evidence-based complementary and...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 789263 12 pageshttpdxdoiorg1011552013789263

Research ArticleChimonanthus nitens var salicifolius AqueousExtract Protects against 5-Fluorouracil InducedGastrointestinal Mucositis in a Mouse Model

Zhenze Liu12 Jun Xi3 Sven Schroumlder4 Weigang Wang3 Tianpei Xie5

Zhugang Wang3 Shisan Bao26 and Jian Fei123

1 School of Life Science and Technology Tongji University Shanghai 200092 China2The Sino-Australia Joint Laboratory Lishui Institute of Traditional Chinese Medicine Tongji UniversityLishui 323000 China

3 Shanghai Research Centre for Model Organisms Shanghai 201203 China4HanseMerkur Centre for Traditional Chinese Medicine at the University Medical Centre Hamburg-EppendorfHaus Ost 55 UKE Campus Martinistraszlige 52 20246 Hamburg Germany

5 Shanghai Standard Biotech Co Ltd Shanghai 201203 China6Discipline of Pathology Bosch Institute and School of Medical Sciences University of Sydney NSW 2006 Australia

Correspondence should be addressed to Shisan Bao bobbaosydneyeduau and Jian Fei jfeitongjieducn

Received 19 June 2013 Revised 8 September 2013 Accepted 16 September 2013

Academic Editor Lorenzo Cohen

Copyright copy 2013 Zhenze Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Gastrointestinal mucositis is a major side effect of chemotherapy leading to life quality reduction in patients and interruptingthe therapy of cancer Chimonanthus nitens var salicifolius (CS) is a traditional Chinese herb for enteral disease Considering theprotective effect of CS on intestine we hypothesize that the aqueous extract of CS could be benefcial to gastrointestinalmucositis Toverify this a mousemucositis model was induced by 5-Fluorouracil (5-Fu) Male BalbCmice were treated with CS aqueous extract(5 10 and 20 gkg) or loperamide (02mgkg) intragastrically for 11 days and the severity of mucositis was evaluated Furthermorethe chemical compounds of CS aqueous extract were also analysed by high-performance liquid chromatography (HPLC) Ourresults demonstrated that CS aqueous extract improved mice body weight diarrhoea and faecal blood maintained the liverfunction and intestinal length alleviated villus shortening and suppressed the apoptosis and inflammation in small intestine Weconcluded that CS could protect mice against 5-Fu inducedmucositis by inhibiting apoptosis and inflammation and this protectiveeffect might be associated with the 3 flavonoids (rutin quercetin and kaempferol) identified in CS aqueous extract

1 Introduction

Chimonanthus nitens var salicifolius (also named Chimonan-thus salicifolius abbreviation CS) belongs to Calycanthaceaefamily and is considered as a unique plant species in ChinaThis plant is generally distributed in the mountain areas ofsoutheast China and it is a semievergreen shrub with solitaryand small yellowish flowers The leaf blade of CS is linear-lanceolate or oblong-lanceolate and has bristle on veins andmargin [1] It is reported that leaves of CS are rich in proteincrude fat fibers minerals vitamin B

2 and C [2] Tradition-

ally the leaves and branchlets of CS are considered as herbs

for treating common cold influenza dyspepsia gastritisenteral disease and insects bites in part of China [3 4] Dryleaves of CS are also boiled as tea by the indigenous peopleand they are thought to have a protective effect on gastroin-testinal track [4]

Most chemotherapeutic agents target rapidly dividingcells indiscriminately either malignant or healthy includingintestinal basal stem cells in crypts [5 6] Mucositis is theresultant damage to gut induced by chemotherapy and thisside effect limits its clinical applications [7] Patients withgastrointestinal mucositis usually show abnormal pain mal-absorption and a range of gastrointestinal symptoms like

2 Evidence-Based Complementary and Alternative Medicine

vomiting diarrhoea ulceration and bleeding [8ndash11] Gas-trointestinal mucositis has been reported to occur in 40 ofpatients receiving standard dose and almost in all patientsreceiving high dose chemotherapy [12] thus represents asignificant clinical and economic burden in oncology [13]

The chemotherapy induced gastrointestinal mucositisinvolves not only the direct injury to intestinal basal stemcells but also a consequence of complex biological eventssuch as reactive oxygen species (ROS) generation immunecells infiltration and proinflammatory cytokines oversecre-tion [14] Therefore drugs with antiapoptosis antioxidativeor anti-inflammatory properties were thought to benefitmucositis patients [15] Over these few decades a variety ofstrategies have been developed for mucositis cure howeverfew of these approaches have shown sufficient safety andefficacy [16] With a growing interest in nature medicineherb is being adopted as a potential strategy for treatingthis adverse effect of chemotherapy [17 18] More recentlyCS has been proved to have an antidiarrhoea effect inmice [19] However its function in chemotherapy inducedgastrointestinal mucositis is unknown

In the current study we utilized 5-fluorouracil (5-Fu) acommonly used chemotherapeutic agent to induce intestinalinjury in BalbC mice which mimics human chemotherapyinduced mucositis Three different doses of CS aqueousextract were administered to mice for 11 days and theirpossible effect was examined by clinical symptoms bloodbiochemical tests intestinal histopathology and expressionanalysis of genes relevant to apoptosis proliferation andinflammation In addition loperamide which is used formanaging chemotherapy induced mild-moderate diarrhoea[20 21] was chosen as a symptomatic drug to compare theefficacy with CS aqueous extract Such data may provide use-ful information for the prevention or treatment of mucositisin patients undergoing chemotherapy

2 Materials and Methods

21 Animals Adult (9 weeks) male BALBc mice obtainedfrom Shanghai SLACLaboratory Animal Co Ltd (ShanghaiChina) were housed under specific pathogen-free environ-ment with food and water ad libitum The environmentwas maintained at 22∘C with a 12 h lightdark cycle Animalwelfare and experimental procedures were carried out strictlyin accordancewith the guidance for care anduse of laboratoryanimals (National Research Council of USA 1996) andapproved by Institutional Animal Care and Use Committeeof Shanghai Research Centre for Model Organisms

22 Preparation of CS Aqueous Extract CS was obtainedfrom Tongji University Lishui Institute of Traditional ChineseMedicine (Lishui Zhejiang China) In brief fresh leaves ofCS were crushed and dehydrated at room temperature withconstant aeration Dry material (100 g) was macerated inwater (06 L) for 2 h and boiled for 10min Subsequentlythe aqueous phase was harvested filtered and centrifugedsupernatant was collected and freeze dried using a freezedryer (Heto Powerdry PL 3000 Thermo Thermo FisherScientific USA)Then the extract was redissolved with sterile

water into 3 different concentrations (025 05 and 1 gmL ofcrude drug) Mice were administrated intragastrically withCS aqueous extract twice a day by 10mLkg body weight(equivalent to 5 10 and 20 gkg)

23 High Performance Liquid Chromatography (HPLC) Anal-ysis Main compounds in CS aqueous extract (01 gmL)wereanalysed usingHPLCmethodThe apparatus system (AgilentTechnologies Germany) was equipped with a binary pumpa diode array detector and an autosampler An extend-C18column (150 nm lowast 46mm Agilent Germany) was used forseparation and the temperature was maintained at 25∘C10 120583L of sample was injected into the system and the flowrate was set at 1mLmin The mobile phase consisted ofmethanol (B) and 01 phosphoric acid in water (A) Thelinear gradient elution started at 30 (B) changed to 40(B) after 15min and changed to 60 after 35min UV-Visabsorption spectra were monitored by diode array detectorat 265 nm For analysis the standard substances of rutinquercetin and kaempferol were used (Sigma-Aldrich USA)Compounds in aqueous extract of CS were quantified onthe basis of their peak areas and calibration curves of thecorresponding standard substance

24 Mucositis Inducement and Treatment 5-Fu was pur-chased fromXuDongHaiPu Pharmaceutical Co Ltd (Shang-hai China) Loperamide (fromXirsquoan Jangsen PharmaceuticalLtd China) was employed to compare the efficacy with CSaqueous extract Mucositis in mice was induced by 3-day 5-FU administration (50mgkg intraperitoneally) on days 5 6and 7 while the controls were treatedwith saline Loperamide(01mgkg) or CS aqueous extract (25 5 and 10 gkg) wasgiven intragastrically from day 1 to 11 twice daily at 900 amand 900 pm The control and 5-Fu groups were treated withvehicle only (normal drinking water)

25 Tissue Collection and Mucositis Assessment Diseaseseverity was assessed by monitoring body weight and scoringfor diarrhoea and faecal blood Diarrhoea score was basedon the consistency of stools using the modified parametersas described before [15 22] 0 normal 1 slightly wet 2moderate wet 3 loose 4 watery stool The faecal blood wasmeasured by a commercial testing paper (BASO diagnosticsInc China) with the following scores 0 normal 1 slightbleeding 2 moderate bleeding 3 severe bleeding and 4visible bleeding

Serum was collected on day 11 for blood biochemicalassays The small and large intestines were harvested andmeasured for their lengths immediately followingmice sacri-fice Then small intestines were flushed with cold phosphatebuffered saline Part of small intestine was fixed in 4paraformaldehyde overnight for histological processing asdescribed previously [22] and another part was snap frozenin liquid nitrogen for further analysis

26 Serum Biochemical Assays Serum used for blood bio-chemical assay was prepared and detected immediately usingChemix-180 automated biochemistry analyzer system (Sys-mex Corp Japan) Liver functiondamage following 5-Fu

Evidence-Based Complementary and Alternative Medicine 3

challenge was evaluated by measuring the serum level of ala-nine aminotransferase (ALT) and aspartate aminotransferase(AST) while the renal functiondamage was monitored bythe urea nitrogen (BUN) and creatinine (CRE)

27 Histological Assessment Paraformaldehyde fixed andparaffin embedded small intestine was sectioned at 5 120583m forhaematoxylin and eosin (HampE) staining The morphologicalassessment was performed by measuring the change in smallintestinal villus heights and crypts depths using a lightmicroscope (Nikon 90i Japan) and the image analysis pro-gram of Nikon Nis-element Thirty villuscrypts were mea-sured in longitudinal tissue sections for each mouse and allassessments were performed by an independent pathologistin a blind fashion The shortening of villus was evaluated bythe average villus-to-crypt ratio

28 Immunohistochemistry Immunohistochemical detec-tion of proliferating cell nuclear antigen (PCNA) was per-formed on small intestine sections as described previously[23] In brief the sections were incubated with a rabbit poly-clonal antibody to PCNA (1 100 Abcam biotechnologyChina) overnight at 4∘C After washing slides were stainedwith a biotin-conjugated secondary antibody (1 200 Bey-otime Institute of Biotechnology China) followed by avidin-biotin-peroxidase complex (VECTASTAIN ABC kit Vector)for 1 hour each at 37∘C The diaminobenzidine was used asthe immunodetection substrate Quantification of PCNAimmunohistochemical strain was processed by an ImagePro-Plus program

29 Gene Expression Analysis Total RNA was isolated fromsnap frozen intestine using a TRIzol A+ reagent (TiangenBiotech China) according to the instructions from the man-ufacturer cDNA was prepared by reverse transcription usinga QuantScript RT kit (Tiangen Biotech China) The geneexpressions of caspase3 PCNA cyclin-D1 tumor necrosisfactor alpha (TNF-120572) interleukin 1 beta (IL-1120573) IL-6 and IL-12b were quantified by real-time polymerase chain reaction(PCR) using the SuperReal PreMix Plus kit (TiangenBiotechChina) and a gradient cycler machine (Eppendorf HamburgGermany) The primer sequences were as follows

caspase3 51015840-CTGACTGGAAAGCCGAAACTC-31015840(forward) 51015840-CGACCCGTCCTTTGAATTTCT-31015840(reverse)PCNA 51015840-TTGCACGTATATGCCGAGACC-31015840(forward) 51015840-GGTGAACAGGCTCATTCATCTCT-31015840 (reverse)cyclin-D1 51015840-TGAGCTTGTTCACCAGAAGCAG-31015840 (forward) 51015840-TGAGCTTGTTCACCAGAAGCA-G-31015840 (reverse)TNF-120572 51015840-CCTGTAGCCCACGTCGTAG-31015840 (for-ward) 51015840-GGGAGTAGACAAGGTACAACCC-31015840(reverse)IL-1120573 51015840-GAAATGCCACCTTTTGACAGTG-31015840(forward) 51015840-TGGATGCTCTCATCAGGACAG-31015840(reverse)

(mAU

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2013-04-11 10-25-02043-0301D)DAD1ASig = 3654 Ref = off (D1200CHEMDATALIUYELAMEI

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Figure 1 Fingerprint analysis of aqueous extract of CS HPLC of CSaqueous extract is plotted at 265 nm using an extend-C18 columnand gradient elution with methanol and 01 phosphoric acid Thepeak identifications are rutin (A) quercetin (B) and kaempferol (C)

IL-6 51015840-ACAACCACGGCCTTCCCTACT-31015840 (for-ward) 51015840-GCCATTGCACAACTCTTTTCTCAT-31015840(reverse)IL-12b 51015840-TGGTTTGCCATCGTTTTGCTG-31015840 (for-ward) 51015840-ACAGGTGAGGTTCACTGTTTCT-31015840(reverse)120573-actin 51015840-ATTGCTGACAGGATGCAGAA-31015840(forward) 51015840-GCTGATCCACATCTGCTGGAA-31015840(reverse)

The120573-actin gene is used as the housekeeping gene and thegene expression is presented as 2minusΔΔCT

210 Statistical Analyses All data are showed as mean plusmnSEM Differences between groups were estimated using one-way or two-way analysis of variance (ANOVA) followed bythe Bonferroni posttest analysis Significant differences wereaccepted when 119875 values lt005 The statistical analysis wascalculated and plotted using GraphPad Prism version 40(GraphPad Software Canada)

3 Results

31 Chemical Characterisation of CS Aqueous Extract CSaqueous extract was prepared and used for chemical anal-ysis by HPLC The fingerprint is represented in Figure 1Three flavonoids were identified and quantified Rutin wasthe most abundant (004088) in CS aqueous extractand kaempferol accounted the second major component(002892) Quercetin was also identified (001427)

32 Effect of CS Aqueous Extract on Body Weight Loss Bodyweight is an objective criterion for determining the severityof mucositis [6 15] due to the decrease in food intake andabsorption capability Gradual bodyweight loss was observedin mice with 5-Fu challenge reaching sim18 reduction on day10 (Figure 2(a)) As expected there was no significant bodyweight change in the mock treated animals CS treatmentinhibited 5-Fu induced body weight loss at all doses Signifi-cant improved body weight was detected in a dose dependentmanner at day 10 (CS 5 10 gkg119875 lt 001 20 gkg119875 lt 0005)


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Figure 2 Symptoms develop during mucositis Bodyweight loss (a) diarrhoea (b) and faecal blood score (c) of mice with vehicle orCSloperamide treatment were recorded daily The inset shows data from the day 10 for a better understanding of the relationships amongthese groups Each point or bar represents the mean plusmn SEM (119899 = 8group) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0005

There was no significant difference between 5-Fu and 5-Fu +loperamide treatments (Figure 2(a))

33 Effect of CS Aqueous Extract on Diarrhoea and FaecalBlood Diarrhoea and faecal blood are common gastroin-testinal symptoms of mucositis following chemotherapy [2425] There is no abnormality observed in the control groupHowever 5-Fu challenge for 3 days caused a sudden elevationof diarrhoea score on day 8 and a progressive increase till day10 CS (10 and 20 gkg) and loperamide treatment showed anantidiarrhoea effect at day 8 (diarrhoea score sim25 119875 lt 005)However only treatment with 10 and 20 gkg CS reducedthe symptom of diarrhoea when it became severe at day 10(diarrhoea score sim35119875 lt 005 Figure 2(b)) Consistent withthe 5-Fu induced diarrhoea faecal blood also appeared onday 8 and the faecal blood score worsened daily till sacrifice(Figure 1(C)) Marked improvement was found in 20 gkg CSaqueous extract treatment group at day 9 (119875 lt 001) and day

10 (119875 lt 0005) However such effect was not found in anyother treatments (Figure 2(c))

34 Effect of CS Aqueous Extract on LiverKidney InjuryBlood biochemical assay is an easy way to evaluate anddiagnose the injury in liver and kidney which are organssusceptible to the cytotoxicity of chemotherapeutic agents Todetermine the protection effect of CS aqueous extract against5-Fu caused liver and kidney injury mice blood biochemicalindexes were assayed including AST ALT BUN and CRE 5-Fu challenged mice showed significant increase in AST bysim14-fold (119875 lt 005) and in ALT by 13-fold (119875 lt 005)compared to that of vehicle treated only CS aqueous extractat all doses (CS 10 gkg 119875 lt 005 5 20 gkg 119875 lt 001) andloperamide (119875 lt 001) treatment suppressed the elevation ofserum ALT indicating a protection effect on liver There wasalso an attenuation of AST increase in 20 gkg CS aqueous


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Figure 3 Changes of blood biochemical markers after 5-Fu administration SerumAST (a) ALT (b) BUN (c) and CRE (d) were determinedfor monitoring liver and kidney functions Each bar represents the mean plusmn SEM (119899 = 8group) lowast119875 lt 005 and lowastlowast119875 lt 001

extract treated mice (119875 lt 005) however loperamide did notshow such function (Figures 3(a) and 3(b))

No significant difference in BUN and CRE was foundamong groups (Figures 3(c) and 3(d)) suggesting that thereis no kidney damage following 5-Fu acute challenge

35 Effect of CS Aqueous Extract on Intestinal Injury Theintestine damage induced by chemotherapy is partially man-ifested by the reduction of intestinal length [6 26] Thereforelengths of large and small intestine with different treatmentwere determined The colon length in the 5-Fu treated groupwas significantly shortened by sim25 compared to that of themock treated mice (119875 lt 0005) while the reduction of colonlength was markedly alleviated by treatment with loperamide(119875 lt 0005) or CS aqueous extract at 20 gkg (119875 lt 005)This result suggested that the 5-Fu inducedmucositis in coloncould be prevented by loperamide or CS aqueous extract(Figure 4(a)) Similar to colon there was also a shortening insmall intestine after 5-Fu challenge (119875 lt 005) and 20 gkgCStreatment attenuated the reduction in small intestine lengthsignificantly (119875 lt 001) however loperamide failed to preventthe small intestine shortening (Figure 4(b))

Furthermore to evaluate the 5-Fu induced mucosaldamage at themicroscopic level small intestineHampE sectionswere examined (Figures 5(a) 5(b) 5(c) and 5(d)) Comparedwith the mock treated mice 5-Fu challenge resulted in villusdeformation loss and atrophy Additionally morphologicalanalysis showed that the ratio of villus heights to cryptsdepths shortened by sim50 in 5-Fu group than control mice(119875 lt 0005) and treatmentwithCS aqueous extract at 20 gkgsignificantly inhibited the shortening effect induced by 5-Fu(119875 lt 0005) In contrast loperamide did not prevent theshortening of villus (Figure 5(e))

36 Effect of CS Aqueous Extract on Apoptosis and Prolifer-ation Intestinal basal stem cell is susceptible to the toxicityof chemotherapeutic drugs for its character of rapid turning-over leading to cell apoptosis in gut tissue and resultingin mucositis Caspase3 is a member of caspase family andplays a central role in the execution-phase of cell apoptosis[27]The expression of caspase3 was quantified and real-timePCR results showed that caspase3 RNA level was increasedby 80 in 5-Fu treated group (119875 lt 001) Such inductionwas inhibited by CS aqueous extract treatment at 20 gkg


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Figure 4 Shortening of colon and small intestine after 5-Fu administration 4 days after 5-Fu challenge tissues from different groups werecollected and the shortening of colon (a) and small intestine (b) was detected to evaluate the severity of mucositis Each bar represents themean plusmn SEM (119899 = 8group) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0005

(119875 lt 005) However no significant inhibition was observedin loperamide treated group (Figure 6(a))

Cell proliferation is involved in the recovery stage ofchemotherapy induced mucositis As expected all groupstreated with 5-Fu showed an elevation of PCNA expressionThe RNA level of 5-Fu mice increased by 80 compared tocontrol mice (119875 lt 0005) and treatment with CS aqueousextract at 20 gkg reduced the expression of PCNA comparedto which with 5-Fu treatment only (119875 lt 001) suggestinglower proliferation in small intestine (Figure 6(b)) Similarresult can be observed in cyclin-D1 (Figure 6(c)) Howeverno significant effects were observed on PCNA and cyclin-D1 expressions in loperamide treated small intestine (Figures6(b) and 6(c)) Additionally the density of PCNA labellingwas quantified using image-Pro plus as described previously[28] The data showed that there is a correlation betweenmRNA and protein for PCNA (Figures 6(d) and 6(e)) Thesefindings showed that CS at 20 gkg significantly suppressedcellular proliferation supporting the effect of CS in ourcurrent treatment

37 Effect of CS Aqueous Extract on Intestinal InflammationInflammation is involved in pathogenesis of mucositis andthe proinflammatory cytokines are considered as importantfactors and potential targets for treatment of mucositis [2930] The gene expressions of cytokines TNF-120572 IL-1120573 andIL-12b were quantified As expected there were significantincreases in TNF-120572 by 15- (119875 lt 0005) IL-1120573 by 14- andIL-12b by 18-fold (119875 lt 001) following 5-Fu challengeTreatment with CS aqueous extract suppressed the elevationof cytokines expression especially at dose of 20 gkg (TNF-120572119875 lt 005 IL-1120573 IL-12b 119875 lt 0005) Loperamide only showedsuppression function on IL-1120573 RNA level (119875 lt 005) but

no inhibitory effect was found in TNF-120572 and IL-12b (Figures7(a) 7(b) and 7(c))

4 Discussions

Gastrointestinalmucositis is a common side effect induced byradio- or chemotherapy leading to interruptions and delaysin tumor treatment [31 32] and it is one of the primarydeterminants of morbidity and mortality in oncology [8]Nowadays there are no broadly effective treatments formucositis and the standard managements are limited topain relief antidiarrhoeal medication and infection control[13] However couples of strategies are already undergoinginvestigation such as keratinocyte growth factor (KGF)[26 33 34] insulin-like growth factor-I (IGF-I) [6] IL-11[35] and glutamine [36] Traditional herbal medicines alsoshow a potential application in mucositis treatment andsome of them have been reported to be able to benefit thegastrointestinal mucositis complicated with chemotherapy[37ndash39] In the present study we demonstrated the protectiveeffect of CS on 5-Fu induced mice intestinal injury andanalysed the fingerprint of CS aqueous extract by HPLC

Our result showed that CS attenuated the 5-Fu inducedbody weight loss in a dose dependent manner indicatingimprovements in food intake and nutrition Additionally CSalso alleviated the diarrhoea in mucositis mice Diarrhoeahappened as high as 50sim80 in patients that receivedchemotherapy [25] and might be life threatening when itbecomes severe [40] The chemotherapy induced diarrhoeamight be associated with the altered gut motility impairingwater absorption and intestinal microflora [24 25] Lop-eramide an opioid drug affecting intestinal smooth muscledirectly was commonly used for clinical diarrhoea control


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Figure 5 Histological and morphological change of small intestine Small intestine from control (a) 5-Fu (b) 5-Fu + loperamide (c) and5-Fu + CS aqueous extract at 20 gkg (d) was prepared for HampE sections Morphological assessment was performed using the image analysisprogram of Nikon Nis-element and villus shortening was calculated by the average villus-to-crypts ratio (e) to evaluate the mucosal damageEach bar represents the mean plusmn SEM (119899 = 8group) lowastlowastlowast119875 lt 0005

[24 41] In the present study loperamide showed an antidiar-rhoea effect on moderate symptom at day 8 (score sim25) butfailed to relieve it when severe episode appeared at day 10(scoresim35)This result is consistentwith the reported clinicalobservations [40 42] Bleeding is usually accompanied withdiarrhoea in patients undergoing chemotherapy [24 25] Inour results a marked elevation of faecal blood score wasfound in 5-Fu challengedmice and high dose of CS treatmentalleviated the stool bleeding This finding suggested that CSalso has a capacity for reducing the ulcerative lesions ingastrointestinal track

Intestinal shortening is a phenomenonwhich is describedboth in chemotherapy inducedmucositis [6] and experimen-tal colitismodel [22] suggesting destruction or inflammationin intestineThe current study demonstrates that CS aqueousextract treatment (20 gkg) maintained the length of smalland large intestine against 5-Fu induced shortening whileloperamide only showed a protective effect on colon but noton small intestine Interestingly compared to colon the 5-Fu induced shortening in small intestine is quite small thusmight suggest a lighter injury in small intestine than colonAlthough there is small significance the treatment course


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Figure 6 Changes of cell apoptosis and proliferation in mice small intestine To determine the expression of apoptosis and proliferationrelevant genes expressions of caspase-3 (a) PCNA (b) and cyclin-D1 (c) were quantified by real-time PCRMoreover small intestine sectionswere prepared for immunohistochemical staining of PCNA (d) and quantified for the immunohistochemical density of PCNA positivecells (e) Each bar represents the mean plusmn SEM (119899 = 8group) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0005

was rather short (one week) and there might be other factorsthat contribute to such difference which will be investigatedin our future experiment Furthermore histopathologicalevidence confirmed that there was also a severe mucosaldamage in small intestine manifested by villus deformationloss and atrophy CS aqueous extract treatment at 20 gkgblocked the villus shortening Such phenomenon can befound in other antimucositis biological agents such as IGF-I [6] and KGF [26] suggesting that CS aqueous extractmight stimulate mucosal growth and optimize the digestivefunction during chemotherapy However loperamide failedto attenuate the atrophy of villus in small intestine

The pathogenesis of mucositis is complex and multifac-torial Chemotherapeutic agents could interrupt the turning-over of basal stem cells in crypts and induce apoptosis Ourresults demonstrated a downregulation of apoptotic gene

caspase3 in CS group (20 gkg) compared with mice treatedwith 5-Fu only This data may indicate a prevention effectof CS on apoptosis In addition cell apoptosis is just onecomponent of the pathogenesis of chemotherapy induced gutdamage Ceramide signalling oxidative stress inflammationand cell cycling are also implicated in mucositis [43] Theprocess of mucositis has been described as a 5-phase modelincluding initiation primary damage and message genera-tion signal amplification ulceration and healing phase [1443] The direct injury to DNA and generation of ROS aretwo characterized events in initiation phase [44] Then aseries of transcription factors were prompted to respond tothe primary damage such as nuclear factor 120581B (NF-120581B)Wnt and p53 [16] which trigger a consequence of moleculessuch as proinflammatory cytokines adhesion molecules andcyclooxygenase-2 (Cox-2) [43] TNF-120572 is a crucial cytokine


4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

lowast

lowast

lowastlowast

lowastlowastlowastTNF-120572

(a)

35

30

25

20

15

10

05

00

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-1120573

lowast

lowastlowast lowastlowastlowast

(b)

5

4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-12b

Con

trol

5-F

u

lowast

lowast


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)

Figure 7 Gene expressions of proinflammatory cytokines in small intestine To examine the inflammatory status in small intestines RNAof TNF-120572 (a) IL-1120573 (b) and IL-12b (c) was quantified by real-time PCR Each bar represents the mean plusmn SEM (119899 = 8group) lowast119875 lt 005lowastlowast

119875 lt 001 and lowastlowastlowast119875 lt 0005

involved in the pathogenesis of mucositis which amplifiesthe NF-120581B signal and initiates mitogen activated proteinkinase (MAPK) pathway [44]Thus in line with our findingsCS aqueous extract treatments suppressed the elevationof TNF-120572 induced by 5-Fu challenge Furthermore othercytokines including IL-1120573 and IL-12b were also inhibited byCS treatment suggesting an anti-inflammatory effect of CS

During the stage of healing epithelium cells prolifer-ate differentiate to repair the destruction of mucosa andreestablish the normal local microflora [11 43] Interestinglyelevations of PCNA and cyclin-D1 were observed in all 5-Fu treated groups suggesting a healing process from theexperimental mucositis However the gene expression islower in CS treated groups compared with that of vehicletreated only It remained unclear if the lower level of PCNAand cyclin-D1 in CS treated groups is caused by a directsuppression effect or due to the mild mucositis resulting inless expression of the genes relevant to crypts regeneration

To analyse the active component CS aqueous extractis prepared for HPLC detection Three flavonoids wereidentified (rutin quercetin and kaempferol) and rutin wasthe most abundant (004088) Rutin is considered as apowerful antioxidant with pharmacological benefits includ-ing antitumor anti-inflammatory and antidiarrhoeal effects[45 46] It has been reported to prevent renal inflammationand apoptosis induced by chemotherapeutic agent cisplatin[45] The other two compounds quercetin (001427) andkaempferol (002892) are also described to have protectivefunctions against tumor oxidative stress and inflammation[47 48] In addition kaempferol is reported to enhance theintestinal barrier function by expression of tight junctionproteins [49] This information might be useful to explainthe biochemical mechanism of CS aqueous extract protectingmice from the development of mucositis

CS aqueous extract showed effects on inhibiting apoptosisand inflammation in the current experiment and which


seems to play a ldquoprotectiverdquo role in chemotherapy howeverits influence on the antitumor activity of 5-Fu has not beenmentioned The pharmacokinetics of 5-Fu has been wellreported in both humans and animals [50 51] Furthermoreit was demonstrated that there was potential interactionbetween chemotherapeutic agents and herbs [52] which mayresult in modification of pharmacokinetics of 5-Fu in theanimals with CS treatment The metabolism and clearanceof 5-Fu combined with CS treatment are currently beinginvestigated Additionally a (gastrointestinal) tumor model[53] is also needed to evaluate the protective role of CS ingastrointestinal mucositis and its mitigating effect againstanticancer chemotherapy Furthermore our present workonly identified 3 flavonoids However there are still a varietyof active components present in the extract but withoutrecognization whichmight play an important role in CS pro-tective functions Subsequent studies would further concernin completing the description of various components in CSaqueous extract and determining the functions of individualcompound andmucositis as well as their potential synergisticactivity Thus would provide a better understanding aboutthe mechanism of CS attenuating chemotherapy inducedgastrointestinal mucositis

5 Conclusion

The current investigation indicated that the aqueous extractof CS benefits mice against 5-Fu induced gastrointestinalmucositis attenuating the subsequent body weight loss diar-rhea and faecal blood reducing the hepatic injury andmain-taining both intestinal length and villus structure Howeverloperamide which is a symptomatic drug for managing mild-moderate diarrhoea induced by chemotherapy only displayedpartial protective function Additionally this antimucositisactivity of CS aqueous extract might be due to inhibitingapoptosis and inflammation in small intestine Chemicalanalysis usingHPLC identified threemajor compounds in CSaqueous extract including rutin quercetin and kaempferolThe possible explanation of CS protecting against mucositismight be associated with the characters of these flavonoidssuch as antioxidative anti-inflammatory antiapoptotic andantidiarrhoeal effect Our results suggest that CS is a promis-ing candidate for treatment of gastrointestinal mucositisassociated with chemotherapy

Abbreviations

CS Chimonanthus nitens var salicifolius5-Fu 5-FluorouracilROS Reactive oxygen speciesHPLC High-performance liquid chromatographyALT Alanine aminotransferaseAST Aspartate aminotransferaseBUN Urea nitrogenCRE CreatinineHampE Hematoxylin and eosinPCR Polymerase chain reactionPCNA Proliferating cell nuclear antigen

TNF Tumor necrosis factorIL InterleukinANOVA Analysis of varianceKGF Keratinocyte growth factorIGF-I Insulin-like growth factor-INF-120581B Nuclear factor 120581BCox-2 Cyclooxygenase-2MAPK Mitogen activated protein kinase

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors thank Ms Wenting WU for the technicalassistanceThis work was supported by the Grant from LishuiMunicipality and National Key Project (2010CB9455012010CB912604) and the E-Institutes of Shanghai MunicipalEducation Commission (E03003)

References

[1] C Y Hu andW F Xiao ldquoBiological characteristic sand cultiva-tion techniques of Chimonanthus salicifoliusrdquo Forest by-Productand Speciality in China vol 76 pp 5ndash6 2005

[2] C Y Hu and X Y Tang ldquoAnalysis of nutritious components inleaves of wild Chimonanthus salicifoliusrdquo Journal of HuangshanUniversity vol 9 pp 91ndash93 2007

[3] L YWang Z D Zhang Z R Zou and Z H U D ldquoAdvances ofstudies on chemical composition and pharmacological activityofChimonanthus lindlrdquo LishizhenMedicine andMateriaMedicaResearch vol 23 pp 3103ndash3106 2012

[4] J X Fang andGD Cheng ldquoOn the characters ofChimonanthussalicifolius and its distribution and utilization in huangshanregionrdquo Journal of Anhui Agricultural Sciences vol 37 pp 1530ndash1531 2009

[5] E P Mitchell ldquoGastrointestinal toxicity of chemotherapeuticagentsrdquo Seminars in Oncology vol 33 no 1 pp 106ndash120 2006

[6] J C Cool J L Dyer C J Xian R N Butler M S Geierand G S Howarth ldquoPre-treatment with insulin-like growthfactor-I partially ameliorates 5-fluorouracil-induced intestinalmucositis in ratsrdquo Growth Hormone and IGF Research vol 15no 1 pp 72ndash82 2005

[7] M Davila and R S Bresalier ldquoGastrointestinal complicationsof oncologic therapyrdquo Nature Clinical Practice Gastroenterologyand Hepatology vol 5 no 12 pp 682ndash696 2008

[8] D M K Keefe ldquoGastrointestinal mucositis a new biologicalmodelrdquo Supportive Care in Cancer vol 12 no 1 pp 6ndash9 2004

[9] D M Keefe D E Peterson and M M Schubert ldquoDevelop-ing evidence-based guidelines for management of alimentarymucositis process and pitfallsrdquo Supportive Care in Cancer vol14 no 6 pp 492ndash498 2006

[10] Y Saegusa T Ichikawa T Iwai et al ldquoChanges in the mucusbarrier of the rat during 5-fluorouracil-induced gastrointestinalmucositisrdquo Scandinavian Journal of Gastroenterology vol 43no 1 pp 59ndash65 2008

[11] S T Sonis ldquoThe pathobiology of mucositisrdquo Nature ReviewsCancer vol 4 no 4 pp 277ndash284 2004


[12] D M K Keefe ldquoMucositis guidelines what have they achievedand where to from hererdquo Supportive Care in Cancer vol 14 no6 pp 489ndash491 2006

[13] J M Bowen R J Gibson and D M K Keefe ldquoAnimal modelsof mucositis implications for therapyrdquo Journal of SupportiveOncology vol 9 no 5 pp 161ndash168 2011

[14] S T Sonis ldquoNew thoughts on the initiation of mucositisrdquo OralDiseases vol 16 no 7 pp 597ndash600 2010

[15] T-YHuangH-C Chu Y-L Lin et al ldquoMinocycline attenuates5-fluorouracil-induced small intestinal mucositis in mousemodelrdquo Biochemical and Biophysical Research Communicationsvol 389 no 4 pp 634ndash639 2009

[16] S T Sonis ldquoMucositis the impact biology and therapeuticopportunities of oral mucositisrdquo Oral Oncology vol 45 no 12pp 1015ndash1020 2009

[17] D M Keefe S T Sonis and J M Bowen ldquoEmerging drugs forchemotherapy-inducedmucositisrdquoExpert Opinion on EmergingDrugs vol 13 no 3 pp 511ndash522 2008

[18] W Lam S Bussom F Guan et al ldquoChemotherapy thefour-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicityrdquo Science Translational Medi-cine vol 2 no 45 article 45ra59 2010

[19] H Wen J Fei and Y Tong ldquoA pilot study on the anti-diarrhealeffect of Chimonanthus salicifoliusrdquo Jingxi Journal of TraditionalChinese Medicine vol 43 pp 62ndash63 2012

[20] A Alimonti A Gelibter I Pavese et al ldquoNew approachesto prevent intestinal toxicity of irinotecan-based regimensrdquoCancer Treatment Reviews vol 30 no 6 pp 555ndash562 2004

[21] O Letari C Booth A Bonazzi et al ldquoEfficacy of CR3294 a newbenzamidine derivative in the prevention of 5-fluorouracil-induced gastrointestinal mucositis and diarrhea in micerdquo Can-cer Chemotherapy and Pharmacology vol 66 no 5 pp 819ndash8272010

[22] S Bao E D J Carr Y-H Xu and N H Hunt ldquoGp91 phoxcontributes to the development of experimental inflammatorybowel diseaserdquo Immunology and Cell Biology vol 89 no 8 pp853ndash860 2011

[23] N N Tavakoli A K Harris D R Sullivan B D Hamblyand S Bao ldquoInterferon-gamma deficiency reduces neointimalformation in a model of endoluminal endothelial injury com-bined with atherogenic dietrdquo International Journal of MolecularMedicine vol 30 pp 545ndash552 2012

[24] R J Gibson and D M K Keefe ldquoCancer chemotherapy-induced diarrhoea and constipation mechanisms of damageand prevention strategiesrdquo Supportive Care in Cancer vol 14no 9 pp 890ndash900 2006

[25] R J Gibson and A M Stringer ldquoChemotherapy-induceddiarrhoeardquo Current Opinion in Supportive and Palliative Carevol 3 no 1 pp 31ndash35 2009

[26] C L Farrell J V Bready K L Rex et al ldquoKeratinocyte growthfactor protects mice from chemotherapy and radiation-inducedgastrointestinal injury and mortalityrdquo Cancer Research vol 58no 5 pp 933ndash939 1998

[27] F An B Gong H Wang et al ldquomiR-15b and miR-16 regulateTNF mediated hepatocyte apoptosis via BCL2 in acute liverfailurerdquo Apoptosis vol 17 no 7 pp 702ndash716 2012

[28] Y Xu H Wang S Bao F Tabassam andW Cai ldquoAmeliorationof liver injury by continuously targeted intervention againstTNFRp55 in rats with acute-on-chronic liver failurerdquo PLoS Onevol 8 Article ID e68757 2013

[29] R J Lindsay M S Geier R Yazbeck R N Butler and GS Howarth ldquoOrally administered emu oil decreases acuteinflammation and alters selected small intestinal parameters ina rat model of mucositisrdquo British Journal of Nutrition vol 104no 4 pp 513ndash519 2010

[30] M L P Melo G A C Brito R C Soares et al ldquoRole ofcytokines (TNF-120572 IL-1120573 andKC) in the pathogenesis ofCPT-11-induced intestinalmucositis inmice effect of pentoxifylline andthalidomiderdquo Cancer Chemotherapy and Pharmacology vol 61no 5 pp 775ndash784 2008

[31] N J Nonzee N A Dandade T Markossian et al ldquoEvaluatingthe supportive care costs of severe radiochemotherapy-inducedmucositis and pharyngitis results from a Northwestern univer-sity costs of cancer program pilot study with head and neck andnonsmall cell lung cancer patients who received care at a countyhospital a veterans administration hospital or a comprehensivecancer care centerrdquo Cancer vol 113 no 6 pp 1446ndash1452 2008

[32] K V I Rolston L S Elting S B Cantor and E B RubensteinldquoThe burdens of cancer therapy clinical and economic out-comes of chemotherapy-induced mucositisrdquo Cancer vol 100no 6 pp 1324ndash1326 2004

[33] C L Farrell K L Rex J N Chen et al ldquoThe effects ofkeratinocyte growth factor in preclinical models of mucositisrdquoCell Proliferation vol 35 supplement 1 pp 78ndash85 2002

[34] Y Kilic K Rajewski and W Dorr ldquoEffect of post-exposureadministration of keratinocyte growth factor (Palifermin) onradiation effects in oral mucosa in micerdquo Radiation and Envi-ronmental Biophysics vol 46 no 1 pp 13ndash19 2007

[35] R J Gibson D M K Keefe F M Thompson J M ClarkeG J Goland and A G Cummins ldquoEffect of interleukin-11 onameliorating intestinal damage after methotrexate treatment ofbreast cancer in ratsrdquoDigestive Diseases and Sciences vol 47 no12 pp 2751ndash2757 2002

[36] J Sun H Wang and H Hu ldquoGlutamine for chemotherapyinduced diarrhea a meta-analysisrdquo Asia Pacific Journal ofClinical Nutrition vol 21 pp 380ndash385 2012

[37] M P Farrell and S Kummar ldquoPhase IIIA randomized study ofPHY906 a novel herbal agent as a modulator of chemotherapyin patients with advanced colorectal cancerrdquo Clinical ColorectalCancer vol 2 no 4 pp 253ndash256 2003

[38] J Prasetyo K Naruse T Kato C Boonchird S Harashimaand E Y Park ldquoBioconversion of paper sludge to biofuel bysimultaneous saccharification and fermentation using a cellu-lase of paper sludge origin and thermotolerant saccharomycescerevisiae TJ14rdquoBiotechnology for Biofuels vol 4 article 35 2011

[39] T HWright R Yazbeck K A Lymn et al ldquoThe herbal extractIberogast improves jejunal integrity in rats with 5-Fluorouracil(5-FU)-induced mucositisrdquo Cancer Biology and Therapy vol 8no 10 pp 923ndash929 2009

[40] L B Saltz ldquoUnderstanding and managing chemotherapy-induced diarrheardquoThe Journal of Supportive Oncology vol 1 no1 pp 35ndash38 2003

[41] J Stern andC Ippoliti ldquoManagement of acute cancer treatment-induced diarrheardquo Seminars in Oncology Nursing vol 19 no 3pp 11ndash16 2003

[42] A B Benson III J A Ajani R B Catalano et al ldquoRec-ommended guidelines for the treatment of cancer treatment-induced diarrheardquo Journal of Clinical Oncology vol 22 no 14pp 2918ndash2926 2004

[43] J M Bowen and D M Keefe ldquoNew pathways for alimentarymucositisrdquo Journal of Oncology vol 2008 Article ID 907892 7pages 2008


[44] S T Sonis ldquoRegimen-related gastrointestinal toxicities in can-cer patientsrdquo Current Opinion in Supportive and Palliative Carevol 4 no 1 pp 26ndash30 2010

[45] W Arjumand A Seth and S Sultana ldquoRutin attenuatescisplatin induced renal inflammation and apoptosis by reducingNF120581B TNF-120572 and caspase-3 expression inwistar ratsrdquo Food andChemical Toxicology vol 49 no 9 pp 2013ndash2021 2011

[46] G Gong Y Qin W Huang S Zhou X Yang and D LildquoRutin inhibits hydrogen peroxide-induced apoptosis throughregulating reactive oxygen species mediated mitochondrialdysfunction pathway in humanumbilical vein endothelial cellsrdquoEuropean Journal of Pharmacology vol 628 no 1-3 pp 27ndash352010

[47] J M Calderon-Montano E Burgos-Moron C Perez-Guerreroand M Lopez-Lazaro ldquoA review on the dietary flavonoidkaempferolrdquo Mini Reviews in Medicinal Chemistry vol 11 pp298ndash344 2011

[48] J V Formica ldquoReview of the biology of quercetin and relatedbioflavonoidsrdquo Food and Chemical Toxicology vol 33 no 12 pp1061ndash1080 1995

[49] T Suzuki S Tanabe and H Hara ldquoKaempferol enhancesintestinal barrier function through the cytoskeletal associationand expression of tight junction proteins in Caco-2 cells 1-3rdquoJournal of Nutrition vol 141 no 1 pp 87ndash94 2011

[50] G D Heggie J-P Sommadossi and D S Cross ldquoClinical phar-macokinetics of 5-fluorouracil and its metabolites in plasmaurine and bilerdquo Cancer Research vol 47 no 8 pp 2203ndash22061987

[51] G Milano and A-L Chamorey ldquoClinical pharmacokinetics of5-fluorouracil with consideration of chronopharmacokineticsrdquoChronobiology International vol 19 no 1 pp 177ndash189 2002

[52] K I Block and C Gyllenhaal ldquoClinical corner herb-drug inter-actions in cancer chemotherapy theoretical concerns regardingdrug metabolizing enzymesrdquo Integrative Cancer Therapies vol1 no 1 pp 83ndash89 2002

[53] M M Taketo ldquoMouse models of gastrointestinal tumorsrdquoCancer Science vol 97 no 5 pp 355ndash361 2006

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


vomiting diarrhoea ulceration and bleeding [8ndash11] Gas-trointestinal mucositis has been reported to occur in 40 ofpatients receiving standard dose and almost in all patientsreceiving high dose chemotherapy [12] thus represents asignificant clinical and economic burden in oncology [13]

The chemotherapy induced gastrointestinal mucositisinvolves not only the direct injury to intestinal basal stemcells but also a consequence of complex biological eventssuch as reactive oxygen species (ROS) generation immunecells infiltration and proinflammatory cytokines oversecre-tion [14] Therefore drugs with antiapoptosis antioxidativeor anti-inflammatory properties were thought to benefitmucositis patients [15] Over these few decades a variety ofstrategies have been developed for mucositis cure howeverfew of these approaches have shown sufficient safety andefficacy [16] With a growing interest in nature medicineherb is being adopted as a potential strategy for treatingthis adverse effect of chemotherapy [17 18] More recentlyCS has been proved to have an antidiarrhoea effect inmice [19] However its function in chemotherapy inducedgastrointestinal mucositis is unknown

In the current study we utilized 5-fluorouracil (5-Fu) acommonly used chemotherapeutic agent to induce intestinalinjury in BalbC mice which mimics human chemotherapyinduced mucositis Three different doses of CS aqueousextract were administered to mice for 11 days and theirpossible effect was examined by clinical symptoms bloodbiochemical tests intestinal histopathology and expressionanalysis of genes relevant to apoptosis proliferation andinflammation In addition loperamide which is used formanaging chemotherapy induced mild-moderate diarrhoea[20 21] was chosen as a symptomatic drug to compare theefficacy with CS aqueous extract Such data may provide use-ful information for the prevention or treatment of mucositisin patients undergoing chemotherapy

2 Materials and Methods

21 Animals Adult (9 weeks) male BALBc mice obtainedfrom Shanghai SLACLaboratory Animal Co Ltd (ShanghaiChina) were housed under specific pathogen-free environ-ment with food and water ad libitum The environmentwas maintained at 22∘C with a 12 h lightdark cycle Animalwelfare and experimental procedures were carried out strictlyin accordancewith the guidance for care anduse of laboratoryanimals (National Research Council of USA 1996) andapproved by Institutional Animal Care and Use Committeeof Shanghai Research Centre for Model Organisms

22 Preparation of CS Aqueous Extract CS was obtainedfrom Tongji University Lishui Institute of Traditional ChineseMedicine (Lishui Zhejiang China) In brief fresh leaves ofCS were crushed and dehydrated at room temperature withconstant aeration Dry material (100 g) was macerated inwater (06 L) for 2 h and boiled for 10min Subsequentlythe aqueous phase was harvested filtered and centrifugedsupernatant was collected and freeze dried using a freezedryer (Heto Powerdry PL 3000 Thermo Thermo FisherScientific USA)Then the extract was redissolved with sterile

water into 3 different concentrations (025 05 and 1 gmL ofcrude drug) Mice were administrated intragastrically withCS aqueous extract twice a day by 10mLkg body weight(equivalent to 5 10 and 20 gkg)

23 High Performance Liquid Chromatography (HPLC) Anal-ysis Main compounds in CS aqueous extract (01 gmL)wereanalysed usingHPLCmethodThe apparatus system (AgilentTechnologies Germany) was equipped with a binary pumpa diode array detector and an autosampler An extend-C18column (150 nm lowast 46mm Agilent Germany) was used forseparation and the temperature was maintained at 25∘C10 120583L of sample was injected into the system and the flowrate was set at 1mLmin The mobile phase consisted ofmethanol (B) and 01 phosphoric acid in water (A) Thelinear gradient elution started at 30 (B) changed to 40(B) after 15min and changed to 60 after 35min UV-Visabsorption spectra were monitored by diode array detectorat 265 nm For analysis the standard substances of rutinquercetin and kaempferol were used (Sigma-Aldrich USA)Compounds in aqueous extract of CS were quantified onthe basis of their peak areas and calibration curves of thecorresponding standard substance

24 Mucositis Inducement and Treatment 5-Fu was pur-chased fromXuDongHaiPu Pharmaceutical Co Ltd (Shang-hai China) Loperamide (fromXirsquoan Jangsen PharmaceuticalLtd China) was employed to compare the efficacy with CSaqueous extract Mucositis in mice was induced by 3-day 5-FU administration (50mgkg intraperitoneally) on days 5 6and 7 while the controls were treatedwith saline Loperamide(01mgkg) or CS aqueous extract (25 5 and 10 gkg) wasgiven intragastrically from day 1 to 11 twice daily at 900 amand 900 pm The control and 5-Fu groups were treated withvehicle only (normal drinking water)

25 Tissue Collection and Mucositis Assessment Diseaseseverity was assessed by monitoring body weight and scoringfor diarrhoea and faecal blood Diarrhoea score was basedon the consistency of stools using the modified parametersas described before [15 22] 0 normal 1 slightly wet 2moderate wet 3 loose 4 watery stool The faecal blood wasmeasured by a commercial testing paper (BASO diagnosticsInc China) with the following scores 0 normal 1 slightbleeding 2 moderate bleeding 3 severe bleeding and 4visible bleeding

Serum was collected on day 11 for blood biochemicalassays The small and large intestines were harvested andmeasured for their lengths immediately followingmice sacri-fice Then small intestines were flushed with cold phosphatebuffered saline Part of small intestine was fixed in 4paraformaldehyde overnight for histological processing asdescribed previously [22] and another part was snap frozenin liquid nitrogen for further analysis

26 Serum Biochemical Assays Serum used for blood bio-chemical assay was prepared and detected immediately usingChemix-180 automated biochemistry analyzer system (Sys-mex Corp Japan) Liver functiondamage following 5-Fu







(mAU

)

350300250200150100500

minus5010 20 30 40

Time (min)

10786

14616

16534

18220

22591 26232

32634

(A)

(B)(C)


10786

16534

18220

22591 262322222222222 32(B)





3 Results




Body weightBo

dy w

eigh

t cha

nge (

)

105

100

95

90

85

110

100

90

80

4 5 6 7 8 9 10

Time (day)


lowast

(a)

Diarrhoea

Dia

rrho

ea sc

ore

0

1

2

3

4

0

1

2

3

4

4 5 6 7 8 9 10

Time (day)


lowast

(b)

Faecal blood

Faec

al b

lood

scor

e

0

1

2

3

4

0

1

2

3

4

4 5 6 7 8 9 10

Time (day)




(c)







175

150

125

100

75

50

ASTA

ST (U

L)

lowast lowast

(a)

45

40

35

30

25

20

15

10

ALT

ALT

(UL

)

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

(b)

12

10

8

6

4

2

0

BUN

BUN

(mm

olL

)



(c)

30

25

20

15

10

CRE


CRE

(120583m

olL

)


(d)








90

85

80

75

70

65

60

Colon lengthC

olon

leng

th (c

m)

lowastlowast

lowastlowastlowast

lowastlowastlowastC

ontro

l

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(a)

48

47

46

45

44

43

42

41

40


Smal

l int

estin

e len

gth

(cm

)

Con

trol

5-F

u

5-F

u +

lope

ram

ide

lowast lowastlowast

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)






4 Discussions




(a) (b)

(c) (d)

5

4

3

2

1

0


Villu

s hei

ghts

cryp

ts de

pths

Con

trol

5-F

u


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(e)





Caspase3 R

elat

ive m

RNA

ex

pres

sion

(fold

)25

20

15

10

05

00

Con

trol

5-F

u

lowastlowastlowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(a)

PCNA

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

4

3

2

1

0


Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)

Cyclin-D1

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

32

24

16

08

00

lowastlowastlowast

Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)



(d)

15

10

5

0

Apoptotic index

Apop

totic

inde

x (times

103)

Con

trol

5-F

u

lowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(e)






4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

lowast

lowast

lowastlowast


(a)

35

30

25

20

15

10

05

00

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-1120573

lowast


(b)

5

4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-12b

Con

trol

5-F

u

lowast

lowast


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)









5 Conclusion


Abbreviations





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















(mAU

)

350300250200150100500

minus5010 20 30 40

Time (min)

10786

14616

16534

18220

22591 26232

32634

(A)

(B)(C)


10786

16534

18220

22591 262322222222222 32(B)





3 Results




Body weightBo

dy w

eigh

t cha

nge (

)

105

100

95

90

85

110

100

90

80

4 5 6 7 8 9 10

Time (day)


lowast

(a)

Diarrhoea

Dia

rrho

ea sc

ore

0

1

2

3

4

0

1

2

3

4

4 5 6 7 8 9 10

Time (day)


lowast

(b)

Faecal blood

Faec

al b

lood

scor

e

0

1

2

3

4

0

1

2

3

4

4 5 6 7 8 9 10

Time (day)




(c)







175

150

125

100

75

50

ASTA

ST (U

L)

lowast lowast

(a)

45

40

35

30

25

20

15

10

ALT

ALT

(UL

)

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

(b)

12

10

8

6

4

2

0

BUN

BUN

(mm

olL

)



(c)

30

25

20

15

10

CRE


CRE

(120583m

olL

)


(d)








90

85

80

75

70

65

60

Colon lengthC

olon

leng

th (c

m)

lowastlowast

lowastlowastlowast

lowastlowastlowastC

ontro

l

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(a)

48

47

46

45

44

43

42

41

40


Smal

l int

estin

e len

gth

(cm

)

Con

trol

5-F

u

5-F

u +

lope

ram

ide

lowast lowastlowast

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)






4 Discussions




(a) (b)

(c) (d)

5

4

3

2

1

0


Villu

s hei

ghts

cryp

ts de

pths

Con

trol

5-F

u


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(e)





Caspase3 R

elat

ive m

RNA

ex

pres

sion

(fold

)25

20

15

10

05

00

Con

trol

5-F

u

lowastlowastlowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(a)

PCNA

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

4

3

2

1

0


Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)

Cyclin-D1

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

32

24

16

08

00

lowastlowastlowast

Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)



(d)

15

10

5

0

Apoptotic index

Apop

totic

inde

x (times

103)

Con

trol

5-F

u

lowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(e)






4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

lowast

lowast

lowastlowast


(a)

35

30

25

20

15

10

05

00

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-1120573

lowast


(b)

5

4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-12b

Con

trol

5-F

u

lowast

lowast


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)









5 Conclusion


Abbreviations





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Body weightBo

dy w

eigh

t cha

nge (

)

105

100

95

90

85

110

100

90

80

4 5 6 7 8 9 10

Time (day)


lowast

(a)

Diarrhoea

Dia

rrho

ea sc

ore

0

1

2

3

4

0

1

2

3

4

4 5 6 7 8 9 10

Time (day)


lowast

(b)

Faecal blood

Faec

al b

lood

scor

e

0

1

2

3

4

0

1

2

3

4

4 5 6 7 8 9 10

Time (day)




(c)







175

150

125

100

75

50

ASTA

ST (U

L)

lowast lowast

(a)

45

40

35

30

25

20

15

10

ALT

ALT

(UL

)

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

(b)

12

10

8

6

4

2

0

BUN

BUN

(mm

olL

)



(c)

30

25

20

15

10

CRE


CRE

(120583m

olL

)


(d)








90

85

80

75

70

65

60

Colon lengthC

olon

leng

th (c

m)

lowastlowast

lowastlowastlowast

lowastlowastlowastC

ontro

l

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(a)

48

47

46

45

44

43

42

41

40


Smal

l int

estin

e len

gth

(cm

)

Con

trol

5-F

u

5-F

u +

lope

ram

ide

lowast lowastlowast

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)






4 Discussions




(a) (b)

(c) (d)

5

4

3

2

1

0


Villu

s hei

ghts

cryp

ts de

pths

Con

trol

5-F

u


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(e)





Caspase3 R

elat

ive m

RNA

ex

pres

sion

(fold

)25

20

15

10

05

00

Con

trol

5-F

u

lowastlowastlowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(a)

PCNA

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

4

3

2

1

0


Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)

Cyclin-D1

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

32

24

16

08

00

lowastlowastlowast

Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)



(d)

15

10

5

0

Apoptotic index

Apop

totic

inde

x (times

103)

Con

trol

5-F

u

lowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(e)






4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

lowast

lowast

lowastlowast


(a)

35

30

25

20

15

10

05

00

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-1120573

lowast


(b)

5

4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-12b

Con

trol

5-F

u

lowast

lowast


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)









5 Conclusion


Abbreviations





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















175

150

125

100

75

50

ASTA

ST (U

L)

lowast lowast

(a)

45

40

35

30

25

20

15

10

ALT

ALT

(UL

)

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

(b)

12

10

8

6

4

2

0

BUN

BUN

(mm

olL

)



(c)

30

25

20

15

10

CRE


CRE

(120583m

olL

)


(d)








90

85

80

75

70

65

60

Colon lengthC

olon

leng

th (c

m)

lowastlowast

lowastlowastlowast

lowastlowastlowastC

ontro

l

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(a)

48

47

46

45

44

43

42

41

40


Smal

l int

estin

e len

gth

(cm

)

Con

trol

5-F

u

5-F

u +

lope

ram

ide

lowast lowastlowast

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)






4 Discussions




(a) (b)

(c) (d)

5

4

3

2

1

0


Villu

s hei

ghts

cryp

ts de

pths

Con

trol

5-F

u


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(e)





Caspase3 R

elat

ive m

RNA

ex

pres

sion

(fold

)25

20

15

10

05

00

Con

trol

5-F

u

lowastlowastlowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(a)

PCNA

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

4

3

2

1

0


Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)

Cyclin-D1

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

32

24

16

08

00

lowastlowastlowast

Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)



(d)

15

10

5

0

Apoptotic index

Apop

totic

inde

x (times

103)

Con

trol

5-F

u

lowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(e)






4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

lowast

lowast

lowastlowast


(a)

35

30

25

20

15

10

05

00

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-1120573

lowast


(b)

5

4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-12b

Con

trol

5-F

u

lowast

lowast


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)









5 Conclusion


Abbreviations





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















90

85

80

75

70

65

60

Colon lengthC

olon

leng

th (c

m)

lowastlowast

lowastlowastlowast

lowastlowastlowastC

ontro

l

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(a)

48

47

46

45

44

43

42

41

40


Smal

l int

estin

e len

gth

(cm

)

Con

trol

5-F

u

5-F

u +

lope

ram

ide

lowast lowastlowast

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)






4 Discussions




(a) (b)

(c) (d)

5

4

3

2

1

0


Villu

s hei

ghts

cryp

ts de

pths

Con

trol

5-F

u


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(e)





Caspase3 R

elat

ive m

RNA

ex

pres

sion

(fold

)25

20

15

10

05

00

Con

trol

5-F

u

lowastlowastlowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(a)

PCNA

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

4

3

2

1

0


Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)

Cyclin-D1

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

32

24

16

08

00

lowastlowastlowast

Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)



(d)

15

10

5

0

Apoptotic index

Apop

totic

inde

x (times

103)

Con

trol

5-F

u

lowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(e)






4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

lowast

lowast

lowastlowast


(a)

35

30

25

20

15

10

05

00

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-1120573

lowast


(b)

5

4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-12b

Con

trol

5-F

u

lowast

lowast


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)









5 Conclusion


Abbreviations





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

5

4

3

2

1

0


Villu

s hei

ghts

cryp

ts de

pths

Con

trol

5-F

u


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(e)





Caspase3 R

elat

ive m

RNA

ex

pres

sion

(fold

)25

20

15

10

05

00

Con

trol

5-F

u

lowastlowastlowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(a)

PCNA

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

4

3

2

1

0


Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)

Cyclin-D1

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

32

24

16

08

00

lowastlowastlowast

Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)



(d)

15

10

5

0

Apoptotic index

Apop

totic

inde

x (times

103)

Con

trol

5-F

u

lowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(e)






4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

lowast

lowast

lowastlowast


(a)

35

30

25

20

15

10

05

00

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-1120573

lowast


(b)

5

4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-12b

Con

trol

5-F

u

lowast

lowast


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)









5 Conclusion


Abbreviations





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Caspase3 R

elat

ive m

RNA

ex

pres

sion

(fold

)25

20

15

10

05

00

Con

trol

5-F

u

lowastlowastlowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(a)

PCNA

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

4

3

2

1

0


Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(b)

Cyclin-D1

Rel

ativ

e mRN

A

expr

essio

n (fo

ld)

32

24

16

08

00

lowastlowastlowast

Con

trol

5-F

u

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)



(d)

15

10

5

0

Apoptotic index

Apop

totic

inde

x (times

103)

Con

trol

5-F

u

lowast

5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)(e)






4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

lowast

lowast

lowastlowast


(a)

35

30

25

20

15

10

05

00

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-1120573

lowast


(b)

5

4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-12b

Con

trol

5-F

u

lowast

lowast


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)









5 Conclusion


Abbreviations





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

lowast

lowast

lowastlowast


(a)

35

30

25

20

15

10

05

00

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-1120573

lowast


(b)

5

4

3

2

1

0

Relat

ive m

RNA

expr

essio

n (fo

ld)

IL-12b

Con

trol

5-F

u

lowast

lowast


5-F

u +

lope

ram

ide

5-Fu

+ C

S (5

gkg

)

5-Fu

+ C

S (1

0 gk

g)

5-Fu

+ C

S (2

0 gk

g)

(c)









5 Conclusion


Abbreviations





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion


Abbreviations





Acknowledgments


References





























































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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