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1、Medical Mycology,2020,0,110doi:10.1093/mmy/myaa053Advance Access Publication Date:0 2020Original ArticleOriginal ArticlePaeonol alleviates dextran sodium sulfate induced colitis involvingCandida albicans-associated dysbiosisYuzhu Ge1,Min Pan1,Chuanfeng Zhang1,Changzhong Wang1,2,3,4,Kelong Ma1,2,3,4,

2、Guiming Yan1,2,3,4,Tianming Wang1,3,4,Daqiang Wu1,2,3,4and Jing Shao1,2,3,4,1Laboratory of Infection and Tumor,College of Integrated Chinese and Western Medicine(College of Life Science),Anhui University of Chinese Medicine,436 Room,Zhijing Building,No.1 Qianjiang Road,Xinzhan District,Hefei230012,A

3、nhui,China,2Institute of Integrated Traditional Chinese and Western Medicine,Anhui Academy of Chi-neseMedicine,ZhijingBuilding,No.1QianjiangRoad,XinzhanDistrict,Hefei230012,Anhui,China,3KeyLaboratoryof XinAn Medicine,Ministry of Education,Anhui Academy of Chinese Medicine,XinAn Building,No.103 Meis-

4、han Road,Shushan District,Hefei 230038,Anhui,China and4Anhui Provincial Key Laboratory for Chinese HerbalCompound,Anhui Academy of Chinese Medicine,Hefei 230012,ChinaTo whom correspondence should be addressed.Prof.Guiming Yan,E-mail:922-;Dr.Jing Shao,E-mail:The authors contribute equally to this pap

5、er.Received 8 January 2020;Revised 21 March 2020;Accepted 10 June 2020;Editorial Decision 8 June 2020AbstractInflammatory bowel disease(IBD),which consists of ulcerative colitis(UC)and Crohn s disease(CD),is achronic inflammatory disorder of the gastrointestinal tract.Occurrence and development of U

6、C have beenassociated with multiple potential causative factors,which include fungal dysbiosis.Growing evidence re-veals that Candida albicans-associated dysbiosis is correlated with clinical deterioration in UC.Paeonol(PAE)is a commonly used traditional medicine with multiple reported properties in

7、cluding effective alleviation ofUC.In this study,a murine UC model was established by colonizing mice with additional C.albicans viagavage prior to dextran sodium sulfate(DSS)administration.Effects of PAE treatment were also assessedat initiation and in preestablished C.albicans-associated colitis.T

8、he results showed that C.albicans sup-plementation could aggravate disease activity index(DAI),compromise mucosal integrity,exacerbate fecaland tissue fungal burdens,increase serum-glucan and anti-Saccharomyces cerevisiae antibody(ASCA)levels,promote serum and colonic tissue pro-inflammatory cytokin

9、e secretion(tumor necrosis factor(TNF)-,interleukin(IL)-1,IL-6,and IL-8)and decrease the anti-inflammatory cytokine IL-10 level.It also stimulatedDectin-1,TLR2 and TLR4 as well as expression of their downstream effector NF-B in colonic tissue.AfterPAE treatment,the adverse impacts of C.albicans on c

10、olitis were relieved,via decreased receptor-associatedlocal and systemic inflammation.Our study suggests that PAE should be a candidate for treatment of fungaldysbiosis-associated UC and may act through the Dectin-1/NF-B pathway in collaboration with TLR2 andTLR4.Lay SummaryCandida albicans is belie

11、ved to be an important stimulator in ulcerative colitice(UC)development.Sup-pressing the growth of intestinal C.albicans can be contributory to the amelioration of UC.Paeonol(PAE)isa commonly used traditional medicine with multiple biological functions.In this study,we observed that PAEcould allevia

12、te symptoms in mice UC model accompanying with burden reduction of C.albicans.Therefore,we suppose that PAE can be a candidate in the treatment of C.albicans-associated UC.Key words:Candida albicans,ulcerative colitis,mycobiota,paeonol,Dectin-1.TheAuthor(s)2020.PublishedbyOxfordUniversityPressonbeha

13、lfofTheInternationalSocietyforHumanandAnimalMycology.All rights reserved.For permissions,please e-mail:1Downloaded from https:/ by Shanghai Jiao Tong University Library user on 01 July 20202Medical Mycology,2020,Vol.00,No.00IntroductionUlcerative colitis(UC),a common type of inflammatory boweldiseas

14、e(IBD),is characterized by persistent and nonspecific in-testinalinflammation,withtypicalclinicalmanifestationsinclud-ing abdominal pain,diarrhea and hematochezia.1Recently,themorbidity and mortality of UC have unfortunately increasedaround the world.2Without timely effective treatment,colorec-tal c

15、ancer(CRC)is likely to occur,as the overall risk of CRCin UC patients is reportedly up to 1.4%.3The etiology of UCis largely unclear at present.However,several factors are consid-ered to play a role in development of UC,including genetics,hostimmune response,environmental triggers and gut microbiota

16、.4,5Although constituting only 0.1%of the whole gut microbiotain healthy individuals,6gut commensal fungi are critically im-portant in maintaining host homeostasis by acting synergisticallywith other microbial communities.Candida albicans,one of themost common opportunistic polymorphic fungi,is resp

17、onsiblefor a series of pathologies ranging from mucous infections to sys-temic infections.7C.albicans is nevertheless a normal commen-sal in the human gastrointestinal tract(GT),but it can transforminto a pathogen in response to external environmental factors.Growing evidence reveals that C.albicans

18、-associated dysbiosisis correlated with high UC occurrence,and C.albicans coloniza-tion is capable of promoting UC,with increased inflammatorycytokine levels.811Traditional Chinese medicine(TCM)is a valuable re-source and several TCMs have demonstrated satisfactory ef-ficacy in UC treatment.1215Paeo

19、nol(PAE,2?-hydroxy-4?-methoxyacetophenone;C9H10O3)is a natural phenolic com-pound from Moutan Cortex,the root bark of Paeonia moutanSims,and has diverse biological effects including analgesic,16neuroprotective,17anti-allergic,18anti-atherosclerosis,19anti-osteoclastogenesis and anti-inflammatory act

20、ivities.20,21More-over,there are several lines of evidence that support anti-coliticeffects for PAE.2224Therefore,it is pertinent to explore the po-tential of PAE and its associated mechanisms in the treatment ofUC involving C.albicans-associated dysbiosis.In this study,a DSS-induced UC model with C

21、.albicans pre-colonization in mice was established.Post PAE treatment,it wasfound that fecal and organ fungal burdens were reduced,localand systemic infections were ameliorated,the activity of tissuemacrophages was suppressed,and the expression of Dectin-1,TLR2,TLR4 and NF-B were decreased.MethodsSt

22、rains and cultivationC.albicans SC5314 was a gift from Professor Yuanying Jiang,School of Pharmacy,Second Military Medical University,Shang-hai,China.The isolate was routinely maintained in liquidsabouraud medium(Hope Biotechology,Qingdao,Shangdong,China)at 37C for 1216 hours to reach exponential ph

23、ase(In-vitrogen,Carlsbad,CA,USA).AnimalsFifty mice(C57BL6/J,female,810 weeks)were purchasedfrom Pengyue Experimental Animal Breeding Co.Ltd(License:SCXK20190003,Jinan,China)and fed with sterilized chow andwater ad libitum.They were maintained in the Animal Center ofCollege of Integrated Chinese and

24、Western Medicine(College ofLife Science),Anhui University of Chinese Medicine,and accli-mated for 710 days prior to initiation of the UC model.All ani-mal protocols were approved by the Experimental Animal EthicsCommittee of the Anhui University of Chinese Medicine.Establishment of UC modelThe proce

25、dures for establishment of the UC model were mod-ified from several reported protocols.25,26Briefly,animals werefirst gavaged for 4 consecutive days with C.albicans SC5314(1 108cfu/mouse/day)suspended in sterile phosphate-bufferedsaline(PBS)and then given 3%dextran sodium sulfate(DSS;MP Biomedicals,

26、LLC,Aurora,OH,USA)in distilled water adlibitum for 7 days continuously to induce colitis.Some groupswere treated with PAE(400 mg/kg/d)or mesalazine(MES,200mg/kg/d)alongsideDSSadministration.Vehiclecontrolmicewere given physiological saline.Feces were collected at day 1,3,5,7,9,and11forfungalenumerat

27、ion.Allmiceweresacrificedbycervicaldislocationatday12toharvestbloodandorgans(colon,liver,spleen,kidney and stomach).Tissue samples were stored at20C until use.Colitis severity was assessed using DAI,calcu-lated based on weight loss,stool consistency and occult blood,as previously described.27Fungal

28、burden assayC.albicans enumeration was performed according to previouslyreported methods with minor modifications.28Feces and organswere weighed,suspended,or homogenized in sterile PBS(pH 7.4,Boster Biological Technology,Wuhan,China)and then plated onCHROMagar(cat.no.K08A,Shanghai Central Bio-engine

29、ering,Shanghai,China)containing 100 U/ml penicillin and 0.1 mg/mlstreptomycin(Beyotime Biotechnology,Shanghai,China)priorto incubation for 48 hours at 37C.Green colonies of C.albicanswere counted and recorded for each group.F4/80 stainingStaining for macrophages was carried out according to reported

30、techniqueswithminormodifications.29Briefly,colonictissuewassliced and fixed in acetone for 20 minutes.After washing 3 timesfor 10 min in PBS,the slices were blocked with 10%goat serumat 37C for 45 min.After three rinses with PBS,the tissuesDownloaded from https:/ by Shanghai Jiao Tong University Lib

31、rary user on 01 July 2020Ge et al.3were incubated with anti-F4/80 antibodies(0.2 mg/mL,cat.no.123 110,BioLegend,San Diego,CA,USA)at 4C overnight.Af-terafurther3washeswithPBS,DAPI(100ng/ml,cat.no.C1005,Beyotime,Shanghai,China)was added for a 4 min incubation.Staining was observed and recorded using a

32、 fluorescent micro-scope(Olympus IX81,Japan).Assays for cytokines,-glucan,andanti-Saccharomyces cerevisiae antibodies(ASCA)Levels of tumor necrosis factor(TNF)-(cat.no.CK-E20852),interleukin(IL)-1(cat.no.CK-E20174),IL-6(cat.no.CK-E20188),IL-8(cat.no.CK-E20191),and IL-10(cat.no.CK-E20162)were measure

33、d using ELISA kits(Ruixin Biotechnol-ogy,Quanzhou,Fujian,China).Serum-glucan content was de-termined by a commercial ELISA kit(Associates of Cape CodInc,Beijing,China).Serum ASCA levels were also measured by acommercial ELISA kit(cat.no.CK-E22222,Ruixin Biotechnol-ogy,Quanzhou,Fujian,China)according

34、 to the manufacturer sinstructions.Hematoxylin-eosin(HE)stainingHE staining was performed in line with described methodswith minor adjustments.30In brief,colonic tissue was fixed in10%neutral formalin(cat.no.ZP1101,Zhenwo Bicmedical,Guangzhou,Guangdong,China),then embedded in paraffin andcut into 4

35、m-thick sections.Sections were then stained withhematoxylin and eosin(cat.no.DH006,Leagene,Beijing,China)and pathological changes observed using an optical microscope(Olympus BX51,Japan).ImmunohistochemistryColonic tissue was sectioned for immunohistochemical anal-ysis using reported procedures with

36、 minor modulation.31Briefly,slices were deparaffinized by treatment in xylene(cat.no.20190610JN,Shanghai Richjoint Chemical Reagents,Shang-hai,China)3 times for 15 min each time and then hy-drated with 100%,95%,90%,80%,and 75%gradientethanol(Guanhua Chemical,Suzhou,Anhui,China).After rins-ing with P

37、BS for 2 min,sections were boiled in sodium cit-rate buffer for 20 min(0.01 M,pH 6.0,Leagene,Beijing,China)for antigen repair.After cooling to room temperature,sections were incubated in 3%H2O2in methanol(cat.no.13L26C08,Boster Biological Technology,Wuhan,China)for15 min to inhibit endogenous peroxi

38、de activity.Following rins-ing with PBS,sections were incubated with anti-TLR2(cat.no.10q2471,Affinity Bioscience,OH,USA),anti-TLR4(cat.no.16c5074,Affinity Bioscience,OH,USA)or anti-Dectin-1(cat.no.ab140039,Abcam,USA)antibodies at 4C overnight.After a further three washes in PBS(5 min/time),goat ant

39、i-mouse immunoglobulin G(IgG)secondary antibody(cat.no.1919D0919,Zhong Shan-Golden Bridge Biological Technol-ogy,Beijing,China)was added for 30 min at 37C.Finally,3,3-diaminobenzidin(DAB,cat.no.K186621D,Zhong Shan-Golden Bridge Biological Technology,Beijing,China)was addedat room temperature in the

40、dark for 5 min.Colorization wasstopped with distilled water.Sections were then dehydratedwith an ethanol gradient(75%,80%,90%,95%,and 100%)and xylene to transparency,sealed with neutral resin(Shang-hai Specimen and Model Factory,Shanghai,China),and ob-served with an optical microscope(Olympus BX51,J

41、apan),with data calculated using Image J(National Institutes ofHealth).ImmunoblottingTotal protein preparations were mixed with phenylmethanesul-fonyl fluoride(PMSF,cat.no.P0100,Solarbio,Beijing,China),prepared by solution in RIPA lysis buffer(cat.no.P10013B,Beyotime,Shanghai,China)to a final concen

42、tration of 1 mM(30 min at 4C).Following centrifugation at 10 000 g for 10 min,the protein concentrations were determined using BCA pro-tein analysis kits(cat.no.0912A19,Leagene,Beijing,China).Extracted proteins were separated by SDS-PAGE,transferredto PVDF membranes(cat.no.IPVH00010,Merck MilliporeL

43、td,China)and blocked with 5%skimmed milk powder(In-ner Mongolia Yili Industrial Group,Neimeng,China)dissolvedin TBST buffer(prepared by mixing 0.05 M TBS(pH 7.4,Biosharp Life Sciences,Hefei,Anhui,China)with 1 ml Tween20(Runhua Chemistry,Guangzhou,China)at room temper-ature for 2 hours.Membranes were

44、 then incubated with anti-Dectin-1,anti-TLR2,anti-TLR4,or anti-NF-Bp65(cat.no.26j7543,Affinity Bioscience,OH,USA)antibodies overnightat 4C.Horseradish peroxidase-conjugated anti-rabbit IgG(cat.no.56j9958,Affinity Bioscience,Cincinatti,OH,USA)as sec-ondary antibody was added to membranes,at room temp

45、era-ture for 2 hours.An enhanced chemiluminescence detection kit(cat.no.P0018S,Beyotime Institute of Biotechnology,Shanghai,China)was used for development of protein bands which werescanned with a protein imaging system(LAS4000,GE,Pitts-burgh,PA,USA)and quantified with Image J.32Statistical analysis

46、All experiments were performed in triplicate.The results wereexpressed as mean standard deviation and analyzed by SPSS17.0(SPSS Inc,Chicago,IL,USA).Groups were compared byone-way ANOVA with the least significance difference(LSD)method;P .05 was considered statistically significant.ResultsPAE amelior

47、ates colitis and fungal burdenEstablishment of the UC model is summarized in Figure 1A.After precolonization with C.albicans,mice showed greaterDownloaded from https:/ by Shanghai Jiao Tong University Library user on 01 July 20204Medical Mycology,2020,Vol.00,No.00Figure 1.Paeonol(PAE)ameliorates Can

48、dida albicans aggravated colitis.(A)Schema of dextran sodium sulfate(DSS)induced colitis with C.albicans supple-mentation.(B)Body weight assessment.(C)Disease activity index(DAI)calculation based on weight loss,fecal consistency and blood.(D,E)Colon lengthmeasurement.(F)Colon tissue H&E staining.The

49、 control group was mice fed with normal saline throughout the experiment.The DSS group was mice given3%DSS orally from day 5 to 11 following normal saline feeding from day 1 to 4.The model group was mice orally administered with C.albicans from day 1 to 4followed by 3%DSS from day 5 to 11.The PAE gr

50、oup included mice treated with 400 mg/kg/d PAE along with the same exposure to C.albicans and DSS as themodel group.MES group refers to mice treated with mesalazine instead of PAE with the same protocol as the PAE group.*P .05,*P .01,*P 5 105cfu at day 1 to 80 cfu per 10 mgfeces at day 11(Fig.2A).Ap