Research Article Preventive Effect of Dihydromyricetin...

Research ArticlePreventive Effect of Dihydromyricetin against Cisplatin-InducedNephrotoxicity In Vitro and In Vivo

Fei Wu1 Yi Li2 Haibo Song1 Yuan Zhang1 Yuzhu Zhang1 Miao Jiang1 Fang Wang1

Qian Mu1 Wen Zhang1 Liang Li1 and Dongqi Tang1

1Center for Stem Cell amp Regenerative Medicine The Second Hospital of Shandong University Jinan 250012 China2Department of Joint Surgery Shandong Provincial Hospital Affiliated to Shandong University Jinan Shandong 250021 China

Correspondence should be addressed to Dongqi Tang dqtang1030126com

Received 15 April 2016 Accepted 27 July 2016

Academic Editor Roberto K N Cuman

Copyright copy 2016 Fei Wu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Nephrotoxicity is a frequent severe side effect of cisplatin chemotherapy limiting its clinical use despite being one of the mostpotent chemotherapy drugsDihydromyricetin is a highly abundant compoundpurified from the leaves ofAmpelopsis grossedentataPrevious studies have demonstrated the anti-inflammatory and antioxidative effects of Dihydromyricetin both in vitro and in vivobut little is known about the effects of Dihydromyricetin on cisplatin-induced nephrotoxicity and its underlying mechanismsIn the present study we investigated its potential renoprotective effect and found that Dihydromyricetin ameliorated the renalfunctional impairment and structural damage caused by cisplatin Moreover Dihydromyricetin markedly attenuated cisplatin-induced oxidative stress as well as protecting against cisplatin-induced inflammation and apoptotic cell death in mouse kidneytissues These results collectively highlight the potential of DMY as a rational renoprotective agent against cisplatin

1 Introduction

Cisplatin which belongs to platinum-based antineoplasticdrugs is one of the major antineoplastic and highly effectivedrugs which has been demonstrated to have one of thehighest cure rates [1] Cisplatin in combination withrelated platinum-based therapeutics has been used forthe treatment of bladder testicular ovarian cervical headand neck small-cell and non-small cell lung carcinomaand many other types of cancers [2ndash4] However thesevere side effects in normal tissues especially in kidneysgreatly limit its chemotherapeutic efficacy [1 5] It hasbeen reported that approximately one-third of patientsreceiving high-dose cisplatin treatment experience cisplatinnephrotoxicity leading to a subclinical but permanentreduction of renal function [3 6] For decades numerousstudies have focused on investigating the mechanisms ofnephrotoxicity development and it has been demonstratedthat inflammation oxidative stress injury mitochondrial

dysfunction and direct cytotoxicity to the tubular epithelialcells may partially explain this process [3 7]

Dihydromyricetin (DMY) also known as ampelopsin isa natural flavonoid compound extracted from the leaves ofampelopsis grossedentata which is commonly known as vinetea Vine tea is considered to have numerous pharmacologicalfunctions is used in traditional Chinese medicine to acceler-ate detoxification of ethanol and treat fever and cough andpossesses antioxidative and antimicrobial properties [8 9]As the most abundant and important bioactive ingredient invine tea DMY has been reported to exert anti-inflammatoryantioxidant and anticancer effects in previous research [10ndash12] Zhu et al have demonstrated that DMY exerts a cardio-protective effect against adriamycin-induced injury in ICRmice [13] However little is known about the effect of DMYin cisplatin-induced nephrotoxicity

In the current study we investigated the renoprotectiveeffect of DMY in a cisplatin-treated mouse model by measur-ing the renal functional parameters of serum creatinine (SCr)

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 7937385 9 pageshttpdxdoiorg10115520167937385

2 Evidence-Based Complementary and Alternative Medicine

and blood urea nitrogen (BUN) levels and histological exam-ination using periodic acid-Schiff (PAS) staining We foundthat DMY decreased the malondialdehyde (MDA) level andincreased the catalase activity (CAT) and superoxide dismu-tase (SOD) activities in mouse kidney tissues after treatmentwith cisplatin for 3 days We also observed decreased mRNAexpression of several proinflammatory cytokines MoreoverDMY mediated a protective effect against cisplatin-inducednephrotoxicity by ameliorating apoptotic cell death in HK-2cells

2 Materials and Methods

21 Animals Adult male C57BL6J mice (6ndash8 weeks old 20ndash25 g body weight) were used in this study These mice werepurchased fromShanghai SLACLaboratoryAnimal Co Ltdand housed at 22 plusmn 2∘C and 60 plusmn 5 humidity on a 10ndash12 h light-dark cycle under a pathogen-free condition Allanimals had free access to sterile tap water and food duringthe experiments All of the animal experimental protocolswere performed in accordance with the NIH Guide for Careand Use of Laboratory Animals and were approved by theInstitutional Animal Care and Use Committee at ShandongUniversity China

22 Cisplatin-Induced Nephrotoxicity DMY (Aladdin) wassuspended in sesame oil and administrated by gavage toC57BL6J mice Cisplatin (Sigma-Aldrich) was dissolvedin 09 saline at a concentration of 2mgmL and micewere given a single intraperitoneal injection The mice wererandomly divided into a control group 30mgkg cisplatin-treated group 30mgkg cisplatin and 100mgkg DMY-treated group 30mgkg cisplatin and 300mgkg DMY-treated group and 30mgkg cisplatin and 500mgkg DMY-treated group The DMY treatment was started 4 days priorto a single ip injection of cisplatin and continued for anadditional 3 days

23 Sample Collection Micewere anesthetized and sacrificed3 days after cisplatin injection Blood samples were collectedthrough tail vein and serum was separated and used forthe assessment of serum creatinine concentration (SCr) andblood urea nitrogen (BUN) Next hearts were cannulatedwith 09 NaCl at 90mmHg via the left ventricular apexto clear the kidneys until being whitish Both kidneys wereremoved and stored in liquid nitrogen or fixed in 4paraformaldehyde and embedded in paraffin for histologicalanalysis

24 Assessment of Renal Function Blood samples were trans-ferred to 15mL tubes and allowed to clot Then serumwas separated by centrifugation at 4000 rpm for 15min SCrand BUN were measured using a Beckman Coulter Dxc800biochemical analyzer (Beckman Coulter)

25 Evaluation of Histology and ImmunohistochemistryFormalin-fixed and paraffin-embedded kidney tissues weresectioned at 4 120583mand stainedwith periodic acid-Schiff (PAS)

for histological examination Apoptotic cells were detectedusing the TdT-mediated dUTP nick-end labeling (TUNEL)assay using an in situ apoptosis detection kit (Roche AppliedScience) according to the manufacturerrsquos protocol Immuno-histochemical staining for 8-hydroxydeoxyguanosine (8-OHdG) was performed using anti-8-OHdG antibody (SantaCruz Biotechnology) In the evaluation of hematoxylin andeosin (HE) stained sections interstitial polymorphonuclearleukocyte (PMN) infiltration was scored by a renal pathol-ogist who was blinded to the experimental groups PMNinfiltration per high power (times200) field was graded semi-quantitatively according to the following schema 0 0-1 1 2ndash10 2 11ndash20 3 21ndash40 4 gt40 or too many to count [14]

26 Measurement of Oxidative Stress Markers Oxidativestress markers were measured in kidney tissues of the miceKidney tissues were homogenized in lysis buffer and thesupernatant was used for the measurement of malondialde-hyde (MDA) superoxide dismutase (SOD) and catalaseactivity (CAT)

Determination of kidney tissue MDA concentrationwas measured using a Lipid Peroxidation MDA Assay kit(Beyotime Biotechnology) The concentration of MDA wasdetected by reacting with thiobarbituric acid (TBA) Briefly01mL of the kidney tissue supernatant was mixed with200120583LMDAworking solution heated in a heat block (100∘C)for 15min and then cooled down to room temperatureAfter centrifugation at 1000 g for 10min the supernatant wasmeasured at a wavelength of 532 nm

The assay for SOD activity was based on the abilityof SOD to inhibit nitroblue tetrazolium (NBT) reductionby superoxide Briefly 20 120583L of the tissue supernatant wasmixed with 160 120583L NBT working solution and 20 120583L reactingsolution and then incubated in 37∘C for 30min Finally theoptical density was measured at a wavelength of 560 nm

The catalase activity was detected using a Catalase Anal-ysis Kit (Beyotime Biotechnology) according to the man-ufacturerrsquos instructions Briefly the tissue supernatant wastreated with excess hydrogen peroxide for decompositionfor an indicated time The remaining hydrogen peroxidecoupled with a substrate was treated with peroxidase toproduce N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquino-nemonoimine which absorbs maximally at a wavelength of520 nm

27 Cell Culture HK-2 human kidney proximal tubular cellswere purchased from American Type Culture Collectionand cultured in renal epithelial basal medium (Gibco) withmanufacturer provided supplements in an incubator with 5CO2at 37∘C

28 Cell Viability Assay The cytotoxicity of cisplatin wasdetermined by the water-soluble tetrazolium- (WST-) 1method using the WST-1 cell proliferation and cytotoxic-ity assay kit (Beyotime Biotechnology) Briefly HK-2 wereseeded in 96-well plates at 5000 cellswell and incubated at37∘C for 24 h The media were replaced with normal mediain the absence or presence of DMY (25 or 50120583M) After

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primers for Q-PCR

Primers Forward 51015840-31015840 Reverse 51015840-31015840 Product length (bp)IL-6 AAGGAGTGGCTAAGGACCAA GTTTGCCGAGTAGATCTCAAA 89IL-1120573 TTCCTTGTGCAAGTGTCTGAAG CACTGTCAAAAGGTGGCATTT 76TNF-120572 TGACGTGGAACTGGCAGAAGA TGGGCCATAGAACTGATGAGAG 204TGF-120573 CGTGGAAATCAACGGG CAGAAGTTGGCATGGT 267MCP-1 GCTGACCCCAAGAAGGAATG GAAGACCTTAGGGCAGATGCA 110120573-Actin CATGTACGTTGCTATCCAGGC CTCCTTAATGTCACGCACGAT 250

incubation for 24 h cells were exposed to cisplatin (10 20 30or 50120583M) for another 24 h Then WST-1 reagent was addedfor 4 h at 37∘C and absorbance was measured at 450 nmwavelength using an automated microplate reader

29 DAPI Staining Assay HK-2 cells were pretreated withor without DMY for 24 h and then treated with or with-out cisplatin for another 24 h Cells were washed twicewith PBS and then fixed with 4 paraformaldehyde andpermeabilized with 05 triton X-100 for 10min Afterthat cells were incubated in 410158406-diamidino-2-phenylindole(DAPI)PBS (1 5000 dilution Sigma) for 3min at roomtemperature Finally the nuclei were photographed usingfluorescence microscopy

210 Western Blot Analysis Equal amounts of total proteinwere separated by sodium dodecyl sulfate polyacrylamidegels (SDS-PAGE) and subsequently transferred to polyvinyli-dene fluoride (PVDF) membranes After blocking in 5nonfat dry milk for 1 h membranes were incubated with pri-mary antibodies against Bcl-2 (1 1000 dilution) Bax (1 1000dilution) and GAPDH (1 1000 dilution) overnight at 4∘CMembranes were washed three times in 001M Tris BufferedSaline with 01 Tween-20 (TBST) and then incubatedin HRP-labeled secondary antibody (1 5000 dilution anti-mouse) for 1 h and detected using enhanced chemilumines-cence (ECL) solution

211 Quantitative Real-Time-Polymerase Chain Reaction (Q-PCR) For Q-PCR analysis total RNA was extracted usingthe TRIzol reagent (Invitrogen) cDNA was prepared usingRevert Aid First Strand cDNA Synthesis kit (Thermo Sci-entific) according to the manufacturerrsquos protocol Real-timePCR in triplicate was performed with SYBR Green MasterMix (Applied Biosystems) A standard amplification protocolwas used for real-time PCR analysis 95∘C for 10 s followed by40 cycles of 95∘C for 5 s 60∘C for 30 s and a final extensionat 72∘C for 3min Primers that were used for Q-PCR aresummarized in Table 1

212 Statistical Analysis Data are presented as mean plusmn SDStatistical analysis was conducted using the SPSS softwareAll experiments were repeated at least three times to obtainthe data Statistical evaluation among multiple groups wasperformed by one-way ANOVA followed by Studentrsquos t-test

for analysis of differences in each group A P value lt 005wasconsidered as statistically significant

3 Results

31 Effect of Dihydromyricetin on Cisplatin-Induced GeneralToxicity and Nephrotoxicity in Mice In order to evaluatewhether pretreatment of DMY could ameliorate renal func-tional impairment induced by cisplatin serum creatinine(SCr) and blood urea nitrogen (BUN) levels weremeasured asa marker of renal function in mice as described in Section 2As shown in Figures 1(a) and 1(b) the cisplatin-treated groupexhibited a significant increase in SCr and BUN comparedwith the control group The enhanced levels of SCr andBUN were significantly decreased in the cisplatin with DMYgroups in a dose-dependentmannerTheDMYconcentrationof 500mgkg showed a greater protective effect and was usedfor all following experiments

Furthermore periodic acid-Schiff (PAS) staining wasperformed for histological examination (Figure 1(c)) Asexpected the cisplatin-treated group showed severe loss ofbrush border and cast formation On the contrary DMYtreatment significantly attenuated these pathological changesinduced by cisplatin

Using TUNEL staining cisplatin-administered mice alsoexhibited a significantly higher number of positive nucleicompared with control while the DMY-treated group indi-cated a decrease in apoptotic cells (Figure 2)

32 Effect of Dihydromyricetin on Cisplatin-Induced Cell Via-bility and Apoptosis in HK-2 Cells Since it is commonly rec-ognized that nephrotoxicity caused by cisplatin is attributedto cell injury and death in renal tubules [15ndash17] we nextexamined the effect of DMY on HK-2 cell apoptosis inducedby cisplatin As shown in Figure 3(a) cell viability wasinhibited by cisplatin in a concentration-dependent manner(10 20 30 or 50 120583M) while DMY effectively abated theinhibitory effects caused by cisplatin To further validate thatDMY significantly attenuated cisplatin-induced cell apopto-sis in HK-2 DAPI staining was performed in the followingexperiment The results demonstrated that extensive nuclearfragmentation was clearly observed in the cisplatin-treatedgroup On the contrary after pretreatment with DMY for24 h these nuclear changes were decreased (Figure 3(b))To further validate the impact of DMY on cisplatin-induced


200

150

100

50

0

BUN

(mg

dL)

CisplatinDMY (mgkg)

_ + +

_

+

_ 300 500+

100

lowastlowast

lowastlowast

(a)

20

15

10

05

00

Seru

m cr

eatin

ine (

mg

dL)

_ _ 300 500100Cisplatin

DMY (mgkg)_ + + ++

lowastlowast

lowastlowast

(b)

Cisplatin

DMY

Cisplatin+DMY

Control

(c)

Figure 1 Dihydromyricetin protected against cisplatin-induced general toxicity and nephrotoxicity in vivo (a and b) BUN and SCrmeasurement (c) Periodic acid-Schiff (PAS) staining Kidney sections from the cisplatin-treated group showed severe cast formation andsevere tubular injury Cisplatin with DMY (500mgkg) showed less tubular destruction and mild casts (indicated with arrows) Data arepresented as mean plusmn SD lowastlowast119875 lt 001 versus cisplatin treatment group lowast119875 lt 005 versus cisplatin treatment group and

119875 lt 001 versusnondrug treatment group

apoptosis we measured the expression of the proapoptoticfactor Bax and the antiapoptotic factor Bcl-2 Western blotanalysis indicated that the decreased expression of Bcl-2caused by cisplatin was partially upregulated by the pretreat-ment of DMY and Bax expression was downregulated in theDMY coadministration group (Figures 3(c) and 3(d))

33 Effect of Dihydromyricetin on Oxidative Stress in KidneyTissues Formany years oxidative stress has been recognizedas one of the central players in the pathophysiology ofcisplatin-induced acute kidney injury (AKI) [15 18] To inves-tigate the protectivemechanism ofDMYon cisplatin nephro-toxicity we next measured the level of several oxidative stressmarkers in nephridium Three days after cisplatin treatmenta significant increase in tissue MDA level and decrease ofCAT and SOD activities occurred compared with the control

group while pretreatment of DMY partially blocked theincrease of MDA level and attenuated the reduction of theseantioxidant levels (Table 2) Moreover we also assessed 8-OHdG a commonly used marker for the evaluation of DNAoxidization to confirm the free radical-induced oxidativelesions in each group As shown in Figure 4 the numbersof 8-OHdG-positive cells were much greater in the cisplatin-treated group when compared to the control mice Also asignificant decrease of the positive cell numbers was observedin the cisplatin with Dihydromyricetin group

34 Effect of Dihydromyricetin on Proinflammatory Media-tors in Kidney Tissues Although many studies indicate thatoxidative stress plays critical roles in the pathogenesis ofcisplatin-induced development of kidney damage [14 19]AKI caused by cisplatin is also associated with a severe


Table 2 Effect of Dihydromyricetin on oxidative stress in different experimental groups

Groups MDA (120583Mg tissue) SOD (Umg protein) CAT (Umg protein)Normal 421 plusmn 160 2127 plusmn 199 997 plusmn 147

DMY 500 415 plusmn 107 2378 plusmn 382 955 plusmn 215

Cis 2457 plusmn 473lowastlowast

880 plusmn 248lowastlowast


Cis+DMY 500 1520 plusmn 377

1648 plusmn 159

603 plusmn 115

Cis cisplatin DMY 500 DMY 500mgkg Cis+DMY 500 cisplatin+DMY 500mgkg Data is expressed as mean plusmn SD of 4 mice per group MDAmalondialdehyde SOD superoxide dismutase CAT catalase activity lowastlowast119875 lt 001 versus normal group 119875 lt 001 versus cisplatin treatment group 119875 lt 005versus cisplatin treatment group

Cisplatin

DMY

Cisplatin+DMY

Control

(a)

Num

ber o

f TU

NEL

(pos

itive

cells

fiel

d)

60

40

20

0

lowast

CisplatinDMY (mgkg)

+ +

_

_ _

_ 500500

(b)

Figure 2 TUNEL-positive that is apoptotic nuclei were stained with green spot (a) TUNEL staining demonstrated cisplatin-induced cellapoptosis in the nondrug treatment group DMY treatment group cisplatin treatment group and cisplatin with DMY (500mgkg) treatmentgroup Scale bar 50120583m (b)The TUNEL-positive cells were quantified using Imagine J software at 200x magnification Data are presented asmean plusmn SD lowast119875 lt 005 versus cisplatin treatment group

inflammatory response [20 21] To measure the potentialinflammatory mechanisms we measured the semiquantita-tive score of PMN infiltration by HE staining The cisplatin-treated group had greater PMN infiltration (score 325plusmn025versus 250 plusmn 019) when compared with the DMY coad-ministration group (Figures 5(a) and 5(b)) FurthermoremRNA expression of several proinflammatory cytokineswas also examined (Figure 5(c)) The quantitative real-timePCR results showed that gene expression of IL-1120573 IL-6TNF-120572 and MCP-1 was significantly higher after cisplatinadministration and this effect was partially mitigated by thepretreatment with DMY however the lower gene expressionlevels of IL-6 showed no significant differences when com-pared with the cisplatin group

4 Discussion

As one of the major antineoplastic drugs for the treatmentof solid tumors cisplatin has been used for various types of

cancers since it was approved for the treatment of testicularand ovarian cancers in 1978 However in the past 30 yearsthe application of cisplatin has been seriously limited due toits serious side effect of dose-dependent renal toxicity [22]Nearly 20 of the patients who received high-dose cisplatintreatment developed severe renal insufficiency Until nowthere was no specific treatment for cisplatin-induced renaldysfunction or injury It is generally believed that cisplatinbinding to DNA can not only cause cytotoxicity of tumorsbut also damage other proliferating cells and nephrotoxicityprimarily occurs in proximal tubule epithelial cells

As a traditional Chinese medicine DMY is the primarynatural product extracted from ampelopsis grossedentataand has long been credited with scavenging free radicalprotecting liver and preventing some cancers In the cur-rent study we explored the antioxidation effect of DMYon cisplatin-induced renal toxicity and found that it canperform protective roles both in vitro and in vivo Usingthe mouse model of cisplatin-induced kidney lesion DMY


Inhi

bitio

n ra

tio (

)

0

25

50

75

100

lowastlowast

0 10 20 30 50Cisplatin (120583M)

DMY 0120583MDMY 50120583M

(a)

Control DMY

Cisplatin Cisplatin+DMY

(b)

Bcl-2

Bax

GAPDH

Control Cisplatin Cisplatin+DMY

(c)

0

05

10

15

Bcl-2

GA

PDH

Bax

GA

PDH

0

1

2

3

4

lowast lowastlowast

CisplatinDMY

++

+

___

CisplatinDMY

++

+

___

(d)

Figure 3Dihydromyricetin reduced cell apoptosis caused by cisplatin inHK-2 cells Inhibition rate (a) and cell apoptosis (b) were determinedby WST-1 and DAPI staining (indicated with arrows scale bar 50 120583m) after HK-2 cells were treated with DMY (pretreated 24 h earlier thancisplatin) and cisplatin (c) HK-2 cells were pretreated with DMY (50120583M) for 24 h followed by cisplatin treatment for an additional 24 h thenthe Bcl-2 Bax and GAPDH protein expression was analyzed by Western Blot (d) Protein bands were quantitated by densitometric analysisData are presented as the mean plusmn SD lowastlowast119875 lt 001 versus cisplatin treatment group lowast119875 lt 005 versus cisplatin treatment group and

119875 lt 001

versus nondrug treatment group


Control

Cisplatin

DMY

Cisplatin+DMY

(a)

Num

ber o

f 8-O

HdG

(pos

itive

cells

fiel

d)

40

30

20

10

0

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

lowast

(b)

Figure 4 Dihydromyricetin attenuates cisplatin-induced oxidative stress (a) Immunohistochemical staining was performed for 8-OHdGScale bar 50 120583m 8-OHdG-positive nuclei are stained with dense brown spots (b) The 8-OHdG-positive cells were quantified using ImagineJ software at 400x magnification Data are presented as mean plusmn SD lowast119875 lt 005 versus cisplatin treatment group

could effectively ameliorate the renal functional impairmentand structural damage as well as improving the animalsrsquosurvival rate In addition it mediated a protective effectagainst cisplatin-induced cell apoptosis in HK-2 cells andupregulated antiapoptotic Bcl-2 levels together with down-regulated proapoptotic Bax levels Moreover we found thatDMY decreased the MDA level and increased the CATand SOD activities in mouse kidney tissues after cisplatintreatment and also decreased the gene expression of severalproinflammatory cytokineswhich included IL-1120573 IL-6 TNF-120572 and MCP-1 To the best of our knowledge the effect ofDMY on preventing cisplatin-induced nephrotoxicity mighthave potential clinical application for cancer treatment

For over a decade oxidative stress has been regardedas one of the most widely accepted factors that contributeto cisplatin nephrotoxicity Cisplatin treatment can lead toaccumulation of endogenous ROS and oxidative stress withinthe renal tubular cells and kidney slices as well as in vivoin whole animals Meanwhile during cisplatin treatment arobust inflammatory reaction occurs and inflammasomes arealso stimulated further exacerbating renal tissue damage Inour present study we found that the pretreatment of DMYsignificantly attenuated cisplatin-induced nephrotoxicity notonly by its antioxidation effect but also by decreasing theexpressions of several proinflammatory cytokines

Moreover since the key histopathological features incisplatin nephrotoxicity are renal tubular cell injury anddeath the apoptosis of HK-2 cells was also investigated inour study The Bax and Bcl-2 proteins belong to the Bcl-2family which regulate and execute many cellular intrinsicapoptotic pathways and are the best-characterized group of

apoptosis-regulating factors [23] Bcl-2 has been regarded tofunction as an inhibitor of apoptosis while Bax is associ-ated with apoptotic cell death process We found that thetreatment with DMY decreased cisplatin-induced apoptosisand necrosis of HK-2 cells The underlying mechanism wasassociated with the effect of DMY upregulating Bcl-2 levelsand downregulating Bax levels

5 Conclusions

DMY within our range of observed concentrations sig-nificantly ameliorated cisplatin-induced nephrotoxicity viaattenuating cisplatin-induced oxidative stress protectedagainst cisplatin-induced apoptotic cell death and alleviatedinflammatory reactions in mouse kidney tissues Althoughwe studied several underlying mechanisms involved in thisprocess further studies are required to explore the othermechanism(s) of the responses evoked by DMY

Competing Interests

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

Acknowledgments

This work was supported by Taishan Scholar Special Fund-ing of Shandong Province (Grant no 21300084963048) toDongqi Tang


Control

Cisplatin

DMY

Cisplatin+DMY

(a)

4

3

2

1

0Sem

i-qua

ntita

tive s

core

(PM

N in

filtr

atio

n)

lowastlowast

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

(b)

15

10

IL-11205738

0

6

4

2

IL-6

TNF-120572

0

5

MCP-1

Control

DMY

Cisplatin

Cisplatin+DMY

Control

DMY

Cisplatin

Cisplatin+DMY

lowastlowast

lowastlowast

lowast

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

(c)

Figure 5 Dihydromyricetin decreased PMN infiltration and expression of several proinflammatory cytokines (a) Hematoxylin and eosin(HE) staining was performed for the analysis of PMN infiltration Scale bar 50 120583m (b) The semiquantitative score of PMN infiltration wasmeasured according to Section 2 (c) q-PCR analysis of IL-1120573 IL-6 TNF-120572 and MCP-1 mRNA expression in the nondrug treatment groupDMY treatment group cisplatin treatment group and cisplatin with DMY (500mgkg) treatment group (119899 = 5) Data are presented as meanplusmn SD lowastlowast119875 lt 001 versus cisplatin treatment group lowast119875 lt 005 versus cisplatin treatment group


References

[1] N Pabla and Z Dong ldquoCisplatin nephrotoxicity mechanismsand renoprotective strategiesrdquo Kidney International vol 73 no9 pp 994ndash1007 2008

[2] D Wang and S J Lippard ldquoCellular processing of platinumanticancer drugsrdquo Nature Reviews Drug Discovery vol 4 no 4pp 307ndash320 2005

[3] G-S Oh H-J Kim A Shen et al ldquoCisplatin-induced kidneydysfunction and perspectives on improving treatment strate-giesrdquo Electrolyte and Blood Pressure vol 12 no 2 pp 55ndash652014

[4] I Arany and R L Safirstein ldquoCisplatin nephrotoxicityrdquo Semi-nars in Nephrology vol 23 no 5 pp 460ndash464 2003

[5] X Yao K Panichpisal N Kurtzman and K Nugent ldquoCisplatinnephrotoxicity a reviewrdquo The American Journal of the MedicalSciences vol 334 no 2 pp 115ndash124 2007

[6] J Beyer O Rick S Weinknecht D Kingreen K Lenz and WSiegert ldquoNephrotoxicity after high-dose carboplatin etoposideand ifosfamide in germ-cell tumors incidence and implicationsfor hematologic recovery and clinical outcomerdquo Bone MarrowTransplantation vol 20 no 10 pp 813ndash819 1997

[7] J Y Park D Lee H-J Jang et al ldquoProtective effect of Artemisiaasiatica extract and its active compound eupatilin againstcisplatin-induced renal damagerdquo Evidence-Based Complemen-tary and Alternative Medicine vol 2015 Article ID 483980 6pages 2015

[8] T K Hyun S H Eom C Y Yu and T Roitsch ldquoHoveniadulcismdashan Asian traditional herbrdquo Planta Medica vol 76 no10 pp 943ndash949 2010

[9] X Hou Q Tong W Wang W Xiong C Shi and J FangldquoDihydromyricetin protects endothelial cells from hydrogenperoxide-induced oxidative stress damage by regulating mito-chondrial pathwaysrdquo Life Sciences vol 130 Article ID 14330 pp38ndash46 2015

[10] Y-S Zhang Z-X Ning S-Z Yang and H Wu ldquoAntioxidationproperties and mechanism of action of dihydromyricetin fromAmpelopsis grossedentatardquo Yao Xue Xue Bao vol 38 no 4 pp241ndash244 2003

[11] S Wu B Liu Q Zhang et al ldquoDihydromyricetin reduced Bcl-2 expression via p53 in human hepatoma HepG2 cellsrdquo PLoSONE vol 8 no 11 Article ID e76886 2013

[12] J Ye Y Guan S Zeng and D Liu ldquoAmpelopsin preventsapoptosis induced by H

2

O2

in MT-4 lymphocytesrdquo PlantaMedica vol 74 no 3 pp 252ndash257 2008

[13] H Zhu P Luo Y Fu et al ldquoDihydromyricetin preventscardiotoxicity and enhances anticancer activity induced byadriamycinrdquo Oncotarget vol 6 no 5 pp 3254ndash3267 2015

[14] M Liu D N Grigoryev M T Crow et al ldquoTranscriptionfactor Nrf2 is protective during ischemic and nephrotoxic acutekidney injury in micerdquo Kidney International vol 76 no 3 pp277ndash285 2009

[15] W Lieberthal V Triaca and J Levine ldquoMechanisms of deathinduced by cisplatin in proximal tubular epithelial cells apop-tosis vs necrosisrdquo American Journal of Physiology vol 270 no4 pp F700ndashF708 1996

[16] S Li A Basnakian R Bhatt et al ldquoameliorates acute renalfailure by reducing cisplatin-induced increased expression ofrenal endonuclease Grdquo American Journal of PhysiologymdashRenalPhysiology vol 287 pp F990ndashF998 2004

[17] Q Wei G Dong J Franklin and Z Dong ldquoThe pathologicalrole of Bax in cisplatin nephrotoxicityrdquo Kidney Internationalvol 72 no 1 pp 53ndash62 2007

[18] R Baliga N Ueda P D Walker and S V Shah ldquoOxidantmechanisms in toxic acute renal failurerdquo Drug MetabolismReviews vol 31 no 4 pp 971ndash997 1999

[19] H S Lee B K Kim Y Nam et al ldquoProtective role ofphosphatidylcholine against cisplatin-induced renal toxicityand oxidative stress in ratsrdquo Food and Chemical Toxicology vol58 pp 388ndash393 2013

[20] R P Miller R K Tadagavadi G Ramesh and W B ReevesldquoMechanisms of cisplatin nephrotoxicityrdquo Toxins vol 2 no 11pp 2490ndash2518 2010

[21] T Kimura T Nojiri H Hosoda et al ldquoProtective effects of C-type natriuretic peptide on cisplatin-induced nephrotoxicity inMicerdquo Cancer Chemotherapy and Pharmacology vol 75 no 5pp 1057ndash1063 2015

[22] P J Loehrer and L H Einhorn ldquoDrugs five years laterCisplatinrdquo Annals of Internal Medicine vol 100 no 5 pp 704ndash713 1984

[23] J-Y Xu Q-H Meng Y Chong et al ldquoSanguinarine inhibitsgrowth of human cervical cancer cells through the inductionof apoptosisrdquo Oncology Reports vol 28 no 6 pp 2264ndash22702012

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and blood urea nitrogen (BUN) levels and histological exam-ination using periodic acid-Schiff (PAS) staining We foundthat DMY decreased the malondialdehyde (MDA) level andincreased the catalase activity (CAT) and superoxide dismu-tase (SOD) activities in mouse kidney tissues after treatmentwith cisplatin for 3 days We also observed decreased mRNAexpression of several proinflammatory cytokines MoreoverDMY mediated a protective effect against cisplatin-inducednephrotoxicity by ameliorating apoptotic cell death in HK-2cells

2 Materials and Methods

21 Animals Adult male C57BL6J mice (6ndash8 weeks old 20ndash25 g body weight) were used in this study These mice werepurchased fromShanghai SLACLaboratoryAnimal Co Ltdand housed at 22 plusmn 2∘C and 60 plusmn 5 humidity on a 10ndash12 h light-dark cycle under a pathogen-free condition Allanimals had free access to sterile tap water and food duringthe experiments All of the animal experimental protocolswere performed in accordance with the NIH Guide for Careand Use of Laboratory Animals and were approved by theInstitutional Animal Care and Use Committee at ShandongUniversity China

22 Cisplatin-Induced Nephrotoxicity DMY (Aladdin) wassuspended in sesame oil and administrated by gavage toC57BL6J mice Cisplatin (Sigma-Aldrich) was dissolvedin 09 saline at a concentration of 2mgmL and micewere given a single intraperitoneal injection The mice wererandomly divided into a control group 30mgkg cisplatin-treated group 30mgkg cisplatin and 100mgkg DMY-treated group 30mgkg cisplatin and 300mgkg DMY-treated group and 30mgkg cisplatin and 500mgkg DMY-treated group The DMY treatment was started 4 days priorto a single ip injection of cisplatin and continued for anadditional 3 days

23 Sample Collection Micewere anesthetized and sacrificed3 days after cisplatin injection Blood samples were collectedthrough tail vein and serum was separated and used forthe assessment of serum creatinine concentration (SCr) andblood urea nitrogen (BUN) Next hearts were cannulatedwith 09 NaCl at 90mmHg via the left ventricular apexto clear the kidneys until being whitish Both kidneys wereremoved and stored in liquid nitrogen or fixed in 4paraformaldehyde and embedded in paraffin for histologicalanalysis

24 Assessment of Renal Function Blood samples were trans-ferred to 15mL tubes and allowed to clot Then serumwas separated by centrifugation at 4000 rpm for 15min SCrand BUN were measured using a Beckman Coulter Dxc800biochemical analyzer (Beckman Coulter)

25 Evaluation of Histology and ImmunohistochemistryFormalin-fixed and paraffin-embedded kidney tissues weresectioned at 4 120583mand stainedwith periodic acid-Schiff (PAS)

for histological examination Apoptotic cells were detectedusing the TdT-mediated dUTP nick-end labeling (TUNEL)assay using an in situ apoptosis detection kit (Roche AppliedScience) according to the manufacturerrsquos protocol Immuno-histochemical staining for 8-hydroxydeoxyguanosine (8-OHdG) was performed using anti-8-OHdG antibody (SantaCruz Biotechnology) In the evaluation of hematoxylin andeosin (HE) stained sections interstitial polymorphonuclearleukocyte (PMN) infiltration was scored by a renal pathol-ogist who was blinded to the experimental groups PMNinfiltration per high power (times200) field was graded semi-quantitatively according to the following schema 0 0-1 1 2ndash10 2 11ndash20 3 21ndash40 4 gt40 or too many to count [14]

26 Measurement of Oxidative Stress Markers Oxidativestress markers were measured in kidney tissues of the miceKidney tissues were homogenized in lysis buffer and thesupernatant was used for the measurement of malondialde-hyde (MDA) superoxide dismutase (SOD) and catalaseactivity (CAT)

Determination of kidney tissue MDA concentrationwas measured using a Lipid Peroxidation MDA Assay kit(Beyotime Biotechnology) The concentration of MDA wasdetected by reacting with thiobarbituric acid (TBA) Briefly01mL of the kidney tissue supernatant was mixed with200120583LMDAworking solution heated in a heat block (100∘C)for 15min and then cooled down to room temperatureAfter centrifugation at 1000 g for 10min the supernatant wasmeasured at a wavelength of 532 nm

The assay for SOD activity was based on the abilityof SOD to inhibit nitroblue tetrazolium (NBT) reductionby superoxide Briefly 20 120583L of the tissue supernatant wasmixed with 160 120583L NBT working solution and 20 120583L reactingsolution and then incubated in 37∘C for 30min Finally theoptical density was measured at a wavelength of 560 nm

The catalase activity was detected using a Catalase Anal-ysis Kit (Beyotime Biotechnology) according to the man-ufacturerrsquos instructions Briefly the tissue supernatant wastreated with excess hydrogen peroxide for decompositionfor an indicated time The remaining hydrogen peroxidecoupled with a substrate was treated with peroxidase toproduce N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquino-nemonoimine which absorbs maximally at a wavelength of520 nm

27 Cell Culture HK-2 human kidney proximal tubular cellswere purchased from American Type Culture Collectionand cultured in renal epithelial basal medium (Gibco) withmanufacturer provided supplements in an incubator with 5CO2at 37∘C

28 Cell Viability Assay The cytotoxicity of cisplatin wasdetermined by the water-soluble tetrazolium- (WST-) 1method using the WST-1 cell proliferation and cytotoxic-ity assay kit (Beyotime Biotechnology) Briefly HK-2 wereseeded in 96-well plates at 5000 cellswell and incubated at37∘C for 24 h The media were replaced with normal mediain the absence or presence of DMY (25 or 50120583M) After










3 Results






200

150

100

50

0

BUN

(mg

dL)

CisplatinDMY (mgkg)

_ + +

_

+

_ 300 500+

100

lowastlowast

lowastlowast

(a)

20

15

10

05

00

Seru

m cr

eatin

ine (

mg

dL)

_ _ 300 500100Cisplatin

DMY (mgkg)_ + + ++

lowastlowast

lowastlowast

(b)

Cisplatin

DMY

Cisplatin+DMY

Control

(c)















1648 plusmn 159

603 plusmn 115


Cisplatin

DMY

Cisplatin+DMY

Control

(a)

Num

ber o

f TU

NEL

(pos

itive

cells

fiel

d)

60

40

20

0

lowast

CisplatinDMY (mgkg)

+ +

_

_ _

_ 500500

(b)



4 Discussion





Inhi

bitio

n ra

tio (

)

0

25

50

75

100

lowastlowast

0 10 20 30 50Cisplatin (120583M)

DMY 0120583MDMY 50120583M

(a)

Control DMY


(b)

Bcl-2

Bax

GAPDH


(c)

0

05

10

15

Bcl-2

GA

PDH

Bax

GA

PDH

0

1

2

3

4

lowast lowastlowast

CisplatinDMY

++

+

___

CisplatinDMY

++

+

___

(d)


119875 lt 001



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

Num

ber o

f 8-O

HdG

(pos

itive

cells

fiel

d)

40

30

20

10

0

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

lowast

(b)






5 Conclusions


Competing Interests


Acknowledgments



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

4

3

2

1

0Sem

i-qua

ntita

tive s

core

(PM

N in

filtr

atio

n)

lowastlowast

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

(b)

15

10

IL-11205738

0

6

4

2

IL-6

TNF-120572

0

5

MCP-1

Control

DMY

Cisplatin

Cisplatin+DMY

Control

DMY

Cisplatin

Cisplatin+DMY

lowastlowast

lowastlowast

lowast

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

(c)



References













2

O2


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3 Results






200

150

100

50

0

BUN

(mg

dL)

CisplatinDMY (mgkg)

_ + +

_

+

_ 300 500+

100

lowastlowast

lowastlowast

(a)

20

15

10

05

00

Seru

m cr

eatin

ine (

mg

dL)

_ _ 300 500100Cisplatin

DMY (mgkg)_ + + ++

lowastlowast

lowastlowast

(b)

Cisplatin

DMY

Cisplatin+DMY

Control

(c)















1648 plusmn 159

603 plusmn 115


Cisplatin

DMY

Cisplatin+DMY

Control

(a)

Num

ber o

f TU

NEL

(pos

itive

cells

fiel

d)

60

40

20

0

lowast

CisplatinDMY (mgkg)

+ +

_

_ _

_ 500500

(b)



4 Discussion





Inhi

bitio

n ra

tio (

)

0

25

50

75

100

lowastlowast

0 10 20 30 50Cisplatin (120583M)

DMY 0120583MDMY 50120583M

(a)

Control DMY


(b)

Bcl-2

Bax

GAPDH


(c)

0

05

10

15

Bcl-2

GA

PDH

Bax

GA

PDH

0

1

2

3

4

lowast lowastlowast

CisplatinDMY

++

+

___

CisplatinDMY

++

+

___

(d)


119875 lt 001



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

Num

ber o

f 8-O

HdG

(pos

itive

cells

fiel

d)

40

30

20

10

0

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

lowast

(b)






5 Conclusions


Competing Interests


Acknowledgments



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

4

3

2

1

0Sem

i-qua

ntita

tive s

core

(PM

N in

filtr

atio

n)

lowastlowast

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

(b)

15

10

IL-11205738

0

6

4

2

IL-6

TNF-120572

0

5

MCP-1

Control

DMY

Cisplatin

Cisplatin+DMY

Control

DMY

Cisplatin

Cisplatin+DMY

lowastlowast

lowastlowast

lowast

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

(c)



References













2

O2


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















200

150

100

50

0

BUN

(mg

dL)

CisplatinDMY (mgkg)

_ + +

_

+

_ 300 500+

100

lowastlowast

lowastlowast

(a)

20

15

10

05

00

Seru

m cr

eatin

ine (

mg

dL)

_ _ 300 500100Cisplatin

DMY (mgkg)_ + + ++

lowastlowast

lowastlowast

(b)

Cisplatin

DMY

Cisplatin+DMY

Control

(c)















1648 plusmn 159

603 plusmn 115


Cisplatin

DMY

Cisplatin+DMY

Control

(a)

Num

ber o

f TU

NEL

(pos

itive

cells

fiel

d)

60

40

20

0

lowast

CisplatinDMY (mgkg)

+ +

_

_ _

_ 500500

(b)



4 Discussion





Inhi

bitio

n ra

tio (

)

0

25

50

75

100

lowastlowast

0 10 20 30 50Cisplatin (120583M)

DMY 0120583MDMY 50120583M

(a)

Control DMY


(b)

Bcl-2

Bax

GAPDH


(c)

0

05

10

15

Bcl-2

GA

PDH

Bax

GA

PDH

0

1

2

3

4

lowast lowastlowast

CisplatinDMY

++

+

___

CisplatinDMY

++

+

___

(d)


119875 lt 001



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

Num

ber o

f 8-O

HdG

(pos

itive

cells

fiel

d)

40

30

20

10

0

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

lowast

(b)






5 Conclusions


Competing Interests


Acknowledgments



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

4

3

2

1

0Sem

i-qua

ntita

tive s

core

(PM

N in

filtr

atio

n)

lowastlowast

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

(b)

15

10

IL-11205738

0

6

4

2

IL-6

TNF-120572

0

5

MCP-1

Control

DMY

Cisplatin

Cisplatin+DMY

Control

DMY

Cisplatin

Cisplatin+DMY

lowastlowast

lowastlowast

lowast

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

(c)



References













2

O2


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























1648 plusmn 159

603 plusmn 115


Cisplatin

DMY

Cisplatin+DMY

Control

(a)

Num

ber o

f TU

NEL

(pos

itive

cells

fiel

d)

60

40

20

0

lowast

CisplatinDMY (mgkg)

+ +

_

_ _

_ 500500

(b)



4 Discussion





Inhi

bitio

n ra

tio (

)

0

25

50

75

100

lowastlowast

0 10 20 30 50Cisplatin (120583M)

DMY 0120583MDMY 50120583M

(a)

Control DMY


(b)

Bcl-2

Bax

GAPDH


(c)

0

05

10

15

Bcl-2

GA

PDH

Bax

GA

PDH

0

1

2

3

4

lowast lowastlowast

CisplatinDMY

++

+

___

CisplatinDMY

++

+

___

(d)


119875 lt 001



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

Num

ber o

f 8-O

HdG

(pos

itive

cells

fiel

d)

40

30

20

10

0

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

lowast

(b)






5 Conclusions


Competing Interests


Acknowledgments



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

4

3

2

1

0Sem

i-qua

ntita

tive s

core

(PM

N in

filtr

atio

n)

lowastlowast

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

(b)

15

10

IL-11205738

0

6

4

2

IL-6

TNF-120572

0

5

MCP-1

Control

DMY

Cisplatin

Cisplatin+DMY

Control

DMY

Cisplatin

Cisplatin+DMY

lowastlowast

lowastlowast

lowast

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

(c)



References













2

O2


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Inhi

bitio

n ra

tio (

)

0

25

50

75

100

lowastlowast

0 10 20 30 50Cisplatin (120583M)

DMY 0120583MDMY 50120583M

(a)

Control DMY


(b)

Bcl-2

Bax

GAPDH


(c)

0

05

10

15

Bcl-2

GA

PDH

Bax

GA

PDH

0

1

2

3

4

lowast lowastlowast

CisplatinDMY

++

+

___

CisplatinDMY

++

+

___

(d)


119875 lt 001



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

Num

ber o

f 8-O

HdG

(pos

itive

cells

fiel

d)

40

30

20

10

0

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

lowast

(b)






5 Conclusions


Competing Interests


Acknowledgments



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

4

3

2

1

0Sem

i-qua

ntita

tive s

core

(PM

N in

filtr

atio

n)

lowastlowast

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

(b)

15

10

IL-11205738

0

6

4

2

IL-6

TNF-120572

0

5

MCP-1

Control

DMY

Cisplatin

Cisplatin+DMY

Control

DMY

Cisplatin

Cisplatin+DMY

lowastlowast

lowastlowast

lowast

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

(c)



References













2

O2


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

Cisplatin

DMY

Cisplatin+DMY

(a)

Num

ber o

f 8-O

HdG

(pos

itive

cells

fiel

d)

40

30

20

10

0

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

lowast

(b)






5 Conclusions


Competing Interests


Acknowledgments



Control

Cisplatin

DMY

Cisplatin+DMY

(a)

4

3

2

1

0Sem

i-qua

ntita

tive s

core

(PM

N in

filtr

atio

n)

lowastlowast

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

(b)

15

10

IL-11205738

0

6

4

2

IL-6

TNF-120572

0

5

MCP-1

Control

DMY

Cisplatin

Cisplatin+DMY

Control

DMY

Cisplatin

Cisplatin+DMY

lowastlowast

lowastlowast

lowast

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

(c)



References













2

O2


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

Cisplatin

DMY

Cisplatin+DMY

(a)

4

3

2

1

0Sem

i-qua

ntita

tive s

core

(PM

N in

filtr

atio

n)

lowastlowast

CisplatinDMY (mgkg)

+ +

__ _

_ 500500

(b)

15

10

IL-11205738

0

6

4

2

IL-6

TNF-120572

0

5

MCP-1

Control

DMY

Cisplatin

Cisplatin+DMY

Control

DMY

Cisplatin

Cisplatin+DMY

lowastlowast

lowastlowast

lowast

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

8

0

6

4

2

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

Relat

ive m

RNA

expr

essio

n(ta

rget

gen

e120573

-act

in)

(c)



References













2

O2


















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References













2

O2


















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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