding monoterpene glycosides (albiflorin and paeoni-florin) flavonoids (liquiritin isoliquiritin and isoliquirit-igenin) and triterpene saponins (glycyrrhizic acid) [51]Meanwhile UPLC method has been described for the deter-mination of albiflorin and paeoniflorin in rat plasma [52] andit has also been used to simultaneously determine the concen-trations of paeoniflorin liquiritin and glycyrrhizic acid in thetransport samples [53] Moreover pharmacokinetic profilesof monoterpene glycosides (albiflorin oxypaeoniflorin andpaeoniflorin) flavonoids (liquiritin isoliquiritin liquiriti-genin isoliquiritigenin and ononin) and triterpene saponins(glycyrrhizin and glycyrrhetinic acid) in rat after oral admin-istration of SGD extracts by HPLC-MSMS have beenrevealed [54 55] In the present study UFLC profiles of twosingle herbs and SGD were performed for quality control ofSGD A simple and reliable UFLC method was developed forthe simultaneous determination of paeoniflorin and liquiritinin SGD Paeoniflorin is one representative monoterpeneglycoside in Paeoniae Radix Liquiritin is a kind of flavonoidsthat is extracted from Glycyrrhizae Radix Furthermore inorder to reveal the active constituents and their contributionto the effect of SGD we also examined the antiadenomyosiseffects of its 2 major ingredients paeoniflorin and liquiritinon human adenomyosis-derived cells Results showed thatboth paeoniflorin and liquiritin significantly reduced prolif-eration accelerated apoptosis and suppressed ER-120572 mRNAand OTR mRNA expression in human adenomyosis-derivedcells Paeoniflorin can also decrease production of PGE

2and

PGF2120572 These findings suggested that the antiadenomyosis

effects of SGD on human adenomyosis-derived cells would


be partially achieved by these 2 components In previousstudies it was reported that paeoniflorin possessed anal-gesic [56] anti-inflammatory [57] immunoregulatory [58]cognition-enhancing [59] neuromuscular-blocking [60 61]and antihyperglycemic [62] activities could relax vascularsmoothmuscle [63] and suppressed rat adjuvants arthritis byreducingCOX-2 expression [64] Liquiritin exhibited an anti-inflammatory effect in some models of inflammation [65]could induce apoptosis in stomach cancer cell [66] and is fre-quently used to treat injury or swelling for its life-enhancingproperties as well as detoxification in traditional Orientalmedicine [67] These evidences may partially support ourpresent findings Taken together our findings suggested thatpaeoniflorin and liquiritin might be bioactive componentsof SGD However further research is needed to evaluate thebiological and pharmacological effects of other constituentsof SGD

5 Conclusion

In conclusion our study showed that SGD and its major con-stituents (paeoniflorin and liquiritin) displayed a differentialability to inhibit proliferation and induce apoptosis of humanadenomyosis-derived cells SGD and paeoniflorin effectivelydecreased the production of PGE

2 PGF

2120572 Meanwhile

SGD paeoniflorin and liquiritin significantly suppressedthe expression of ER-120572 mRNA and OTR mRNA in humanadenomyosis-derived cells The results of the present studysuggest that SGDand itsmajor constituents (paeoniflorin andliquiritin) possess active antiadenomyosis activities

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

Yong-Ge Guan and Jin-Bin Liao contributed equally to thiswork

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (Project no 81173296 Project no 81202716and Project no 81473715) andTheSpecial Funds fromCentralFinance of China in Support of the Development of LocalColleges and University (Educational finance Grant no 276(2014))

References

[1] A Ferenczy ldquoPathophysiology of adenomyosisrdquoHuman Repro-duction Update vol 4 no 4 pp 312ndash322 1998

[2] R Azziz ldquoAdenomyosis current perspectivesrdquo Obstetrics andGynecology Clinics of North America vol 16 no 1 pp 221ndash2351989

[3] L Zhao S Zhou L Zou and X Zhao ldquoThe expression andfunctionality of stromal caveolin 1 in human adenomyosisrdquoHuman Reproduction vol 28 no 5 pp 1324ndash1338 2013

[4] X Mao Y Wang A V Carter X Zhen and S-W Guo ldquoTheretardation of myometrial infiltration reduction of uterinecontractility and alleviation of generalized hyperalgesia inmice with induced adenomyosis by levo-tetrahydropalmatine(l-THP) and andrographoliderdquo Reproductive Sciences vol 18no 10 pp 1025ndash1037 2011

[5] X Liu and S-W Guo ldquoA pilot study on the off-label use ofvalproic acid to treat adenomyosisrdquo Fertility and Sterility vol89 no 1 pp 246ndash250 2008

[6] C Bergeron F Amant andA Ferenczy ldquoPathology and physio-pathology of adenomyosisrdquo Best Practice and Research ClinicalObstetrics and Gynaecology vol 20 no 4 pp 511ndash521 2006

[7] D R Grow and R B Filer ldquoTreatment of adenomyosis withlong-term GnRH analogues a case reportrdquo Obstetrics amp Gyne-cology vol 78 no 3 part 2 pp 538ndash539 1991

[8] O Abulafia and D M Sherer ldquoTranscatheter uterine arteryembolization for the management of symptomatic uterineleiomyomasrdquo Obstetrical and Gynecological Survey vol 54 no12 pp 745ndash753 1999

[9] S C Goodwin B McLucas M Lee et al ldquoUterine artery em-bolization for the treatment of uterine leiomyomata midtermresultsrdquo Journal of Vascular and Interventional Radiology vol10 no 9 pp 1159ndash1165 1999

[10] X M Wang ldquoThe application of Shaoyao-Gancao Doction inthe treatment of gynecological disordersrdquo Journal of ModernMedicine Health vol 24 no 14 pp 2145ndash2146 2008

[11] T Tanaka ldquoA novel anti-dysmenorrhea therapy with cyclicadministration of two Japanese herbal medicinesrdquo Clinical andExperimental Obstetrics and Gynecology vol 30 no 2-3 pp 95ndash98 2003

[12] J Gao ldquoChinese medicine therapy for uterine fibroidsrdquo ForeignMedical Sciences (Obstet Gynecol Fascicle) vol 30 no 2 pp 128ndash129 2003

[13] Y G Guan Y W He K Y Li and Y Song ldquoEffects of Shaoyao-Gancao dectionon on the expresion of Ras Raf and Mek-2 in human adenomyosis-derived cellsrdquo Liaoning Journal ofTraditional Chinese Medicine vol 41 no 7 pp 1530ndash1532 2014

[14] A Imai S Horibe S Fuseya K Iida H Takagi and T TamayaldquoPossible evidence that the herbal medicine shakuyaku-kanzo-to decreases prostaglandin levels through suppressing arachi-donate turnover in endometriumrdquo Journal of Medicine vol 26no 3-4 pp 163ndash174 1995

[15] Y Sato T Akao J-X He et al ldquoGlycycoumarin from Gly-cyrrhizae Radix acts as a potent antispasmodic through inhi-bition of phosphodiesterase 3rdquo Journal of Ethnopharmacologyvol 105 no 3 pp 409ndash414 2006

[16] L Y Feng S Q Yan S Q Wu and L J Zhang ldquoExperimentalstudy on the analgesia mechanism of Paeornia and GlycyrrhizadecoctionrdquoChinese Journal of Experimental TraditionalMedicalFormulae vol 25 no 1 pp 23ndash25 2002

[17] A J Zhu B W Fang X Z Wu Z S Tian S D Guo and D HLi ldquoStudy on anti-inflammation effect of shaogan decoctionrdquoTianjin Medical Journal vol 37 no 2 pp 120ndash123 2009

[18] T Mori S Sakamoto M Matsuda et al ldquoSuppression of spon-taneous development of uterine adenomyosis and mammaryhyperplastic alveolar nodules by Chinese herbal medicines inmicerdquoThe American Journal of Chinese Medicine vol 21 no 3-4 pp 263ndash268 1993

[19] L Shen Y Feng D-S Xu X Lin and L Zhang ldquoPharmacoki-netic study on characteristic ingredients from Shaoyao beforeand after compatibilityrdquoChinese Pharmaceutical Journal vol 43no 23 pp 1774ndash1776 2008


[20] M Matsuda H Sasabe Y Adachi T Suzuki and T MorildquoIncreased invasion activity of endometrial stromal cells andelevated expression of matrix metalloproteinase messengerRNA in the uterine tissues of mice with experimentally inducedadenomyosisrdquoThe American Journal of Obstetrics and Gynecol-ogy vol 185 no 6 pp 1374ndash1380 2001

[21] T-C Chang E A Wentzel O A Kent et al ldquoTransactivationof miR-34a by p53 broadly influences gene expression andpromotes apoptosisrdquoMolecular Cell vol 26 no 5 pp 745ndash7522007

[22] P J Ferguson E Kurowska D J Freeman A F Chambersand D J Koropatnick ldquoA flavonoid fraction from cranberryextract inhibits proliferation of human tumor cell linesrdquo Journalof Nutrition vol 134 no 6 pp 1529ndash1535 2004

[23] H Suzuki-Kakisaka T Murakami T Hirano Y Terada N Yae-gashi and K Okamura ldquoEffects of photodynamic therapyusing 5-aminolevulinic acid on cultured human adenomyosis-derived cellsrdquo Fertility and Sterility vol 87 no 1 pp 33ndash38 2007

[24] Y-Y Li Z Zhang Z-H Wang H-W Wang L Zhang and LZhu ldquorBTI induces apoptosis in human solid tumor cell linesby loss in mitochondrial transmembrane potential and caspaseactivationrdquo Toxicology Letters vol 189 no 2 pp 166ndash175 2009

[25] X-Y Song Y-D Li Y-P Shi L Jin and J Chen ldquoQuality con-trol of traditional Chinese medicines a reviewrdquo Chinese Journalof Natural Medicines vol 11 no 6 pp 596ndash607 2013

[26] J Schmahl and B Capel ldquoCell proliferation is necessary forthe determination of male fate in the gonadrdquo DevelopmentalBiology vol 258 no 2 pp 264ndash276 2003

[27] G CooperThe Cell A Molecular Approach Sinauer AssociatesSunderland Mass USA 2nd edition 2000 httpwwwncbinlmnihgovbooksNBK9963

[28] Y-J Chen H-Y Li Y-L Chang et al ldquoSuppression ofmigratoryinvasive ability and induction of apoptosis inadenomyosis-derived mesenchymal stem cells by cyclooxygen-ase-2 inhibitorsrdquo Fertility and Sterility vol 94 no 6 pp 1972ndash1979 2010

[29] A M Propst B J Quade A R Gargiulo R A Nowak andE A Stewart ldquoAdenomyosis demonstrates increased expressionof the basic fibroblast growth factor receptorligand systemcompared with autologous endometriumrdquo Menopause vol 8no 5 pp 368ndash371 2001

[30] H Okada and T W Mak ldquoPathways of apoptotic and non-apoptotic death in tumour cellsrdquo Nature Reviews Cancer vol4 no 8 pp 592ndash603 2004

[31] K Kokawa T Shikone andRNakano ldquoApoptosis in the humanuterine endometrium during the menstrual cyclerdquo The Journalof Clinical Endocrinology and Metabolism vol 81 no 11 pp4144ndash4147 1996

[32] S Elmore ldquoApoptosis a review of programmed cell deathrdquoToxicologic Pathology vol 35 no 4 pp 495ndash516 2007

[33] J-H Yang M-Y Wu C-D Chen M-J Chen Y-S Yang andH-N Ho ldquoAltered apoptosis and proliferation in endometrialstromal cells of women with adenomyosisrdquo Human Reproduc-tion vol 22 no 4 pp 945ndash952 2007

[34] J-H Kim S-H Jung Y-I Yang et al ldquoArtemisia leaf extractinduces apoptosis in human endometriotic cells through regu-lation of the p38 and NF120581B pathwaysrdquo Journal of Ethnopharma-cology vol 145 no 3 pp 767ndash775 2013

[35] S-H Han M-H Hur J Buckle J Choi and M S Lee ldquoEffectof aromatherapy on symptoms of dysmenorrhea in college stu-dents a randomized placebo-controlled clinical trialrdquo Journal

of Alternative and Complementary Medicine vol 12 no 6 pp535ndash541 2006

[36] S Kissler S Zangos J Kohl et al ldquoDuration of dysmenorrhoeaand extent of adenomyosis visualised by magnetic resonanceimagingrdquo European Journal of Obstetrics Gynecology and Repro-ductive Biology vol 137 no 2 pp 204ndash209 2008

[37] K J Sales and H N Jabbour ldquoCyclooxygenase enzymes andprostaglandins in pathology of the endometriumrdquo Reproduc-tion vol 126 no 5 pp 559ndash567 2003

[38] H Koike H Egawa T Ohtsuka M Yamaguchi T Ikenoueand N Mori ldquoCorrelation between dysmenorrheic severityand prostaglandin production in women with endometriosisrdquoProstaglandins Leukotrienes and Essential Fatty Acids vol 46no 2 pp 133ndash137 1992

[39] K J Sales V Grant and H N Jabbour ldquoProstaglandin E2 andF2120572 activate the FP receptor and up-regulate cyclooxygenase-2 expression via the cyclic AMP response elementrdquo Molecularand Cellular Endocrinology vol 285 no 1-2 pp 51ndash61 2008

[40] J R Vane Y S Bakhle and R M Botting ldquoCyclooxygenases 1and 2rdquo Annual Review of Pharmacology and Toxicology vol 38pp 97ndash120 1998

[41] M A Lumsden RW Kelly and D T Baird ldquoPrimary dysmen-orrhoea the importance of both prostaglandins E

2and F

2120572rdquo

British Journal of Obstetrics amp Gynaecology vol 90 no 12 pp1135ndash1140 1983

[42] T Shibata T Morimoto A Suzuki H Saito and T Yanai-hara ldquoThe effect of Shakuyaku-kanzo-to on prostaglandinproduction in human uterine myometriumrdquo Acta Obstetrica etGynaecologica Japonica vol 48 no 5 pp 321ndash327 1996

[43] J Kitawaki ldquoAdenomyosis the pathophysiology of an oestro-gen-dependent diseaserdquo Best Practice and Research ClinicalObstetrics and Gynaecology vol 20 no 4 pp 493ndash502 2006

[44] E Trukhacheva Z Lin S Reierstad Y-H Cheng M Miladand S E Bulun ldquoEstrogen receptor (ER) 120573 regulates ER120572expression in stromal cells derived from ovarian endometrio-sisrdquo The Journal of Clinical Endocrinology and Metabolism vol94 no 2 pp 615ndash622 2009

[45] S Wedren L Lovmar K Humphreys et al ldquoEstrogen receptoralpha gene polymorphism and endometrial cancer riskmdasha case-control studyrdquo BMC Cancer vol 8 article 811 2008

[46] X Li X Liu and S-W Guo ldquoClinical profiles of 710 pre-menopausal women with adenomyosis who underwent hys-terectomyrdquo Journal of Obstetrics and Gynaecology Research vol40 no 2 pp 485ndash494 2014

[47] J Kitawaki HObayashi H Ishihara et al ldquoOestrogen receptor-alpha gene polymorphism is associated with endometriosisadenomyosis and leiomyomatardquo Human Reproduction vol 16no 1 pp 51ndash55 2001

[48] S-W Guo X Mao Q Ma and X Liu ldquoDysmenorrhea andits severity are associated with increased uterine contractilityand overexpression of oxytocin receptor (OTR) in women withsymptomatic adenomyosisrdquo Fertility and Sterility vol 99 no 1pp 231ndash240 2013

[49] J Nie X Liu and S-W Guo ldquoImmunoreactivity of oxytocinreceptor and transient receptor potential vanilloid type 1 andits correlation with dysmenorrhea in adenomyosisrdquo AmericanJournal of Obstetrics and Gynecology vol 202 no 4 pp 346e1ndash346e8 2010

[50] H Zhang S Chen F Qin X Huang P Ren and X GuldquoSimultaneous determination of 12 chemical constituents in thetraditional Chinese Medicinal Prescription Xiao-Yao-San-Jia-Wei byHPLC coupledwith photodiode array detectionrdquo Journal


of Pharmaceutical and Biomedical Analysis vol 48 no 5 pp1462ndash1466 2008

[51] Q Yin PWang A Zhang H Sun XWu and XWang ldquoUltra-performance LC-ESIquadrupole-TOFMS for rapid analysis ofchemical constituents of Shaoyao-Gancao decoctionrdquo Journal ofSeparation Science vol 36 no 7 pp 1238ndash1246 2013

[52] P Gan M Zhong X Huang et al ldquoPharmacokinetic compar-isons of albiflorin and paeoniflorin after oral administrationof Shaoyao-Gancao-Tang and single herb Paeony decoction toratsrdquo Planta Medica vol 78 no 3 pp 237ndash243 2012

[53] Y Chen J Wang L Wang L Chen and Q Wu ldquoAbsorptionand interaction of the main constituents from the traditionalchinese drug pair shaoyao-gancao via a caco-2 cell monolayermodelrdquoMolecules vol 17 no 12 pp 14908ndash14917 2012

[54] Y Wang C Xu P Wang et al ldquoPharmacokinetic comparisonsof different combinations of Shaoyao-Gancao-Decoction inrats simultaneous determination of ten active constituents byHPLC-MSMSrdquo Journal of Chromatography vol 932 pp 76ndash872013

[55] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[56] E Sugishita S Amagaya and Y Oghihara ldquoStudies on the com-bination of glycyrrhizae radix in shakuyakukanzo-tordquo Journal ofPharmacobio-Dynamics vol 7 no 7 pp 427ndash435 1984

[57] J Yamahara T Yamada H Kimura T Sawada and HFujimura ldquoBiologically active principles of crude drugs IIAnti-allergic principles in ldquoShoseiryu-Tordquo anti-inflammatoryproperties of paeoniflorin and its derivativesrdquo Journal ofPharmacobio-Dynamics vol 5 no 11 pp 921ndash929 1982

[58] J Liang A Zhou M Chen and S Xu ldquoNegatively regulatoryeffects of paeoniflorin on immune cellsrdquo European Journal ofPharmacology vol 183 no 3 pp 901ndash902 1990

[59] HOhta J-WNi KMatsumotoHWatanabe andM ShimizuldquoPeony and its major constituent paeoniflorin improve radialmaze performance impaired by scopolamine in ratsrdquo Pharma-cology Biochemistry and Behavior vol 45 no 3 pp 719ndash7231993

[60] M Kimura I Kimura K Takahashi et al ldquoBlocking effects ofblended paeoniflorin or its related compounds with gly-cyrrhizin on neuromuscular junctions in frog and mouserdquoJapanese Journal of Pharmacology vol 36 no 3 pp 275ndash2821984

[61] M Kimura I Kimura M Muroi T Nakamura and S ShibataldquoDepolarizing effects of glycyrrhizin-derivatives relating to theblend effects with paeoniflorin in mouse diaphragm musclerdquoJapanese Journal of Pharmacology vol 41 no 2 pp 263ndash2651986

[62] F-L Hsu C-W Lai and J-T Cheng ldquoAntihyperglycemic effectsof paeoniflorin and 8-debenzoylpaeoniflorin glucosides fromthe root of Paeonia lactiflorardquo Planta Medica vol 63 no 4 pp323ndash325 1997

[63] S N Jin J F Wen T T Wang D G Kang H S Lee and K WCho ldquoVasodilatory effects of ethanol extract of Radix PaeoniaeRubra and its mechanism of action in the rat aortardquo Journal ofEthnopharmacology vol 142 no 1 pp 188ndash193 2012

[64] Y-Q Zheng W Wei L Zhu and J-X Liu ldquoEffects and mech-anisms of Paeoniflorin a bioactive glucoside from paeony rooton adjuvant arthritis in ratsrdquo Inflammation Research vol 56 no5 pp 182ndash188 2007

[65] C S Seo J-A Lee D Jung et al ldquoSimultaneous determinationof liquiritin hesperidin and glycyrrhizin byHPLC-photodiodearray detection and the anti-inflammatory effect of Pyungwi-sanrdquoArchives of Pharmacal Research vol 34 no 2 pp 203ndash2102011

[66] H Hibasami H Iwase K Yoshioka and H Takahashi ldquoGly-cyrrhizin induces apoptosis in human stomach cancer KATOIII and human promyelotic leukemiaHL-60 cellsrdquo InternationalJournal of Molecular Medicine vol 16 no 2 pp 233ndash236 2005

[67] Z-A Chen J-L Wang R-T Liu et al ldquoLiquiritin potentiateneurite outgrowth induced by nerve growth factor in PC12cellsrdquo Cytotechnology vol 60 no 1ndash3 pp 125ndash132 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


prescription was sourced from the Chinese Medical ClassicstextmdashShanghan lun in 210 CEThe herbal prescription whichis made up of two herbs (Paeoniae Radix and GlycyrrhizaeRadix ldquoShaoyaordquo and ldquoGancaordquo in Chinese resp) is com-monly used in the treatment of gynecological disorders inChina including dysmenorrheal [10 11] menorrhagia [12]and infertility [10] Clinically SGD has been used veryefficaciously and widely to treat adenomyosis in women intraditional Chinese medical practice since it has the advan-tage of noninvasive and lessno side effects [13 14] Similarherbal prescription has also been used in Japan and otherOriental countries [15] In additionmodern pharmacologicalstudies have demonstrated that SGD possesses analgesic [16]and anti-inflammatory [17] effects Moreover SGD treatmentresulted in a significantly low incidence of adenomyosis in anexperimental animal model [18]

However little is known about the potential mechanismsof the antiadenomyosis effect of SGD In this study we ex-amined the effects of SGD and itsmajor constituents (paeoni-florin and liquiritin) on the proliferation and apoptosis ofhuman adenomyosis-derived cells In addition their effectson prostaglandin E

2(PGE2) and prostaglandin F

2120572(PGF2120572)

production and estrogen receptor-120572 (ER-120572) mRNA andoxytocin receptor (OTR) mRNA expression were also inves-tigated

2 Materials and Methods

21 Materials Acetonitrile (Merk Darmstadt Germany)Paeoniflorin and liquiritin were obtained fromThe NationalInstitute for the Control of Pharmaceutical and BiologicalProducts (Beijing China) 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide (MTT) Apoptosis DetectionKit (Catalog no V13241) sodium dodecyl (lauryl) sulphate-hydrochloride (SDS-HCl) and Trizol were purchased fromInvitrogen Dulbeccorsquos modified Eaglersquos medium (DMEM)Collagenase I 025 trypsin fetal bovine serum (FBS) andphosphate buffered saline (PBS) were obtained from GibcoM-MLV RTase cDNA Synthesis Kit and SYBR Premix Ex Taqwere obtained from Takara (Dalian China) PGE

2enzyme-

linked immunosorbent assay (ELISA) kit was obtained fromRampD Systems (Minneapolis USA) PGF

2120572ELISA kit was

purchased from ENZO Life Sciences

22 Single Herb and SGD Preparation Paeoniae Radix andGlycyrrhizae Radix were purchased from Caizhilin MaterialsCompany of Guangzhou and authenticated by professor Zi-Ren Su in the School of Pharmacy of Guangzhou Universityof Chinese Medicine

According to the preparation method of SGD by Shenet al with slight modifications [19] extract solutions of twosingle herbs and SGD were prepared in the following proce-dure Two single herbs and SGD were macerated for 30minwith eight times (vw) distilled water The medical solutionswere heated to boiling and then decocted for 40min and thefiltrates were collected The residues were decocted again for30min with eight times (vw) distilled water then collected

filtrates again and were mixed with previous collected fil-trates condensed under reduced pressure at 80∘C to obtain aconcentration equivalent to 1 gmL crude herb materials andstored at minus20∘C

23 Ultra-Fast Liquid Chromatography (UFLC) AnalysisUltra-fast liquid chromatography (UFLC) profiles of twosingle herbs and SGD were performed as the followingprocedures extract solutions of Paeoniae Radix (02mL)Glycyrrhizae Radix (008mL) and SGD (028mL) with aconcentration equivalent to 1 gmL crude herbmaterials weredried and then extractedwithmethanol-water (10mL 50 50vv) under ultrasonication for 30min The solutions werefiltered and then determined for UFLC analysis

UFLC equipment was controlled with LC-20AD pump(Shimadzu Co Kyoto Japan) using a YMC-UltraHT ProC18

column (50mm times 20mm ID 2 120583m YMC Inc) Theelution gradient was carried out with binary solvent systemconsisting of 05 acetic acid in H

2O (solvent A) and MeCN

(solvent B) A linear gradient profile with the followingproportions (vv) of solvent B was applied gradient profile0 to 10min and 5 of B 10 to 20min and 5 to 15 of B 20to 30min and 15 to 30 of B 30 to 40min and 30 to 50of B and 40 to 45min and 50 to 70 of BThe flow rate wascontrolled with LC 20AD at 04mLmin injection volume5 120583L and the column temperature was maintained at 28∘CThe effluents from the column were detected at absorbenciesranging from 200 to 400 nm using a photodiode-array detec-torThe three-dimensional data collection and processing forpeak analysis were accomplished with CLASS-LC10 SystemAnalysis Software (Shimadzu Co Kyoto Japan)

24 Tissue Preparation Adenomyosis specimens were ob-tained at the time of laparoscopy or laparotomy performedfor adenomyosis treatment from eight Chinese women aged20ndash45 years All patients had regular menstrual cycles Nopatients were undergoing hormone therapy prior to surgeryAll biopsy specimens were collected at the First AffiliatedHospital of Guangzhou University of Traditional ChineseMedicine between November 2009 and August 2010 withthe permission of the local ethics committee and writteninformed consent was obtained from each patient

25 Cell Isolation and Culture The biopsy specimens wereimmediately placed in ice-cold DMEM for transport to thelaboratory The tissues were then dissociated as previouslydescribed with some modifications [20] The biopsy speci-mens were washed once with PBS and twice with DMEMThe biopsy specimens were then minced into small piecesand incubated at 37∘C for 5 to 6 hours in Hankrsquos balanced saltsolution supplemented with 01 type I collagenase DMEMcontaining 10 FBS was added The cell suspensions werefiltered through a 200mesh stainless steel screen and the fil-trates were centrifuged (1000 rpm 8min 3 times) Cells weresuspended at a concentration of 5 times 105 cellmL in DMEMsupplemented with 10FBS then transferred to a tissueculture dish and incubated at 37∘C In order to accumulateenough cells for further study cultured cells of individual













3 Results








0 10 20 30 40(min)

0

10

20

30

40

50

60

70

(mAU

)

O

O

OHOHHO

OHO

O

HO

OO

OHOHHO

O OH

Paeoniflorin

LiquiritinHOH

O

OHO

254nm 4nm (100)

(a)

0 10 20 30 40(min)

Paeoniflorin

0

10

20

30

40

50

60

70

80

(mAU

)

254nm 4nm (100)

(b)

0 10 20 30 40

Liquiritin

0102030405060708090

100110

(mAU

)

(min)

254nm 4nm (100)

(c)





100

80

60

40

20

00 100 200 300 400 500


Cel

l sur

viva

l (

)

(a)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 2 4 6 8 10


PALQ

(b)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 20 40 60 80 100


(c)








2and PGF






2and

PGF2120572


2and PGF

2120572might be



102 103101

100

100

102

103

101

PI-P

MT4

Log



Control

102 103101

100

100

102

103

101

SGD 10

102 103101

100

100

102

103

101

SGD 20

102 103101

100

100

102

103

101

PA 025

100

102

103

101

102 103101100

LQ 025

100

102

103

101

PI-P

MT4

Log

102 103101100

PA 05

100

102

103

101

102 103101100

LQ 05

100

102

103

101

102 103101100

MIF 10

(a)


Apop

tosis

inde

x (

)

35

30

25

20

15

10

5

0

Control 10 20 025 05 025 05 10

lowast

lowast

lowast

lowastlowast


(b)








E 2co

ncen

trat

ion

(pg106

cells

)

(mgmL)

1800

1600

1400

1200

1000

800

600

400

200

0


lowastlowast

lowastlowast

SGD PA LQControl 10 20 025 05 025 05

(a)

PGF 2

120572co

ncen

trat

ion

(pg106

cells

)

3500

3000

2500

2000

1500

1000

500

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(b)





2and PGF

2120572


2and PGF


Relat

ive e

xpre

ssio

n of

ER-120572

mRN

A

045

040

035

002

001

000

lowastlowast


lowastlowast


(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(a)

Relat

ive e

xpre

ssio

n of

OTR

mRN

A

010

008

006

004

002

000


lowastlowast


lowast

(mgmL)

SGD PA LQ

Control 10 20 025 05 025 05

(b)


4 Discussion








2



2and PGF









2and PGF

2120572was




2

and PGF2120572


2

and PGF2120572






2and





5 Conclusion


2 PGF

2120572 Meanwhile






Acknowledgments


References












































2and F

2120572rdquo



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























3 Results








0 10 20 30 40(min)

0

10

20

30

40

50

60

70

(mAU

)

O

O

OHOHHO

OHO

O

HO

OO

OHOHHO

O OH

Paeoniflorin

LiquiritinHOH

O

OHO

254nm 4nm (100)

(a)

0 10 20 30 40(min)

Paeoniflorin

0

10

20

30

40

50

60

70

80

(mAU

)

254nm 4nm (100)

(b)

0 10 20 30 40

Liquiritin

0102030405060708090

100110

(mAU

)

(min)

254nm 4nm (100)

(c)





100

80

60

40

20

00 100 200 300 400 500


Cel

l sur

viva

l (

)

(a)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 2 4 6 8 10


PALQ

(b)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 20 40 60 80 100


(c)








2and PGF






2and

PGF2120572


2and PGF

2120572might be



102 103101

100

100

102

103

101

PI-P

MT4

Log



Control

102 103101

100

100

102

103

101

SGD 10

102 103101

100

100

102

103

101

SGD 20

102 103101

100

100

102

103

101

PA 025

100

102

103

101

102 103101100

LQ 025

100

102

103

101

PI-P

MT4

Log

102 103101100

PA 05

100

102

103

101

102 103101100

LQ 05

100

102

103

101

102 103101100

MIF 10

(a)


Apop

tosis

inde

x (

)

35

30

25

20

15

10

5

0

Control 10 20 025 05 025 05 10

lowast

lowast

lowast

lowastlowast


(b)








E 2co

ncen

trat

ion

(pg106

cells

)

(mgmL)

1800

1600

1400

1200

1000

800

600

400

200

0


lowastlowast

lowastlowast

SGD PA LQControl 10 20 025 05 025 05

(a)

PGF 2

120572co

ncen

trat

ion

(pg106

cells

)

3500

3000

2500

2000

1500

1000

500

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(b)





2and PGF

2120572


2and PGF


Relat

ive e

xpre

ssio

n of

ER-120572

mRN

A

045

040

035

002

001

000

lowastlowast


lowastlowast


(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(a)

Relat

ive e

xpre

ssio

n of

OTR

mRN

A

010

008

006

004

002

000


lowastlowast


lowast

(mgmL)

SGD PA LQ

Control 10 20 025 05 025 05

(b)


4 Discussion








2



2and PGF









2and PGF

2120572was




2

and PGF2120572


2

and PGF2120572






2and





5 Conclusion


2 PGF

2120572 Meanwhile






Acknowledgments


References












































2and F

2120572rdquo



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 10 20 30 40(min)

0

10

20

30

40

50

60

70

(mAU

)

O

O

OHOHHO

OHO

O

HO

OO

OHOHHO

O OH

Paeoniflorin

LiquiritinHOH

O

OHO

254nm 4nm (100)

(a)

0 10 20 30 40(min)

Paeoniflorin

0

10

20

30

40

50

60

70

80

(mAU

)

254nm 4nm (100)

(b)

0 10 20 30 40

Liquiritin

0102030405060708090

100110

(mAU

)

(min)

254nm 4nm (100)

(c)





100

80

60

40

20

00 100 200 300 400 500


Cel

l sur

viva

l (

)

(a)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 2 4 6 8 10


PALQ

(b)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 20 40 60 80 100


(c)








2and PGF






2and

PGF2120572


2and PGF

2120572might be



102 103101

100

100

102

103

101

PI-P

MT4

Log



Control

102 103101

100

100

102

103

101

SGD 10

102 103101

100

100

102

103

101

SGD 20

102 103101

100

100

102

103

101

PA 025

100

102

103

101

102 103101100

LQ 025

100

102

103

101

PI-P

MT4

Log

102 103101100

PA 05

100

102

103

101

102 103101100

LQ 05

100

102

103

101

102 103101100

MIF 10

(a)


Apop

tosis

inde

x (

)

35

30

25

20

15

10

5

0

Control 10 20 025 05 025 05 10

lowast

lowast

lowast

lowastlowast


(b)








E 2co

ncen

trat

ion

(pg106

cells

)

(mgmL)

1800

1600

1400

1200

1000

800

600

400

200

0


lowastlowast

lowastlowast

SGD PA LQControl 10 20 025 05 025 05

(a)

PGF 2

120572co

ncen

trat

ion

(pg106

cells

)

3500

3000

2500

2000

1500

1000

500

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(b)





2and PGF

2120572


2and PGF


Relat

ive e

xpre

ssio

n of

ER-120572

mRN

A

045

040

035

002

001

000

lowastlowast


lowastlowast


(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(a)

Relat

ive e

xpre

ssio

n of

OTR

mRN

A

010

008

006

004

002

000


lowastlowast


lowast

(mgmL)

SGD PA LQ

Control 10 20 025 05 025 05

(b)


4 Discussion








2



2and PGF









2and PGF

2120572was




2

and PGF2120572


2

and PGF2120572






2and





5 Conclusion


2 PGF

2120572 Meanwhile






Acknowledgments


References












































2and F

2120572rdquo



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

80

60

40

20

00 100 200 300 400 500


Cel

l sur

viva

l (

)

(a)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 2 4 6 8 10


PALQ

(b)

100

80

60

40

20

0

Cel

l sur

viva

l (

)

0 20 40 60 80 100


(c)








2and PGF






2and

PGF2120572


2and PGF

2120572might be



102 103101

100

100

102

103

101

PI-P

MT4

Log



Control

102 103101

100

100

102

103

101

SGD 10

102 103101

100

100

102

103

101

SGD 20

102 103101

100

100

102

103

101

PA 025

100

102

103

101

102 103101100

LQ 025

100

102

103

101

PI-P

MT4

Log

102 103101100

PA 05

100

102

103

101

102 103101100

LQ 05

100

102

103

101

102 103101100

MIF 10

(a)


Apop

tosis

inde

x (

)

35

30

25

20

15

10

5

0

Control 10 20 025 05 025 05 10

lowast

lowast

lowast

lowastlowast


(b)








E 2co

ncen

trat

ion

(pg106

cells

)

(mgmL)

1800

1600

1400

1200

1000

800

600

400

200

0


lowastlowast

lowastlowast

SGD PA LQControl 10 20 025 05 025 05

(a)

PGF 2

120572co

ncen

trat

ion

(pg106

cells

)

3500

3000

2500

2000

1500

1000

500

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(b)





2and PGF

2120572


2and PGF


Relat

ive e

xpre

ssio

n of

ER-120572

mRN

A

045

040

035

002

001

000

lowastlowast


lowastlowast


(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(a)

Relat

ive e

xpre

ssio

n of

OTR

mRN

A

010

008

006

004

002

000


lowastlowast


lowast

(mgmL)

SGD PA LQ

Control 10 20 025 05 025 05

(b)


4 Discussion








2



2and PGF









2and PGF

2120572was




2

and PGF2120572


2

and PGF2120572






2and





5 Conclusion


2 PGF

2120572 Meanwhile






Acknowledgments


References












































2and F

2120572rdquo



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















102 103101

100

100

102

103

101

PI-P

MT4

Log



Control

102 103101

100

100

102

103

101

SGD 10

102 103101

100

100

102

103

101

SGD 20

102 103101

100

100

102

103

101

PA 025

100

102

103

101

102 103101100

LQ 025

100

102

103

101

PI-P

MT4

Log

102 103101100

PA 05

100

102

103

101

102 103101100

LQ 05

100

102

103

101

102 103101100

MIF 10

(a)


Apop

tosis

inde

x (

)

35

30

25

20

15

10

5

0

Control 10 20 025 05 025 05 10

lowast

lowast

lowast

lowastlowast


(b)








E 2co

ncen

trat

ion

(pg106

cells

)

(mgmL)

1800

1600

1400

1200

1000

800

600

400

200

0


lowastlowast

lowastlowast

SGD PA LQControl 10 20 025 05 025 05

(a)

PGF 2

120572co

ncen

trat

ion

(pg106

cells

)

3500

3000

2500

2000

1500

1000

500

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(b)





2and PGF

2120572


2and PGF


Relat

ive e

xpre

ssio

n of

ER-120572

mRN

A

045

040

035

002

001

000

lowastlowast


lowastlowast


(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(a)

Relat

ive e

xpre

ssio

n of

OTR

mRN

A

010

008

006

004

002

000


lowastlowast


lowast

(mgmL)

SGD PA LQ

Control 10 20 025 05 025 05

(b)


4 Discussion








2



2and PGF









2and PGF

2120572was




2

and PGF2120572


2

and PGF2120572






2and





5 Conclusion


2 PGF

2120572 Meanwhile






Acknowledgments


References












































2and F

2120572rdquo



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















E 2co

ncen

trat

ion

(pg106

cells

)

(mgmL)

1800

1600

1400

1200

1000

800

600

400

200

0


lowastlowast

lowastlowast

SGD PA LQControl 10 20 025 05 025 05

(a)

PGF 2

120572co

ncen

trat

ion

(pg106

cells

)

3500

3000

2500

2000

1500

1000

500

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(b)





2and PGF

2120572


2and PGF


Relat

ive e

xpre

ssio

n of

ER-120572

mRN

A

045

040

035

002

001

000

lowastlowast


lowastlowast


(mgmL)SGD PA LQ

Control 10 20 025 05 025 05

(a)

Relat

ive e

xpre

ssio

n of

OTR

mRN

A

010

008

006

004

002

000


lowastlowast


lowast

(mgmL)

SGD PA LQ

Control 10 20 025 05 025 05

(b)


4 Discussion








2



2and PGF









2and PGF

2120572was




2

and PGF2120572


2

and PGF2120572






2and





5 Conclusion


2 PGF

2120572 Meanwhile






Acknowledgments


References












































2and F

2120572rdquo



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2



2and PGF









2and PGF

2120572was




2

and PGF2120572


2

and PGF2120572






2and





5 Conclusion


2 PGF

2120572 Meanwhile






Acknowledgments


References












































2and F

2120572rdquo



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion


2 PGF

2120572 Meanwhile






Acknowledgments


References












































2and F

2120572rdquo



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































2and F

2120572rdquo



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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