Research Article Protection Effect of Zhen-Wu-Tang on ...Research Article Protection Effect of Zhen-Wu-Tang on Adriamycin-Induced Nephrotic Syndrome via Inhibiting Oxidative Lesions

Research ArticleProtection Effect of Zhen-Wu-Tang onAdriamycin-Induced Nephrotic Syndrome via InhibitingOxidative Lesions and Inflammation Damage

Chun-ling Liang Jun-biao Wu Jie-mei Lai Shu-fang Ye Jin Lin Hui OuyangJanis Ya-xian Zhan and Jiu-yao Zhou

School of Chinese Materia Medica Guangzhou University of Chinese Medicine Guangzhou Guangdong 510006 China

Correspondence should be addressed to Janis Ya-xian Zhan zyxgzucmeducn and Jiu-yao Zhou zhoujiuyaotomcom

Received 5 January 2014 Revised 23 February 2014 Accepted 24 February 2014 Published 9 April 2014

Academic Editor Karl Wah-Keung Tsim

Copyright copy 2014 Chun-ling Liang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Zhen-wu-tang (ZWT) a well-known formula in China is widely used to treat chronic kidney diseases However very littleinformation on ZWTrsquos mechanism of action is currently available In this study we investigated the possible protective roleand underlying mechanism of ZWT on nephrotic syndrome (NS) induced by Adriamycin (intravenous injection 60mgkg)in rats using biochemical and histopathological approaches ZWT decreased urine protein excretion and the serum levels oftotal cholesterol triglycerides blood urea nitrogen and creatinine significantly in diseased rats A decrease in plasma levels oftotal protein and albumin was also recorded in nephropathic rats Pathological results show an improved pathological state andrecovering glomerular structure in ZWT treatment groups ZWT decreased renal IL-8 level but increased renal IL-4 level Inaddition rats subjected to ZWT exhibited less IgG deposition in glomerulus compared with model group RT-PCR results showedthat ZWT decreased the mRNA expression of NF-120581B p65 and increased the mRNA expression of I120581B Furthermore ZWT reducedthe level of MDA and increased SOD activity These results demonstrated that ZWT ameliorated Adriamycin-induced NS inrats possibly by inhibiting Adriamycin-induced inflammation damage enhancing bodyrsquos antioxidant capacity thereby protectingglomerulus from injury

1 Introduction

Nephrotic syndrome (NS) is the triad of proteinuria lowserumalbumin and edemaThedisease is seenmostly in boyswith predominance in children from the Asian subcontinent[1] The global pandemic of NS is progressing at an alarmingrate and severely diminishes the quality of life for millions[2] Despite the changing face of NS treatment options andresources remain woeful The first line of treatment uses oldbut important approaches to reduce kidney tissue damageby trying to control the underlying conditions of NS Forexample patients with hypercholesterolemia secondary toNS always treated with lip-lowering agents and with edemasecondary to salt and water retention can be controlledwith the judicious use of diuretics Venous complicationsassociated with NS can be treated with anticoagulants [3] Of

all the therapeutics hormone therapy is the most commonlyused but it has many side effects and is easy to recrudesceIf the treatment fails the patient begins to experience kidneyfailure Traditional Chinese prescription has been commonlyrecognized as safe and effective in the treatment of chronickidney disorders in China and Japan with its various effectsmade by a variety of chemical components multilinked andmultitargeted in the body

NS has been associated with a wide variety of abnor-malities in the immune response especially in cell mediatedimmunity which supposed that soluble factors secreted byT cells may form the critical upstream signal for initiatingglomerular changes in NS [4] The occurrence of NS pro-teinuria and renal pathologic changes are associated withthe change in Th1Th2 in favor of Th2 lymphocytes [5]The reiterative active renal inflammation is associated with

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 131604 11 pageshttpdxdoiorg1011552014131604

2 Evidence-Based Complementary and Alternative Medicine

the development of proteinuria renal dysfunction glomeru-lar cell proliferation and extracellular matrix componentsexpansion and prolonged renal fibrosis [6 7] Thereforeinhibiting the activation of renal inflammation could bethe therapeutic target of protecting renal lesion in NS NF-120581B a ubiquitous transcription factor governs the expressionof genes encoding for cytokines chemokines growth fac-tors adhesion molecules and other factors involved in theimmune and inflammatory response A series of correlationsbetween the activation of NF-120581B and onset of both immune-and nonimmune-mediated glomerular injury implicate dys-regulation of the NF-120581B pathway in its pathogenesis [8ndash10] Therefore we postulated possible involvement of NF-120581Bin the pathogenesis of NS Antioxidant therapy is anotherimportant strategy for NS treatment and is considered tobe an important pathogenic mechanism Reactive oxygenspecies (ROS) are continuously produced physiologically andplay an important role in the expression of cell functions suchas transmission of impulse information However excessiveproduction of ROS would act as mediators of adverse eventssuch as inflammation necrosis and apoptosis In the kidneyROS are important causes of acute and subacute renalfailure in most cases Also in certain animal models ofNS superoxide anions and hydroxyl radicals are the maincauses of nephrotoxicity [11] It has also been reported thatrenal failure that results from increased ROS and decreasedantioxidant enzyme can be prevented by antioxidant therapy[12 13]

Zhen-wu-tang (ZWT) is a classic prescription to treatchronic kidney disease (CKD) with the paths of Wen yangand LiWater which is recorded in Treatise on Febrile Diseases[14] As we know edema is the most common presenting fea-ture in CKD It becomes detectable when the fluid retentionexceeds 3 of theweight of the body [15] ZWThas been usedas a remedy for various kidney diseases with the efficacy torelieve relative symptoms manifested by edema dysuria andoliguria in clinics in China and Japan [16] Furthermore thedecrease in levels of collagen IV fibronectin and laminin inthe extracellularmatrix of the fetal glomerularmesangial cellshas been observed after treatment by ZWT [17] Howeverthe effect of ZWT on Adriamycin- (ADR-) induced NS hasnot been well explored In this study we will investigate theimmune regulation and antioxidation effects of ZWT on ratswith NS induced by ADR The aims of the present study areto investigate the protective effects of ZWT on ADR-inducedNS and to discuss possible underlying mechanism of actionin rat model

2 Methods and Materials

21 Drugs and Drug Treatment

211 Plants Material and Drugs ZWT is composed of fiveherbal medicines Aconiti Lateralis Radix Praeparata (thelateral radix of Aconitum carmichaelii Debx) ZingiberisRhizoma Recens (rhizome of Zingiber officinale Rosc)Atractylodis Macrocephalae Rhizoma (radix of AtractylodesmacrocephalaKoidz) Paeoniae Radix Alba (radix of Paeonia

lactiflora Pall) and Poria (sclerotium of Poria cocos (Schw)Wolf) The dried raw materials of the herbs were purchasedfrom the Zhixin Medicinal Materials Company GuangzhouChina (Lot YPA110001 120301 120301 and 120401 resp) Allherbal medicines were authenticated by Professor Qiu-ZhenZhang at Guangdong Chinese Medicine Museum To assurethe quality control the materials were validated according tothe Chinese Pharmacopeia Dexamethasone was purchasedfrom the Second Affiliated Hospital Guangzhou Universityof Chinese Medicine Guangzhou China (Lot 111001)

212 Preparation of ZWT Amounts of Aconiti LateralisRadix Praeparata Poria Atractylodis Macrocephalae Rhi-zoma Paeoniae Radix Alba and Zingiberis Rhizoma Recenswere mixed according to a ratio of 3 3 2 3 3 All materialswere soaked with eight times distilled water for one hour andthen boiled with distilled water for 2 h For second extractionthe residue from the first extractionwas filtered and the sameextracting condition was applied After being repeated forthree times the mixture of the filtrates was concentrated tothe concentration of 24 g raw materials per milliliter Theextract was kept at 4∘C and dissolved in distilled water beforeuse

22 Chemicals and Reagents ADR was purchased fromShenzhen Main Luck Pharmaceuticals Inc (Lot 0901E)Urinalysis Reagent Strip fromAcon Biotech (Hangzhou) CoLtd (Lot 201109144) Coomassie (Bradford) Protein AssayKits from Nanjing Jiancheng Bioengineering Institute (Lot20120413 and 20120604) Interleukin-4 and Interleukin-8ELISAkits for rats fromAbcam (HK) Ltd (Lot ISC10330EIA-2121R and -2135-R resp) Rabbit Anti-Rat IgGFITC fromBeijing Biosynthesis Biotechnology Co Ltd (Lot 990603)SOD and MDA Assay Kits from Nanjing Jiancheng Bio-engineering Institute (Lot 20120523 and 20120324) Trizol(9108 Lot number AK8106) RT Reagent Kit (RR047A LotAK2003) and SYBR Premix ExTaq II (RR820A Lot numberAK4401) were from Takara Dalian China the primers forNF-120581B p65 I120581B and GAPDH were synthesized by SangonBiotech (Shanghai) Co Ltd

23 Animals and Treatments Fifty adult male specialpathogen free Sprague Dawley rats (Guangzhou Universityof Chinese Medicine Research Center for ExperimentalAnimal) which weighed (200 plusmn 20) g with urine proteinqualitative tests showing negative results (Urinalysis ReagentStrip) (Certificate number SCXK (Guangdong) 2008-0020)were used in the experiments The rats were housed in anair-conditioned room at 25plusmn 2∘C and 65 humidity witha 12 h light12 h dark cycle (Certificate number 2008-0085)During the experiments all animals were given ad libitumto standard laboratory rats chow and water After 1 weekof acclimatization the rats were divided into five groups(ten animals in each group) control group model groupZWT groups (ADR + ZWT 240 gkg and ADR + ZWT120 gkg) DXM group (ADR + DXM) the dosages of ZWTwere chosen referring to the clinical dosage and the result

Evidence-Based Complementary and Alternative Medicine 3

of our preliminary experiment The rats were intravenouslyinjected with ADR (60mgkg dissolved in C) The normalgroup was injected with saline (10mL100 g) only One weekafter injection the rats were administrated distilled water(control group and model group) or ZWT by an oral gavagemethod once daily for 28 days All experiments conformed tothe European Community Guidelines and the regulations ofthe National Institute of Health of US

24 Measurement of Uric Protein To measure urine proteinlevels 24 h urine samples were collected using metaboliccages on days 0 7 14 21 28 and 35 All rats were forbiddenfrom food and free access to water during the course ofsample collection Urinary protein was determined by colori-metric method

25 Blood Sampling and Tissue Removal On day 35 allanimals were sacrificed for blood sample and renal tissueTheblood samples were obtained from abdominal aorta one hourafter gavage administration under chloral hydrate anesthesiaSerum was separated by centrifugation at 4∘C at 3500 rpmfor 15min and stored at minus20∘C for biochemical analysisAfter exsanguination the kidneys were rapidly removed andweightedThe two kidneys were individually divided into twoparts The upper pole of the left kidney was treated for lightmicroscopy the lower pole of the left kidney was divided intofour parts quickly frozen with liquid nitrogen and kept inminus80∘C for the assay of IL-4 and IL-8 The upper pole of theright kidney was frozen with liquid nitrogen and then kept inminus80∘C for immunofluorescence assay of IgG The lower poleof the left kidney was divided into four parts one was forelectron microscopy and the rest were quickly frozen withliquid nitrogen and kept in minus80∘C for gene analysis

26 Measurement of Blood Biochemical Parameters and IL-4and IL-8 in Renal Tissue Serum cholesterol (CHOL) triglyc-erides (TRIG) serum albumin (ALB) serum total protein(TP) creatinine (Scr) and blood urea nitrogen (BUN) levelswere analyzed using automatic clinical chemistry analyzerPlasma SOD and MDA levels were tested according toinstructions of assay kits Renal tissue was homogenizedin saline (100mg tissuemL) with homogenizer and thencentrifuged at 4∘C at 3000 g for 20min The supernatant waskept at minus20∘C for the assay of interleukin-4 and interleukin-8levels using ELISA kits

27 Determination of Renal IgG Renal cortical tissues werecut into 5 120583m sections using freezing microtome and thenoperated as the following steps they were fixed in acetonefor 10min washed in phosphate buffer saline for 3minstained with 1 50 diluted rabbit anti-rat IgG antibody (FITClabelled) at room temperature for 30min washed in phos-phate buffer saline twice 5min each time washed in distilledwater for 1min and wet mounted with 50 glycerin Thesefinished sections were examined under inverted fluorescencemicroscopy and eight micrographs were obtained at randomwith magnification of 400x The fluorescence signal wasquantified using image processing software (Image J 147)

with eight micrographs for each section and six sectionsfor each group The result was expressed as average density(pixel) (average density = integrated densityarea)

28 Light Microscopy The cortical tissues were fixed with10 neutral formalin phosphate buffer dehydrated througha graded alcohol series and embedded in paraffin andthen they were cut into 5 120583m sections and stained withhematoxylin and eosin (HampE) and examined under the lightmicroscope (TE2000 Nikon Japan)

29 Electron Microscopy A portion of cortical tissues wascut into 1mm cubes fixed in 25 glutaraldehyde andpostfixed in 1 osmium tetroxide The samples were dehy-drated through a graded alcohol series and embedded inEpon 812 Four ultrathin sections (60 nm) were cut with adiamond knife continuously for each sample and stainedwithuranyl acetate and lead citrate The sections were examinedunder the electronmicroscope (JEM100CX-a Japan) at 60 kvtimes5000 magnification

210 NF-120581B p65 and I120581B Genes mRNA DeterminationThe total RNAs from different experimental groups wereobtained by Trizol method The concentration of RNA wasdetermined by an absorbance at 260 nm and the purity ofthe RNA was evaluated by measuring the A260A280 ratioRNA was reverse transcribed to cDNA using the Takarareverse transcription reagent with gDNA Eraser All RNAsamples performed gDNA removal step (42∘C 30min)Reverse transcription was performed at 37∘C 15min and85∘C 5 s PCR was performed with the SYBR Green PCRMaster Mix using the following oligonucleotide primersNF-120581B (51015840-ACGATCTGTTTCCCCTCATCT-31015840 antisense51015840-TGGGTGCGTCTTAGTGGTATC-31015840) I120581B (51015840-GAG-GAAATACCCCTCTCCATCT-31015840 antisense 51015840-GCCCTG-GTAGGTTACTCTGTT-31015840) GAPDH (51015840-ACAGCAACA-GGGTGGTGGAC-31015840 antisense 51015840-TTTGAGGGTGCA-GCGAACTT-31015840) The real-time quantitative PCR usedABI7500 (Applied Biosystems USA) and the cyclingprogram was set at 1 cycle of predenaturation at 95∘C for 30 sand then 40 cycles at 95∘C for 10 s 60∘C for 34 s and thenmelting curve was analyzed All the RT-PCR experimentswere conducted strictly according to the rules of the MIQE

211 Statistical and Analysis Datas are presented as mean plusmnSD Statistical analysis was performed with the independent-samples 119905-test by SPSS 170 software Differences were consid-ered significant when 119875 lt 005

3 Results

31 General Conditions On the third day of tail intravenousinjection of ADR proteinuria in the rats was increasedgradually Rats in model group presented lower food intakeand less activity compared with those in control groupHowever rats in treatment groups showed a better mentalcondition raisedmovement and food intake and a better hairsheen


0 1 2 3 4 5

Time (week)

0

100

200

300

400

500

600

Urin

ary

prot

ein

(mg24

h)

Control groupModel groupZWT (24 gkg)

ZWT (12 gkg)DXM group

lowastlowastlowastlowast

lowastlowast

lowastlowast

Figure 1 ZWT decreased the increased 24 h urinary proteininduced by ADR Data were expressed as mean plusmn SD 119899 = 8 Verticalbars represent standard errors of themeans and asterisks and poundsigns designate significant differences

119875 lt 005 and 119875 lt 001

versus model and lowast119875 lt 005 and lowastlowast119875 lt 001 versus control

32 Urinary ProteinAnalysis Proteinuria is one of the impor-tant characteristics in ADR-induced rat nephrotic syndromeIn our study we demonstrated that a single injection of ADRat 60mgkg increased 24 h uric protein excretion three daysafter the injection and the proteinuria reached a maximumof three weeks after the injection Interestingly treating ratswith ZWT at the dosages of 240 gkgd and 12 gkgd for 3weeks resulted in significant declines in the levels of 24 h uricprotein in parallel to the model group (119875 lt 001) (Figure 1)

33 Blood Biochemical Parameters As depicted in Table 1compared with control group the model group displayedincreased serum CHOL and TRIG Scr and BUN (119875 lt 001)but decreased levels of serum TP and ALB (119875 lt 005)These phenotypes were similar to the clinical symptoms ofnephrotic syndrome Furthermore ZWT at the dosages of240 gkgd and 120 gkgd for 28 days significantly preventedthe increase in serum CHOL TRIG Scr and BUN and thedecrease in serum TP and ALB

34 Changes in the Kidney Shape Long-term NSmay inducechanges in the conditions of kidney In the present studykidney of ADR-treated rats showed severe edema and lessluster compared with the control group The kidney in ZWTtreated group (240 gkgd and 120 gkgd) presented moredense shape and redder look (Figure 2) Moreover as shownin Table 2 the ration of absolute kidney weight (KW) toBW was increased in ADR model rats Treatment with ZWTprevented the increase of KWBW

35 IL-4 and IL-8 in Renal Tissue and SOD and MDA inPlasma In our study we found that treatment with ZWT

at the dosages of 240 gkgd and 120 gkgd for 28 dayscould significantly prevent the increase of renal IL-8 and thedecrease of renal IL-4 induced by ADR (Figures 3(a) and3(b)) ZWT could boost antioxidant power and decrease freeradicals damage in NS rat raise the plasma SOD activity andlower the content of plasma MDA (Figures 3(c) and 3(d))

36 Pathologic Changes of Kidney

361 Determination of Renal IgG Using immunofluorescentassay we found that the control group showed extremelyweak fluorescence but ADR-treated rats showed strong fluo-rescence at low magnification (119875 lt 001) Further treatmentwith ZWT decreased the strong fluorescence induced byADR (119875 lt 001) (Figure 4)

362 Light Microscopy and Electron Microscopy Renalpathological examination is a fast clear and direct method indiagnosis of nephrotic syndrome In model group glomeru-lar extracellular matrix (ECM) accumulation and glomerularmesangial cell (GMC) proliferation and base-membranethickness were observed Further foot processes were wideeffacement and the width of foot process was much largerthan that of normal control rats (Figure 5) Under the lightmicroscopy renal tubule in model group was dropsical andBowmanrsquos space was larger than that in control group Andthere were less cells in glomerulus in model group thanthose of control group At high magnification the lympho-cytes and neutrophils cells are seen around a renal tubuleTreatment of NS rats with ZWT reduced the edema andinflammatory cell infiltration ameliorated the effacement offoot processes reduced the lysosomes deposition in footprocess and reduced the edema of glomerulus (Figure 6)

37 Effects of ZWT on NF-120581B p65 and I120581B mRNA Expres-sions NF-120581B plays a key role in the regulation of cytokineexpressions Because most cytokines levels in NS are partlyor predominantly regulated byNF-120581B we postulated possibleinvolvement of NF-120581B in NS In the study the results showedthat the mRNA expression of NF-120581B p65 in the renal tissue ofADR-induced NS rats was highly upregulated and that of I120581Bwas highly downregulated compared with the control groupHowever ZWT could decrease the expression of renal NF-120581Bp65 but increase the expression of I120581B (119875 lt 001) (Table 3)

4 Discussion

ZWT is a blended traditional Chinese medicine specificallyfor the treatment of various renal diseases The present studydemonstrated that ZWT improved ADR-induced nephroticsyndrome NS is a series of clinical symptoms includingproteinuria hypoalbuminemia edema and hyperlipidemia[18] Proteinuria is a hallmark risk factor for that causesmost of the subsequent symptoms of NS [19] Podocytemodel group is a kind of highly differentiated cells formingmultiple interdigitating foot processes It is interconnectedby the slit diaphragms and covers the glomerular basementmembrane surface [20] It is recognized that the dysfunction


(a) (b) (c)

(d) (e)

Figure 2 Macroscopic morphology of the kidneys among five groups (a) control group (treated with saline) (b) model group (treated withADR) (c) ZWT group (treated with ZWT 240 gkg) (d) ZWT group (treated with ZWT 120 gkg) (e) DXM group (treated with DXM09mgkg)

Table 1 Improvement of blood biochemical parameters by ZWT (119899 = 8 mean plusmn SD)

Groups CHOL(mmolL)

TRIG(mmolL)

TP(gL)

ALB(gL)

BUN(120583molL)

Scr(mmolL)

Control 080 plusmn 020 053 plusmn 022 2931 plusmn 169 3085 plusmn 252 449 plusmn 086 2696 plusmn 800

Model 346 plusmn 090

291 plusmn 086

2680 plusmn 330

2795 plusmn 337

750 plusmn 0774758 plusmn 520

ZWT (240 gkg) 228 plusmn 082lowastlowast

146 plusmn 039lowastlowast



633 plusmn 086lowast








DXM (09mgkg) 176 plusmn 066lowastlowast





3641plusmn800lowastlowast

119875 lt 005 and

119875 lt 001 versus control lowast119875 lt 005 and lowastlowast119875 lt 001 versus model CHOL serum total cholesterol TRIG serum triglycerides TP serum totalprotein ALB serum albumin BUN blood urea nitrogen Scr serum creatinine

of glomerular podocyte and the subsequently cellular deathact as the driving forces behind disease initiation and pro-gression respectively [21] Extensive effacement of podocytefoot processes is the keymorphologic change noted inNS andpodocyte damage can result in serious proteinuriaTherefore

it is a central target to inhibit the podocyte frombeing injuredto maintain renal function [22] In the present study thefoot processes of podocyte were severely effaced in ADR-treated rats at week 5 and proteinuria appeared three daysafter injection of ADR treatment with ZWT significantly


0

50

100

150

200

IL-4

in re

nal t

issue

(pg

mg)

lowastlowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(a)

lowastlowast

0

100

200

300

IL-8

in re

nal t

issue

(pg

mg)

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(b)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

20

40

60

80

100

120

Plas

ma S

OD

(Um

L)

(c)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

2

4

6

8

10

12

Plas

ma M

DA

(nm

olm

L)

(d)

Figure 3 The effects of ZWT (gkg) on IL-4 and IL-8 in renal tissue and antioxidant index in plasma (a) IL-4 in renal tissue (b) IL-8 inrenal tissue (c) plasma SOD (d) plasma MDA DMX dexamethasone (09mgkg) data were expressed as mean plusmn SD 119899 = 8 Vertical barsrepresent standard errors of the means Asterisks and pound signs designate significant differences

119875 lt 005 and 119875 lt 001 versus model

and lowast119875 lt 005 and lowastlowast119875 lt 001 versus control

Table 2 ZWTdecrease the ration of absolute kidney weight to bodyweight (119899 = 10 mean plusmn SD)

Groups KW (g) BW (g) KWBW (10minus3)Control 236 plusmn 018 3528 plusmn 286 670 plusmn 062

Model 246 plusmn 021 2976 plusmn 322 808 plusmn 091

ZWT (240 gkg) 231 plusmn 021 3272 plusmn 242 710 plusmn 047lowastlowast


DXM (09mgkg) 188 plusmn 018 2581 plusmn 214 703 plusmn 032lowastlowast119875 lt 005 and

119875 lt 001 versus control lowast119875 lt 005 and lowastlowast119875 lt 001 versusmodel KW kidney weight BW body weight

ameliorated the effacement of foot processes and reducedproteinuria These results suggested that ZWT improved thepodocyte injury in ADR-induced nephrotic syndrome

Table 3 Effects of ZWT on NF-120581B p65 and I120581B mRNA expression(119899 = 10 mean plusmn SD)

Groups NF-120581B p65 mRNA I120581B mRNAControl 0316 plusmn 0043

lowastlowast


Model 1 1ZWT (240 gkg) 0560 plusmn 0154

lowastlowast


ZWT (120 gkg) 0736 plusmn 0236lowast




lowast

119875 lt 005 and lowastlowast119875 lt 001 versus model

The renal function is closely associated with urine proteinexcretion level and a direct reflection of diseasersquos progression[23] The concentrations of Scr and BUN which are twoimportant indexes to reflect renal function depend on the


00

01

02

03Ig

G d

epos

ition

in re

nal t

issue

(pi

xel)

lowastlowast

(a) (b) (c) (d) (e)

DXMgroup

Modelgroup

Controlgroup

ZWT(240 gg)

ZWT(120 gkg)

Figure 4 ZWT decrease the IgG deposition in renal tissue Magnification times400 (a) Control group (treated with saline) (b) model group(treatedwithADR) (c) ZWTgroup (treatedwith ZWT 240mgkg) (d) ZWTgroup (treatedwith ZWT 120mgkg) (e) DXMgroup (treatedwith dexamethasone 09mgkg) Data were expressed as mean plusmn SD 119899 = 6 Vertical bars represent standard errors of the means Asterisksand pound signs designate significant differences

119875 lt 005 and 119875 lt 001 versus model and lowast119875 lt 005 and lowastlowast119875 lt 001 versus control

(a) (b) (c)

(d) (e)

Figure 5 Morphology change in glomerulus under light microscopy Transmission light microscope magnification 10 times 40 (a) Controlgroup (treated with saline) (b) model group (treated with ADR) (c) ZWT group (treated with ZWT 240 gkg) (d) ZWT group (treatedwith ZWT 120 gkg) (e) DXM group (treated with dexamethasone 09mgkg)


Control

(a)

Model

(b)

DXM

(c)

ZWT (240 gkg)

(d)

ZWT (120 gkg)

(e)

Figure 6 Morphology change in glomerulus under light microscopy Transmission electron microscope magnification times6000 (a) Controlgroup (treated with saline) (b) model group (treated with ADR) (c) ZWT group (treated with ZWT 240 gkg) (d) ZWT group (treated withZWT 120 gkg) (e) DXM group (treated with dexamethasone 09mgkg)

glomerular filtration rate (GFR) Renal dysfunction reducesthe capability of filtering them and their levels then rise [24]In this study the rats did show a notable increase in the Scrand BUNafter 5weeks following injection of ADR indicatingthat renal filtrating functionwas being destroyed byADRButthe results also showed that ZWT could decrease the levels ofBUN and Scr in serum indicating that ZWT could enhancerenal function by reducing the synthesis or increasing theexcretion of BUN and Scr in ADR-induced NS rats

Dyslipidemia is also one of themain phenotypes in ADR-induced NS in the rats Consistent with previous reports[25] our present study confirmed that ADR increased plasmalevels of TRIG and CHOL in rats The mechanism involvedin the pathogenesis of dyslipidemia is still unclear Hutchisonreported that abnormal glomerular permeability to plasmaproteins and high density lipoprotein increased biosynthesisof CHOL and TRIG in liver and lipid metabolism disor-ders but reduced serum oncotic pressure contributes tohyperlipidemia [26] In the present study treatment withZWT also improved ADR-induced hypertriglyceridemia andhypercholesterolemia Furthermore ZWT increased the levelof serum TP and ALB improving the hypoproteinemiainduced by ADR All the data demonstrated that ZWT pos-sesses a protective effect on ADR-induced NS by preventingproteinuria protecting renal function and ameliorating footprocesses effacement After that we investigated immune

regulation and antioxidation effects of ZWT on ADR-induced NS in rats to reveal its deep therapy mechanism onNS

A possible role for Th2 cytokines in the induction ofproteinuria in minimal change nephropathy (MCN) is sup-ported by clinical observations such as the association ofMCN with atopy and the apparent induction of MCN byallergic events in some patients [27 28] IL-4 a pleiotropiccytokine is produced by appropriately stimulated CD4+Th2 lymphocytes IL-4 is the primary cytokine for thespontaneous production of IgE and IgG by peripheral bloodmononuclear cells which is associatedwith immune complexdepositing at glomerular basement membrane Howeverits role in NS is controversial In some researches it wasreported that IL-4 was increased in NS rats [29] and IL-4decreased transepithelial electrical resistance of monolayersof glomerular visceral epithelial cells in rats dose dependentlywhich suggested that IL-4 could exert specific effects onglomerular visceral epithelial cells function [30] In someclinical investigations of NS patients it was found that IL-4was decreased during relapse and increased in patients withlong-term remission [31] In the present study the resultsshowed a decreased level of renal IL-4 in ADR-treated groupwhich was prevented by ZWTThe differencemay result fromthe different stages of disease For that IL-4 can also actas anti-inflammatory cytokinesThemechanisms responsible


for the anti-inflammatory effects of IL-4 are attributable tothe suppression of tumor necrosis factor-120572 (TNF-120572) produc-tion by macrophages and upregulation of anti-inflammatorycytokines such as TGF-120573 [32 33] We hypothesized that IL-4 may act as protective effect on NS in later stages IL-8 isan important chemokine that acts on various inflammatorymediators including neutrophil granulocyte T-lymphocytesand basophilic granulocyteThemain biological effect of IL-8is accelerating chemotaxis of neutrophil granulocytes and IL-8 activity is positively associated with the inflammatory cellinfiltration in lesions area [34] In the present study the lym-phocytes and neutrophils cells are seen around a renal tubuleunder the light microscopy In addition IL-8 level in renaltissue was increased in ADR-treated group compared withcontrol groupThat was consistent with previous reports thatthe serum IL-8 levels of the nephrotic phasewere significantlylower than those of remission phase and was positively corre-lated with proteinuria [35 36] Further patients of NS alwayshave severe immune deposition in glomerular mesangiumwhich is defined as a late-stage symptom Immune depositioncan stimulate the release of local proteases and activate thecomplement cascade producing C5-9 attack complex dam-aging glomerular structure and producing proteinuria [37]Immunofluorescence stain showed severe IgG depositionin glomerulus in ADR-treated rats Renal IgG depositionin ZWT-treated rats was significantly reduced comparedwith ADR-treated rats These data suggest that ZWT mayameliorate kidney injury at least in part by modulatingthe balance between inflammatory and anti-inflammatoryresponses

NF-120581B is a transcription factor activated by cell surfacereceptor signaling tomeet stress and inflammatory responsesregulating key cellular processes such as inflammation innateand adaptive immunity and cell growth and survival [38]Notably the NF-120581B family of transcription factors has beenshown to regulate various aspects of T-cell including Th1Th2 Th17 Th9 and Tfh cells [39] It is critical in modu-lation of the immune response through the transcriptionalregulation of cytokine (IL-4 IL-2 IL-6 IL-12 IFN-L-6 IL-CSF and G-CSF) and chemokine (IL-8 and C3) expression[8 40] In addition NF-120581B is sequestered in the cytoplasmbound to I120581B phosphorylation of I120581B releases active NF-120581B which translocates to the nucleus to induce an extensiverange of target genes [41] In response to this we askedwhether a direct causal link between NF-120581B activation andADR induced NS and whether ZWT might regulate thispathway To test this we examined NF-120581B (p65) and I120581BmRNA expression in kidneys of a rat NS model induced byADR In our experiment NF-120581B (p65) mRNA expression inmodel group was significantly upregulated but I120581B mRNAexpression was significantly downregulated compared withcontrol group And the results are consistent with what hasbeen reported The results suggest that the activation of NF-120581B pathway was involved in the pathogenesis of NS ZWTcould inhibit the activation of NF-120581B and prevent kidneysfrom being injured

Antioxidant therapy is another strategy for NS treatmentand is considered to be an important pathogenic mechanismOxidative stress develops from an imbalance between oxygen

free raddical (ORF) production and reduced antioxidantdefenses such as SOD CAT glutathione and glutathioneperoxidase [42 43] ORF could result in seriousDNAdamageand lipid peroxidation and thus damage the glomerular filtra-tion barrier and promote renal cell apoptosis and senescencedecreased regenerative ability of cells and fibrosis [44] MDAis an important end-product generated by lipid peroxidationand has been used to demonstrate increased oxidative stressduring chronic kidney disease [45] In our study rats inADR-treated group presented with high level of plasmaMDAwhich indicated an increased lipid peroxide concentration[46 47] ZWT decreased plasma MDA and inhibited thelipid peroxidation A major mechanism of resistance to ROSis antioxidase enzymes like SOD Rat treated with ZWThas increased plasma SOD compared with that of modelgroup We concluded that ZWT could enhance the activityof endogenous antioxidant enzymes to prevent oxygen freeradical damage

5 Conclusions

Taken together we propose that ZWT could ameliorate theproteinuria low serum albumin hypercholesterolemia andloss of kidney function of NS rats These data provide directevidence for ZWT-treated nephrotic syndrome at least inpart by modulating the balance between inflammatory andanti-inflammatory responses enhancing antioxidant capac-ity and the elimination capacity of ROS However the activecompounds in ZWT responsible for its treatment are notrevealed at present and its more protection mechanismsare also not clear Therefore we will continue to investigatefurther Network pharmacology andmembrane immobilizedchromatography would be involved in our follow-up study

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Chun-ling Liang participated in the experiment and draftedthe paper Jun-biaoWu participated in the design of the studyand assisted in the statistical analysis Jiu-yao Zhou conceivedof the study and participated in its design and coordinationand helped to draft the paper Janis Ya-xian Zhan helped todraft the paper and participated in the experiment designJiemei Lai Shu-fang Ye Jin Lin andHui Ouyang participatedin the measurement of uric protein blood sampling andtissue removal All authors read and approved the final paperChun-ling Liang and Jun-biaoWu contributed equally as firstauthors to this work

Acknowledgments

This paper was supported by Grants from the GuangdongProvince Science Foundation (S2011020005170) andDoctoralProgram of the Ministry of Education (20114425110011)


References

[1] M Horvath and E Sulyok ldquoSteroid responsive nephroticsyndrome in Asiansrdquo Archives of Disease in Childhood vol 61no 5 p 528 1986

[2] K U Eckardt J Coresh O Devuyst et al ldquoEvolving importanceof kidney disease from subspecialty to global health burdenrdquoThe Lancet vol 382 no 9887 pp 158ndash169 2013

[3] J B Lewis and E G Neilson Haerisonrsquos Nephrology and Acid-BaseDisorders PekingUniversityMedical Press Beijing China2011

[4] R J Shalhoub ldquoPathogenesis of lipoid nephrosis a disorder ofT cell functionrdquoThe Lancet vol 2 no 7880 pp 556ndash560 1974

[5] S A Shalaby H M Al-Edressi S A El-Tarhouny M FathEl-Bab and M A Zolaly ldquoType 1type 2 cytokine serumlevels and role of interleukin-18 in children with steroid-sensitive nephrotic syndromerdquo Arab Journal of Nephrology andTransplantation vol 6 no 2 pp 83ndash88 2013

[6] W G Couser ldquoBasic and translational concepts of immune-mediated glomerular diseasesrdquo Journal of the American Societyof Nephrology vol 23 no 3 pp 381ndash399 2012

[7] C Zoja P B Garcia and G Remuzzi ldquoThe role of chemokinesin progressive renal diseaserdquo Frontiers in Bioscience vol 14 no5 pp 1815ndash1822 2009

[8] K-H Kim E-S Lee S-H Cha et al ldquoTranscriptional regu-lation of NF-120581B by ring type decoy oligodeoxynucleotide inan animal model of nephropathyrdquo Experimental and MolecularPathology vol 86 no 2 pp 114ndash120 2009

[9] V Audard A Pawlak M Candelier P Lang and D SahalildquoUpregulation of nuclear factor-related kappa b suggests a dis-order of transcriptional regulation inminimal change nephroticsyndromerdquo PLoS ONE vol 7 no 1 Article ID e30523 2012

[10] C K Fujihara G R Antunes A L Mattar D M A CMalheiros J M Vieira Jr and R Zatz ldquoChronic inhibitionof nuclear factor-120581B attenuates renal injury in the 56 renalablation modelrdquo The American Journal of Physiology RenalPhysiology vol 292 no 1 pp F92ndashF99 2007

[11] I Simic M Tabatabaeifar and F Schaefer ldquoAnimal models ofnephrotic syndromerdquo Pediatric Nephrology vol 28 no 11 pp2079ndash2088 2012

[12] M Li A R Qureshi E Ellis and J Axelsson ldquoImpaired post-prandial fibroblast growth factor (FGF)-19 response in patientswith stage 5 chronic kidney diseases is ameliorated followingantioxidative therapyrdquoNephrology Dialysis Transplantation vol28 supplement 4 pp 212ndash219 2013

[13] A Hamada S Yoshioka D Takuma et al ldquoThe effect of Eri-obotrya japonica seed extract on oxidative stress in adriamycin-induced nephropathy in ratsrdquo Biological and PharmaceuticalBulletin vol 27 no 12 pp 1961ndash1964 2004

[14] Z H Hou and Z H Hou ldquoPrescription and syndrome analysisof Zhenwu Decoction in Treaties of Febrile Diseasesrdquo ChinarsquosTraditional Chinese Medicine Emergency vol 3 no 19 p 482010

[15] R G Krishnan ldquoNephrotic syndromerdquo Paediatrics and ChildHealth vol 22 no 8 pp 337ndash340 2012

[16] Y Cai J Chen J Jiang W Cao and L He ldquoZhen-wu-tang ablended traditional Chinese herbal medicine ameliorates pro-teinuria and renal damage of streptozotocin-induced diabeticnephropathy in ratsrdquo Journal of Ethnopharmacology vol 131 no1 pp 88ndash94 2010

[17] J Geng G C S Wang and W B Gu ldquoThe effects of ZhenwuDecoction on the extracellular matrix of rat mesangial cellsrdquo

Journal of Traditional Chinese Medicine vol 41 no 11 pp 686ndash687 2000

[18] Y Kaneko and I Narita ldquoNephritis and nephrotic syndromerdquoNihon Jinzo Gakkai Shi vol 55 no 1 pp 35ndash41 2013

[19] D Li and J Ding ldquoMolecular mechanism of proteinuriacaused progression of chronic renal diseaserdquo Journal of PekingUniversity Health Sciences vol 42 no 5 pp 608ndash611 2010

[20] W Kriz M Elger M Nagata et al ldquoThe role of podocytes inthe development of glomerular sclerosisrdquo Kidney InternationalSupplement no 45 pp S64ndashS72 1994

[21] J Reiser and S Sever ldquoPodocyte biology and pathogenesis ofkidney diseaserdquo Annual Review of Medicine vol 64 pp 357ndash366 2013

[22] W Kriz ldquoPodocyte is the major culprit accounting for theprogression of chronic renal diseaserdquo Microscopy Research andTechnique vol 57 no 4 pp 189ndash195 2002

[23] K Singh R Ray A Sharma R Gupta A Bagga and A KDinda ldquoPeritubular capillaries and renal function in pediatricidiopathic nephrotic syndromerdquo Saudi Journal of Kidney Dis-eases and Transplantation vol 24 no 5 pp 942ndash949 2013

[24] A O Hosten ldquoBUN and creatininerdquo in Clinical Methods TheHistory Physical and Laboratory Examinations H K WalkerW D Hall and J W Hurst Eds Butterworths Butterworth ADivision of Reed Boston Mass USA 1990

[25] F Nakhoul R Ramadan E Khankin et al ldquoGlomerularabundance of nephrin and podocin in experimental nephroticsyndrome different effects of antiproteinuric therapiesrdquo TheAmerican Journal of Physiology Renal Physiology vol 289 no4 pp F880ndashF890 2005

[26] F N Hutchison ldquoProteinuria hyperlipidemia and the kidneyrdquoMineral and Electrolyte Metabolism vol 19 no 3 pp 127ndash1361993

[27] P W Mathieson ldquoImmune dysregulation in minimal changenephropathyrdquoNephrology Dialysis Transplantation vol 18 sup-plement 6 pp vi26ndashvi29 2003

[28] A A Eddy and J M Symons ldquoNephrotic syndrome in child-hoodrdquoThe Lancet vol 362 no 9384 pp 629ndash639 2003

[29] Y Qi H Xiao C Xu X Tian H Wu and W Shen ldquoCypri-nus carpio decoction improves nutrition and immunity andreduces proteinuria through nephrin and CD2AP expressionsin rats with adriamycin-induced nephropathyrdquo Evidence-BasedComplementary and Alternative Medicine vol 2012 Article ID237482 8 pages 2012

[30] J G van den Berg J Aten M A Chand et al ldquoInterleukin-4 and interleukin-13 act on glomerular visceral epithelial cellsrdquoJournal of the American Society of Nephrology vol 11 no 3 pp413ndash422 2000

[31] V Daniel Y Trautmann M Konrad A Nayir and K ScharerldquoT-lymphocyte populations cytokines and other growth factorsin serum and urine of children with idiopathic nephroticsyndromerdquo Clinical Nephrology vol 47 no 5 pp 289ndash297 1997

[32] B KlementievM N Enevoldsen S Li et al ldquoAntiinflammatoryproperties of a peptide derived from interleukin-4rdquo Cytokinevol 64 no 1 pp 112ndash121 2013

[33] P H Hart G F Vitti D R Burgess G A Whitty D S Piccoliand J A Hamilton ldquoPotential antiinflammatory effects ofinterleukin 4 suppression of human monocyte tumor necrosisfactor 120572 interleukin 1 and prostagandin E2rdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 86 no 10 pp 3803ndash3807 1989


[34] P R Harris H C Weber C M Wilcox R T Jensen and P DSmith ldquoCytokine gene profile in gastric mucosa in Helicobacterpylori infection andZollinger-Ellison syndromerdquoTheAmericanJournal of Gastroenterology vol 97 no 2 pp 312ndash318 2002

[35] F Huang S Horikoshi A Kurusu et al ldquoUrinary levels ofinterleukin-8 (IL-8) and disease activity in patients with IgAnephropathyrdquo Journal of Clinical LaboratoryAnalysis vol 15 no1 pp 30ndash34 2001

[36] E H Garin D K Blanchard K Matsushima and J Y DjeuldquoIL-8 production by peripheral blood mononuclear cells innephrotic patientsrdquoKidney International vol 45 no 5 pp 1311ndash1317 1994

[37] M Noris and G Remuzzi ldquoOverview of complement activationand regulationrdquo Seminars in Nephrology vol 33 no 6 pp 479ndash492 2013

[38] M S Hayden and S Ghosh ldquoSignaling to NF-120581Brdquo Genes andDevelopment vol 18 no 18 pp 2195ndash2224 2004

[39] H Oh and S Ghosh ldquoNF-kappaB roles and regulation indifferent CD4+ T-cell subsetsrdquo Immunological Reviews vol 252no 1 pp 41ndash51 2013

[40] M D Turner A Chaudhry and B Nedjai ldquoTumour necrosisfactor receptor trafficking dysfunction opens the TRAPS doorto pro-inflammatory cytokine secretionrdquo Bioscience Reportsvol 32 no 2 pp 105ndash112 2012

[41] P Viatour M-P Merville V Bours and A Chariot ldquoPhospho-rylation of NF-120581B and I120581B proteins implications in cancer andinflammationrdquo Trends in Biochemical Sciences vol 30 no 1 pp43ndash52 2005

[42] A Ece Y Atamer F Gurkan M Bilici and Y KocyigtldquoAnti-oxidant status in relation to lipoproteins leptin andpro-inflammatory cytokines in children with steroid-sensitivenephrotic syndromerdquo Nephrology vol 9 no 6 pp 366ndash3732004

[43] J L Mathew B C Kabi and B Rath ldquoAnti-oxidant vitaminsand steroid responsive nephrotic syndrome in Indian childrenrdquoJournal of Paediatrics and Child Health vol 38 no 5 pp 450ndash454 2002

[44] D M Small J S Coombes N Bennett D W Johnson and GC Gobe ldquoOxidative stress anti-oxidant therapies and chronickidney diseaserdquo Nephrology vol 17 no 4 pp 311ndash321 2012

[45] A Atamer Y Kocyigit S A Ecder et al ldquoEffect of oxidativestress on antioxidant enzyme activities homocysteine andlipoproteins in chronic kidney diseaserdquo Journal of Nephrologyvol 21 no 6 pp 924ndash930 2008

[46] O P Mishra A K Gupta R Prasad et al ldquoAntioxidant statusof children with idiopathic nephrotic syndromerdquo PediatricNephrology vol 26 no 2 pp 251ndash256 2011

[47] S R Ghodake A N Suryakar R D Ankush R V Katkam KShaikh and A V Katta ldquoRole of free radicals and antioxidantstatus in childhood nephrotic syndromerdquo Indian Journal ofNephrology vol 21 no 1 pp 37ndash40 2011

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



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the development of proteinuria renal dysfunction glomeru-lar cell proliferation and extracellular matrix componentsexpansion and prolonged renal fibrosis [6 7] Thereforeinhibiting the activation of renal inflammation could bethe therapeutic target of protecting renal lesion in NS NF-120581B a ubiquitous transcription factor governs the expressionof genes encoding for cytokines chemokines growth fac-tors adhesion molecules and other factors involved in theimmune and inflammatory response A series of correlationsbetween the activation of NF-120581B and onset of both immune-and nonimmune-mediated glomerular injury implicate dys-regulation of the NF-120581B pathway in its pathogenesis [8ndash10] Therefore we postulated possible involvement of NF-120581Bin the pathogenesis of NS Antioxidant therapy is anotherimportant strategy for NS treatment and is considered tobe an important pathogenic mechanism Reactive oxygenspecies (ROS) are continuously produced physiologically andplay an important role in the expression of cell functions suchas transmission of impulse information However excessiveproduction of ROS would act as mediators of adverse eventssuch as inflammation necrosis and apoptosis In the kidneyROS are important causes of acute and subacute renalfailure in most cases Also in certain animal models ofNS superoxide anions and hydroxyl radicals are the maincauses of nephrotoxicity [11] It has also been reported thatrenal failure that results from increased ROS and decreasedantioxidant enzyme can be prevented by antioxidant therapy[12 13]

Zhen-wu-tang (ZWT) is a classic prescription to treatchronic kidney disease (CKD) with the paths of Wen yangand LiWater which is recorded in Treatise on Febrile Diseases[14] As we know edema is the most common presenting fea-ture in CKD It becomes detectable when the fluid retentionexceeds 3 of theweight of the body [15] ZWThas been usedas a remedy for various kidney diseases with the efficacy torelieve relative symptoms manifested by edema dysuria andoliguria in clinics in China and Japan [16] Furthermore thedecrease in levels of collagen IV fibronectin and laminin inthe extracellularmatrix of the fetal glomerularmesangial cellshas been observed after treatment by ZWT [17] Howeverthe effect of ZWT on Adriamycin- (ADR-) induced NS hasnot been well explored In this study we will investigate theimmune regulation and antioxidation effects of ZWT on ratswith NS induced by ADR The aims of the present study areto investigate the protective effects of ZWT on ADR-inducedNS and to discuss possible underlying mechanism of actionin rat model

2 Methods and Materials

21 Drugs and Drug Treatment

211 Plants Material and Drugs ZWT is composed of fiveherbal medicines Aconiti Lateralis Radix Praeparata (thelateral radix of Aconitum carmichaelii Debx) ZingiberisRhizoma Recens (rhizome of Zingiber officinale Rosc)Atractylodis Macrocephalae Rhizoma (radix of AtractylodesmacrocephalaKoidz) Paeoniae Radix Alba (radix of Paeonia

lactiflora Pall) and Poria (sclerotium of Poria cocos (Schw)Wolf) The dried raw materials of the herbs were purchasedfrom the Zhixin Medicinal Materials Company GuangzhouChina (Lot YPA110001 120301 120301 and 120401 resp) Allherbal medicines were authenticated by Professor Qiu-ZhenZhang at Guangdong Chinese Medicine Museum To assurethe quality control the materials were validated according tothe Chinese Pharmacopeia Dexamethasone was purchasedfrom the Second Affiliated Hospital Guangzhou Universityof Chinese Medicine Guangzhou China (Lot 111001)

212 Preparation of ZWT Amounts of Aconiti LateralisRadix Praeparata Poria Atractylodis Macrocephalae Rhi-zoma Paeoniae Radix Alba and Zingiberis Rhizoma Recenswere mixed according to a ratio of 3 3 2 3 3 All materialswere soaked with eight times distilled water for one hour andthen boiled with distilled water for 2 h For second extractionthe residue from the first extractionwas filtered and the sameextracting condition was applied After being repeated forthree times the mixture of the filtrates was concentrated tothe concentration of 24 g raw materials per milliliter Theextract was kept at 4∘C and dissolved in distilled water beforeuse

22 Chemicals and Reagents ADR was purchased fromShenzhen Main Luck Pharmaceuticals Inc (Lot 0901E)Urinalysis Reagent Strip fromAcon Biotech (Hangzhou) CoLtd (Lot 201109144) Coomassie (Bradford) Protein AssayKits from Nanjing Jiancheng Bioengineering Institute (Lot20120413 and 20120604) Interleukin-4 and Interleukin-8ELISAkits for rats fromAbcam (HK) Ltd (Lot ISC10330EIA-2121R and -2135-R resp) Rabbit Anti-Rat IgGFITC fromBeijing Biosynthesis Biotechnology Co Ltd (Lot 990603)SOD and MDA Assay Kits from Nanjing Jiancheng Bio-engineering Institute (Lot 20120523 and 20120324) Trizol(9108 Lot number AK8106) RT Reagent Kit (RR047A LotAK2003) and SYBR Premix ExTaq II (RR820A Lot numberAK4401) were from Takara Dalian China the primers forNF-120581B p65 I120581B and GAPDH were synthesized by SangonBiotech (Shanghai) Co Ltd

23 Animals and Treatments Fifty adult male specialpathogen free Sprague Dawley rats (Guangzhou Universityof Chinese Medicine Research Center for ExperimentalAnimal) which weighed (200 plusmn 20) g with urine proteinqualitative tests showing negative results (Urinalysis ReagentStrip) (Certificate number SCXK (Guangdong) 2008-0020)were used in the experiments The rats were housed in anair-conditioned room at 25plusmn 2∘C and 65 humidity witha 12 h light12 h dark cycle (Certificate number 2008-0085)During the experiments all animals were given ad libitumto standard laboratory rats chow and water After 1 weekof acclimatization the rats were divided into five groups(ten animals in each group) control group model groupZWT groups (ADR + ZWT 240 gkg and ADR + ZWT120 gkg) DXM group (ADR + DXM) the dosages of ZWTwere chosen referring to the clinical dosage and the result












3 Results



0 1 2 3 4 5

Time (week)

0

100

200

300

400

500

600

Urin

ary

prot

ein

(mg24

h)




lowastlowast

lowastlowast


119875 lt 005 and 119875 lt 001











4 Discussion



(a) (b) (c)

(d) (e)



Groups CHOL(mmolL)

TRIG(mmolL)

TP(gL)

ALB(gL)

BUN(120583molL)

Scr(mmolL)



291 plusmn 086

2680 plusmn 330

2795 plusmn 337




















119875 lt 005 and





0

50

100

150

200

IL-4

in re

nal t

issue

(pg

mg)

lowastlowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(a)

lowastlowast

0

100

200

300

IL-8

in re

nal t

issue

(pg

mg)

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(b)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

20

40

60

80

100

120

Plas

ma S

OD

(Um

L)

(c)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

2

4

6

8

10

12

Plas

ma M

DA

(nm

olm

L)

(d)














lowastlowast



lowastlowast






lowast




00

01

02

03Ig

G d

epos

ition

in re

nal t

issue

(pi

xel)

lowastlowast

(a) (b) (c) (d) (e)

DXMgroup

Modelgroup

Controlgroup

ZWT(240 gg)

ZWT(120 gkg)



(a) (b) (c)

(d) (e)



Control

(a)

Model

(b)

DXM

(c)

ZWT (240 gkg)

(d)

ZWT (120 gkg)

(e)











5 Conclusions






Acknowledgments



References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results



0 1 2 3 4 5

Time (week)

0

100

200

300

400

500

600

Urin

ary

prot

ein

(mg24

h)




lowastlowast

lowastlowast


119875 lt 005 and 119875 lt 001











4 Discussion



(a) (b) (c)

(d) (e)



Groups CHOL(mmolL)

TRIG(mmolL)

TP(gL)

ALB(gL)

BUN(120583molL)

Scr(mmolL)



291 plusmn 086

2680 plusmn 330

2795 plusmn 337




















119875 lt 005 and





0

50

100

150

200

IL-4

in re

nal t

issue

(pg

mg)

lowastlowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(a)

lowastlowast

0

100

200

300

IL-8

in re

nal t

issue

(pg

mg)

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(b)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

20

40

60

80

100

120

Plas

ma S

OD

(Um

L)

(c)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

2

4

6

8

10

12

Plas

ma M

DA

(nm

olm

L)

(d)














lowastlowast



lowastlowast






lowast




00

01

02

03Ig

G d

epos

ition

in re

nal t

issue

(pi

xel)

lowastlowast

(a) (b) (c) (d) (e)

DXMgroup

Modelgroup

Controlgroup

ZWT(240 gg)

ZWT(120 gkg)



(a) (b) (c)

(d) (e)



Control

(a)

Model

(b)

DXM

(c)

ZWT (240 gkg)

(d)

ZWT (120 gkg)

(e)











5 Conclusions






Acknowledgments



References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 2 3 4 5

Time (week)

0

100

200

300

400

500

600

Urin

ary

prot

ein

(mg24

h)




lowastlowast

lowastlowast


119875 lt 005 and 119875 lt 001











4 Discussion



(a) (b) (c)

(d) (e)



Groups CHOL(mmolL)

TRIG(mmolL)

TP(gL)

ALB(gL)

BUN(120583molL)

Scr(mmolL)



291 plusmn 086

2680 plusmn 330

2795 plusmn 337




















119875 lt 005 and





0

50

100

150

200

IL-4

in re

nal t

issue

(pg

mg)

lowastlowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(a)

lowastlowast

0

100

200

300

IL-8

in re

nal t

issue

(pg

mg)

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(b)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

20

40

60

80

100

120

Plas

ma S

OD

(Um

L)

(c)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

2

4

6

8

10

12

Plas

ma M

DA

(nm

olm

L)

(d)














lowastlowast



lowastlowast






lowast




00

01

02

03Ig

G d

epos

ition

in re

nal t

issue

(pi

xel)

lowastlowast

(a) (b) (c) (d) (e)

DXMgroup

Modelgroup

Controlgroup

ZWT(240 gg)

ZWT(120 gkg)



(a) (b) (c)

(d) (e)



Control

(a)

Model

(b)

DXM

(c)

ZWT (240 gkg)

(d)

ZWT (120 gkg)

(e)











5 Conclusions






Acknowledgments



References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e)



Groups CHOL(mmolL)

TRIG(mmolL)

TP(gL)

ALB(gL)

BUN(120583molL)

Scr(mmolL)



291 plusmn 086

2680 plusmn 330

2795 plusmn 337




















119875 lt 005 and





0

50

100

150

200

IL-4

in re

nal t

issue

(pg

mg)

lowastlowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(a)

lowastlowast

0

100

200

300

IL-8

in re

nal t

issue

(pg

mg)

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(b)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

20

40

60

80

100

120

Plas

ma S

OD

(Um

L)

(c)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

2

4

6

8

10

12

Plas

ma M

DA

(nm

olm

L)

(d)














lowastlowast



lowastlowast






lowast




00

01

02

03Ig

G d

epos

ition

in re

nal t

issue

(pi

xel)

lowastlowast

(a) (b) (c) (d) (e)

DXMgroup

Modelgroup

Controlgroup

ZWT(240 gg)

ZWT(120 gkg)



(a) (b) (c)

(d) (e)



Control

(a)

Model

(b)

DXM

(c)

ZWT (240 gkg)

(d)

ZWT (120 gkg)

(e)











5 Conclusions






Acknowledgments



References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

IL-4

in re

nal t

issue

(pg

mg)

lowastlowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(a)

lowastlowast

0

100

200

300

IL-8

in re

nal t

issue

(pg

mg)

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

(b)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

20

40

60

80

100

120

Plas

ma S

OD

(Um

L)

(c)

lowast

Controlgroup

Modelgroup

ZWT(240 gg)

ZWT(120 gkg)

DXMgroup

0

2

4

6

8

10

12

Plas

ma M

DA

(nm

olm

L)

(d)














lowastlowast



lowastlowast






lowast




00

01

02

03Ig

G d

epos

ition

in re

nal t

issue

(pi

xel)

lowastlowast

(a) (b) (c) (d) (e)

DXMgroup

Modelgroup

Controlgroup

ZWT(240 gg)

ZWT(120 gkg)



(a) (b) (c)

(d) (e)



Control

(a)

Model

(b)

DXM

(c)

ZWT (240 gkg)

(d)

ZWT (120 gkg)

(e)











5 Conclusions






Acknowledgments



References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

01

02

03Ig

G d

epos

ition

in re

nal t

issue

(pi

xel)

lowastlowast

(a) (b) (c) (d) (e)

DXMgroup

Modelgroup

Controlgroup

ZWT(240 gg)

ZWT(120 gkg)



(a) (b) (c)

(d) (e)



Control

(a)

Model

(b)

DXM

(c)

ZWT (240 gkg)

(d)

ZWT (120 gkg)

(e)











5 Conclusions






Acknowledgments



References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

(a)

Model

(b)

DXM

(c)

ZWT (240 gkg)

(d)

ZWT (120 gkg)

(e)











5 Conclusions






Acknowledgments



References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusions






Acknowledgments



References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References























































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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