1、Contents lists available at ScienceDirectInternational Immunopharmacologyjournal homepage:Rk3 alleviated DSS-induced ulcerative colitis by protectingcolon barrier and inhibiting NLRP3 in ammasome pathwayMi Tian1,Pei Ma1,Yan Zhang,Yu Mi,Daidi Fan Shaanxi Key Laboratory of Degradable Biomedical Materi
2、als,School of Chemical Engineering,Northwest University,Taibai North Road 229,Xian,Shaanxi 710069,ChinaShaanxi R&D Center of Biomaterials and Fermentation Engineering,School of Chemical Engineering,Northwest University,Taibai North Road 229,Xian,Shaanxi710069,ChinaBiotech&Biomed Research Institute,N
3、orthwest University,Taibai North Road 229,Xian,Shaanxi 710069,ChinaARTICLEINFOKeywords:Ginsenosides Rk3Ulcerative colitisIn ammationTight junctionsShort-chain fatty acidsNLRP3ABSTRACTGinsenosides have a variety of pharmacological activities,including immunomodulatory,antitumor and anti-in ammatory a
4、ctivities.However,the e ect of Rk3 on ulcerative colitis has rarely been reported.This studyevaluated the e ect of Rk3 on DSS-induced ulcerative colitis and preliminarily explored the anti-in ammatorymechanisms.Rk3 administration signi cantly attenuated the weight loss,increased DAI scores,colonic s
5、hort-ening,and increased MPO and iNOS activities caused by DSS in mice.Histological improvement was apparent,tight junctions in the colon were restored,and the levels of short-chain fatty acids(acetic acid,butyric acid andisovaleric acid)were increased.In addition,Rk3 reduced the expression of proin
6、 ammatory factors(TNF-,IL-1 and IL-6),NLRP3,ASC,and Caspase-1,indicating blockade of the NLRP3 in ammasome pathway.Theseresults show that Rk3 can improve DSS-induced ulcerative colitis by protecting intestinal barrier function andinhibiting NLRP3 in ammasome expression,indicating that Rk3 could be u
7、sed as a potential drug for treatingulcerative colitis.1.IntroductionIn ammatory bowel disease(IBD)is a chronic recurrent intestinalin ammation that includes two subtypes:Crohns disease(CD)and ul-cerative colitis(UC).Typical clinical features include abdominal pain,diarrhea,and blood in the stool.Wi
8、th the rapid development of in-dustrialization,IBD has become a global disease with a rapidly risingincidence 1,2.Compared with CD,UC is more common and has abimodal age distribution;one peak is 2030 years old,and the other is5080 years old 3.UC cannot be cured but only be maintained fortreatment,an
9、d the disease process is accompanied by onerous compli-cations.Long-term colon in ammation increases the risk of coloncancer.This has become a very large medical burden for patients andcountries 4.Although there are many treatments for UC,such as 5-aminosalicylic acid preparations,corticosteroids,th
10、iopurines,biolo-gical agents and surgery 5,6,serious side e ects and inadequatetreatmentstillremaintobeaddressed.Therefore,itisnecessarytolookfor drugs with higher e cacy and fewer side e ects.The cause of UC is not very clear but is currently thought to berelatedtogeneticsusceptibility,livinghabits
11、,theimmuneresponseandan imbalance in intestinal microorganisms.Epithelial cells are coupledtogether through tight junctions(TJs)to form a tight barrier that pre-vents microbes in the lumen from triggering an immune response 7.Destructionoftheepithelialbarriercausesintestinalmicroorganismstointeract
12、with immune cells,which causes an in ammatory responsethat gradually becomes uncontrolled.The continuous in ammatoryresponse further causes in ammatory damage to the epithelium 8.ThisisasevereviciouscycleandareasonforUCrecurrence.Therefore,repair of the intestinal barrier in UC patients means a real
13、 relief 9.Short-chain fatty acids(SCFAs)fermented by microorganisms are animportant source of energy and nutrition for colonic epithelial cells,which can maintain the integrity of the epithelium 10.For example,https:/doi.org/10.1016/j.intimp.2020.106645Received 19 February 2020;Received in revised f
14、orm 28 April 2020;Accepted 23 May 2020Abbreviations:DAI,Diseaseactivityindex;MPO,Myeloperoxidase;iNOS,Induciblenitricoxidesynthase;TNF-,Tumornecrosisfactor-;IL-1,Interleukin-1-beta;IL-6,Interleukin-6;SCFAs,Short-chain fatty acids;ZO-1,Zonula occluden-1;NLRP 3,Nucleotide-binding oligomerization domai
15、n-like receptor 3;ASC,Apoptosis-associated speck-like protein containing;Caspase-1,Cysteinyl aspartate speci c proteinase 1 Correspondingauthorsat:ShaanxiKeyLaboratoryofDegradableBiomedicalMaterials,SchoolofChemicalEngineering,NorthwestUniversity,TaibaiNorthRoad229,Xian,Shaanxi 710069,China.E-mail a
16、ddresses:mi_(Y.Mi),(D.Fan).1These authors contributed equally to this work.International Immunopharmacology 85(2020)1066451567-5769/2020 Elsevier B.V.All rights reserved.TDownloaded by at 2021-05-15 11:35:30butyrate can enhance intestinal barrier function by regulating tightjunctionproteins11.Relate
17、dstudieshavealsofoundthatthecontentof short-chain fatty acids in mice decreases during colitis and increasessigni cantly during the recovery process 12.The innate immunesystemisanimportantdefensemechanismthatallowsthebodytoresistmicrobes in a nonspeci c manner 13,14.As an important part of theinnate
18、immunesystem,theNLRP3in ammasomehasbeenincreasinglystudied.TheNLRP3in ammasomeisamultiproteincomplexconsistingof nucleotide binding domains and leucine-rich repetitive sequencecontaining proteins(NLRs),the bridging protein ASC and procaspase-115.Following recognition of pathogen-associated molecular
19、 patterns(PAMPs)or damage-associated molecular patterns(DAMPs),NLRP3in ammasomes are assembled,leading to caspase-1 maturation,fol-lowedbythereleaseofIL-1,animportantproin ammatoryfactorthatis a downstream product of caspase-1 16.High levels of IL-1 areclosely related to intestinal in ammation,and I
20、L-1 is one of the im-portant proin ammatory cytokines in the pathogenesis of intestinalin ammation 17,18.In addition,studies have shown that down-regulation of NLRP3 expression improves intestinal in ammation19,20,and Bauer con rmed that NLRP3/mice are protected fromDSS-induced colitis 21.Therefore,
21、the activation of the NLRP3 in-ammasomeiscriticalinthedevelopmentofin ammatoryprocessesinthe colon,which suggests that drugs that inhibit NLRP3 in ammasomeactivation may have the potential to treat in ammatory diseases.Ginseng,a traditional Chinese medicine,has received extensive at-tention from res
22、earchers 22,23.Ginsenosides are the main activecomponent of ginseng,among which Rg1 has the highest content.Rg1has shown many anti-in ammatory properties 24,25.It has been re-ported that Rg1 can inhibit the in ammatory response of macrophagesinduced by LPS in vitro 26 and ameliorate the symptoms of
23、alcoholichepatitisandcolitisinvivo27.TheginsenosideRk3isgeneratedfromRg1 and has a smaller molecular weight.Previous studies have foundthat Rk3 can inhibit in ammation and the oxidative stress response inalcoholic liver injury 28 and reduce cisplatin-induced oxidative da-mage to the kidneys 29.Howev
24、er,there is limited research on colonin ammation.Based on this knowledge,we investigated the e ects of the ginse-noside Rk3 on DSS-induced colitis in mice,including maintenance ofthe intestinal barrier and inhibition of NLRP3 in ammasome signalingpathways.These ndings may provide evidence to support
25、 Rk3 as atreatment for UC.2.Materials and methods2.1.ReagentsThe ginsenoside Rk3(purity 98%)was provided by Puri cationTechnology Development Co.,Ltd.(Chengdu,China).Primary anti-bodies speci c for NLRP3 and ASC were purchased from SignalwayAntibody(College Park,Maryland,USA).Primary antibodies spec
26、i cfor Caspase-1 and horseradish peroxidase(HRP)-conjugated secondaryantibodies were purchased from AdipoGen(San Diego,CA,USA)andAbbkine(Wuhan,China).Primary antibodies speci c for-actin,ZO-1,occludin and Claudin-1 were purchased from Proteintech Group(Wuhan,China).Carboxymethylcellulose sodium(CMC-
27、Na)was pur-chased from Anhui Shanhe Pharmaceutical Accessories Co.,Ltd.(Huainan,China).Dextran sulfate sodium(DSS;3650 kDa)was ob-tained fromMPBiomedicals(Illkirch,France).Myeloperoxidase(MPO)and iNOS activity assay kits were obtained from Nanjing JianchengBioengineering Institute(Nanjing,China).ELI
28、SA kits for IL-1,TNF-and IL-6 were purchased from Shanghai Enzyme-linked BiotechnologyCo.,Ltd.(Shanghai,China).The BCA Protein Assay Kit was purchasedfrom Beijing Solarbio Science&Technology Co.,Ltd.(Beijing,China).2.2.Animals and experimental design6to8-week-oldmaleC57BL/6micewerepurchasedfromChang
29、shaTianqinBiotechnologyCo.,Ltd.(Changsha,China).Animalwelfareandexperimental procedures were carried out strictly in accordance withthe Guide for the Care and Use of Laboratory Animals(NorthwestUniversity,China)and the related ethical regulations of our university.Mice were randomly divided into 5 g
30、roups(n=8/group):normal,model,and Rk3(20,40 and 60 mg/kg).Rk3 was dissolved in 0.5%sodium carboxymethylcellulose(CMC-Na)and administered orallyonce per day for 14 days.In the normal and model groups,the samevolume of CMC-Na was administered orally as a vehicle control.Inductionofexperimentalcolitisi
31、nmicewasachievedbyprovidingdrinking water containing 2.5%(w/v)DSS from day 8 to day 14.Themice in the normal group were supplied with ddH2O without DSS andserved as a negative control.Mice were monitored daily to ensure thatthe experiment ran normally.2.3.Evaluation of the disease activity index(DAI
32、)The DAI score was calculated as the average of weight loss,stoolconsistency,and fecal occult blood scores.Mice were observed daily tomonitor body weight,stool consistency,and fecal occult blood status,and scores were given for the three parameters based on Coopers 30scoring criteria to calculate th
33、e disease activity index of each mouse.The DAI is de ned as shown in Table 1.2.4.Histopathological and immunohistochemical evaluationsA part of the distal colon was immersed in a 10%formalin xationsolution,embedded in para n,cut into 5-m sections with a micro-tome,fully dewaxed and hydrated.A hemato
34、xylin-eosin staining solu-tionwasusedtomakeHEsections.ATUNELstainingsolutionwasusedto generate TUNEL-stained sections.In addition,para n sections wereimmunostained with anti-NLRP3,anti-ASC,and anti-Caspase-1 anti-bodies.Finally,observation and evaluation were performed using amicroscope(Nikon,Tokyo,
35、Japan).2.5.Evaluation of colonic in ammatory cell in ltrationNeutrophil in ltration into the in amed colonic mucosa wasquanti ed by MPO and iNOS activity assessment.One hundred milli-grams of colon tissue was homogenized in 900 L of normal saline tomake a 10%homogenate,which was centrifuged(3,000 rp
36、m)for10 min.For iNOS detection,we took 50 L of supernatant,addedsamples according to the assay instructions,zeroed the reader at530nmwithdistilledwater,andmeasuredtheabsorbanceofeachtube.For MPO detection,we took 500 L of uncentrifuged homogenate,added samples according to the assay instructions,zer
37、oed the reader at530nmwithdistilledwater,andmeasuredtheabsorbanceofeachtube.The total protein concentration of colon tissue supernatants was de-termined with a BCA protein assay kit.2.6.Measurements of cytokine levelsBlood samples were collected,allowed to stand for 20 min andTable 1Evaluation of di
38、sease activity index(DAI).score Weight loss(%)Stool consistency Gross bleeding0 None Normal No blood11 5 Loose stools25 1031 0 20 Diarrhea Gross blood4 2 0M.Tian,et al.International Immunopharmacology 85(2020)1066452Downloaded by at 2021-05-15 11:35:30centrifuged at3000rpmfor15minat4C.Thesupernatant
39、wasstoredat 80 C for testing.Enzyme-linked immunoassays were used toquantifythelevelsofcytokines(TNF-,IL-1andIL-6)usingELISAkitsaccording to the manufacturers protocol.2.7.Quantitative real-time reverse-transcription PCR(qRT-PCR)Total RNA from colon tissue was isolated using TRIzol reagent(Ambion,MA
40、,USA).The quality of the isolated RNA was measuredusing a NanoDrop nucleic acid protein detector(Thermo Scienti c,Wilmington,DE,USA).First-strand cDNA was synthesized using a re-versetranscriptionkit(Thermo,USA).Eachsamplewasevaluatedthreetimes usingSYBR Green,eighttubes anda Bio-Rad CFXReal-Time PC
41、Rsystem.The PCR procedure was as follows:95 C for 5 min and am-pli cation for 45 cycles at 95 C for 10 s,60 C for 10 s,and 72 C for15 s.Relative expression was calculated using the 2 CTmethod.Theprimer sequences used are shown in Table 2.2.8.Western blot assayAccordingtoYinanHongetal.sexperiment31,t
42、heWBprocedurewasasfollows.TissuewasgroundinthepresenceofliquidnitrogenandthenlysedinRIPAlysisbu er(containing10%(v/v)PMSF)for20minon ice.The lysates were then subjected to centrifugation(13,000 rpm)at 4Cfor 20min.Theprotein concentrations of thesupernatants weredetected with a BCA protein assay kit.
43、Then,the same amount of pro-tein was separated by 12%or 10%SDS-PAGE and transferred topolyvinylidene di uoride(PVDF)membranes(Hybond,Sunnyvale,CA,USA)using a semidry transfer system(Bio-Rad,Hercules,CA,USA).The membranes were incubated with speci c antibodies againstNLRP3,ASC,caspase-1,-actin,ZO-1,O
44、ccludin and Claudin-1 over-night at 4 C,and then HRP-conjugated secondary antibodies were in-cubated for 1 h at room temperature(RT).All of the antibodies werediluted in TBS.The protein signals were analyzed using an ECL detec-tion system(Tanon,Nanjing,China).2.9.Relative concentrations of short-cha
45、in fatty acids(SCFAs)Short-chain fatty acids were detected by the internal isotope stan-dard method using a series mass spectrometer,referring to MinxiaZhangs and Yanhui Hans method 32,33.Approximately 50 mg ofsample with 1 mL of ddH2O was ultrasonically extracted for 30 min inan ice bath in a 2.5-m
46、L EP tube and centrifuged at 12,000 r/min for5 min.Then,45 L of concentrated hydrochloric acid and 2.5 mL ofether were added to the collected supernatant,which was shaken,ex-tracted atroomtemperature for10min,and centrifuged at3500 r/minfor 5 min.The upper organic phase was transferred to a new 5-mL
47、 EPtube and ether extracted,and the organic phases were combined and lteredthrougha0.22-m lter.Then,35 Lofextractionsolutionand100 L of isotope internal standard were pipetted into a 96-well plate,which was placed on a shaker at 500 rpm/min for 1 h.The eluent wastransferred to a 96-well micro ltrati
48、on plate and concentrated with anitrogen blower,and 50 L of acetyl chloride:n-butanol(9:1)wasadded,incubated at65 Cfor 30 minand centrifuged at 3000 rpm/minfor 3 min.The ltered sample was tightly covered with a silica gel capto prevent volatilization and tested on a series mass spectrometer(API4000
49、LC/MS/MS,Applied Biosystems,USA).2.10.Statistical analysisTheresults are expressedas the mean sd.Data were statisticallyevaluated by one-way ANOVA followed by Tukeys test to comparedi erent groups.The level of signi cance was set at a P-value of 0.05.Groups with di erent superscript letters in the g
50、ures were sig-ni cantly di erent.Statistical analysis was performed using GraphPadPrism.3.Results3.1.Rk3 attenuated clinical symptoms in DSS-induced colitis miceToevaluatetheanticolitis e ectsofRk3,weusedDSStoestablishamouse model of ulcerative colitis to mimic human UC and determinedthetherapeutice
