分子生物学知识拓展 (14).pdf

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1、ORIGINAL ARTICLECinnamaldehyde targets TLR-4 and inflammatory mediatorsin acetic-acid induced ulcerative colitis modelSaeideh Momtaz1,2,3&Maryam Navabakhsh4&Negin Bakouee4&Mustafa Dehnamaki4&Mahban Rahimifard2&Maryam Baeeri2&Alireza Abdollahi5&Mohammad Abdollahi2&Mohamad Hosein Farzaei6,7&Amir Hosse

2、in Abdolghaffari1,2,3,4Received:5 December 2020/Accepted:19 February 2021#Institute of Molecular Biology,Slovak Academy of Sciences 2021AbstractCinnamon and its bioactive ingredients such as cinnamaldehyde(CA),with broad pharmacological profiles,are important partsof daily diet in many cultures.More

3、over,there are plenty motivating patents on efficacy of nutritional phytochemicals as novelanti-inflammatorydrugs inpatients withinflammatoryboweldisease(IBD).This studyintendedtoevaluatethe effects ofCAoninflammatorybiomarkersinaceticacid-inducedcolitisrats.Colitiswasinducedinallanimals,exceptinsha

4、mgroup,usingaceticacid(4%).Following colitis induction,in3 groups,CA was administrated orallyat 2,4 and 8 mg/kg/day for 2 days(once a day).Other groups were defined as the control(only treated with acetic acid),sham group(normal saline),and a standard group(Dexamethasone).To evaluate the inflammatio

5、n sites,macroscopic and microscopic markers were assessed.Tissue concentra-tions of interleukin(IL)-6 and tumor necrosis factor-alpha(TNF)-,were assessed by ELISA assay kits,while myeloperoxidase(MPO)was measured spectrophotometrically.The mRNA expression of toll like receptor(TLR)-4 in colon tissue

6、 was assessedby Real time-PCR.CA at 4 mg/kg/day and 8 mg/kg/day significantly improved microscopic and macroscopic manifestations ofcolitis tissues.TNF-,MPO,and IL-6 levels were significantly lower in CA treated groups at all the concentrations tested(P 0.001).CA at 4 and 8 mg/kg/day significantly d

7、ownregulated the mucosal gene expression of TLR-4.CA attenuatedexperimental colitis by means of colitis symptoms,reduction in inflammation cytokines,decline of neutrophil infiltration,andsuppression of TLR-4 expression in acetic acid-induced colitis.CA improved colitis in animal model through suppre

8、ssion ofinflammatory parameters and downregulation of TLR-4 mRNA expression.Keywords Cinnamaldehyde(CA).Inflammatoryboweldisease(IBD).Tumornecrosisfactor-alpha(TNF-).Myeloperoxidase(MPO).Tolllikereceptor4(TLR-4).Interleukin6(IL-6)*Mohamad Hosein F*Amir Hossein A1Medicinal Plants Research Center,Inst

9、itute of Medicinal Plants,ACECR,Tehran,Iran2Toxicology and Diseases Group(TDG),Pharmaceutical SciencesResearch Center(PSRC),The Institute of Pharmaceutical Sciences(TIPS),andDepartmentofToxicologyandPharmacology,SchoolofPharmacy,Tehran University of Medical Sciences,Tehran 1417614411,Iran3Gastrointe

10、stinal Pharmacology Interest Group(GPIG),UniversalScientific Education and Research Network(USERN),Tehran,Iran4Department of Toxicology&Pharmacology,Faculty of Pharmacy,Tehran Medical Sciences,Islamic Azad University,Tehran,Iran5Department of Pathology,Imam Hospital,Tehran University ofMedical Scien

11、ces,Tehran,Iran6Pharmaceutical Sciences Research Center,Health Institute,Kermanshah University of Medical Sciences,Kermanshah,Iran7Medical Biology Research Center,Kermanshah University ofMedical Sciences,Kermanshah,IranBiologiahttps:/doi.org/10.1007/s11756-021-00725-wIntroductionGastrointestinal dis

12、orders are very common all around theworld,in which inflammatory bowel diseases(IBD)are quitepredominant.IBD is usually divided to Ulcerative Colitis(UC)and Crohns Disease(CD)(Saaga et al.2014),withknown common features as abdominal cramps,bloody stool(Dodda et al.2014),and frequent diarrhea(McCormi

13、ck et al.2010).TheuncertaintyofdefinitepathophysiologyofIBDledtoongoinginvestigationsondifferentaspectsofthedisease(Saagaetal.2014).Compellingevidenceconfirmedthatgeneticsuscep-tibility and dysregulation of the immune system are playing fun-damental roles in IBD development(Agliata et al.2019).Up to

14、date,severalpro-inflammatoryandinflammatorymediatorssuchas interleukins(ILs),cytokines,and chemokines have beenlinked to inflammatory diseases alike IBD.Macrophages arecrucial components of the immune system that can neutralizethe pathogens and infiltrate into the damaged tissue(Khl et al.2015).Inad

15、dition,IL-dependentalterationsinmacrophagescon-firmthecontributionofthesecytokinesininflammatoryprocess-es(Seo et al.2017).It was documented that cytokines such astumor necrosis factor-alpha(TNF-),IL-6,IL-8,and IL-26 areactively involved in several inflammatory pathways that arelinked to the IBD ini

16、tiation or progression(Spoettl et al.2007;Fujiietal.2017).Ontheotherhand,theactivityofenzymemyeloperoxidase(MPO)hasalsobeenimplicatedinIBD,asthisenzymelocatesatneutrophilsanditwasevidencedthatthenum-ber of neutrophils increases drastically in patients with IBD.Therefore,assessment of MPO is an impor

17、tant marker of IBD(Hansberry et al.2017).Following the identification of above-mentioned factors and their positive correlations with inflamma-tory process,the significance of toll like receptors(TLRs),as apart of the innate immune system,was alsoconfirmed incolonalinflammation progression(Kawasaki

18、and Kawai 2014).TLRs are a family of molecules participating in the innateimmunity.TLRs include homologoussubtypes(approximate-ly 10 in human and 12 in mice),of which,TLR-4 pathway isassumed as one of the important mechanisms involved in thecolonicinflammation.TLR-4isknownasabacteriallipopoly-saccha

19、ride(LPS)endotoxin receptor.This protein mainlymodulates the LPS inflammatory related responses in mam-mals.In a cascade of events,LPS-induced stimulation ofTLR-4,induces the common TLR adaptor that is called mye-loid differentiation factor 88(MyD88).Accumulated datasuggest that the TLR4-MyD88-NF-B

20、pathway mediates theprimarily inflammatory response(Zhou et al.2017).It wasreported that all TLRs,except TLR3,can act through theMyD88-dependent signaling pathway to induce the expres-sion of pro-inflammatory cytokine genes(Sipos et al.2014).Activation of MyD88 triggers the activation of downstreamn

21、uclear factor-B(NF-B)(Kang et al.2011).Consequently,NF-B translocates into the nucleus and stimulates the tran-scription of several genes that play key roles in cytokineproduction(i.e.pro-inflammatory cytokines such as IL-1,IL-6,as well as chemokines such as IL-8).Suppression ofTLR-4 and consequent

22、activation of NF-B leads to thedownregulation of inflammatory cytokine production(i.e.cyclooxygenase-2(COX-2),TNF-,IL-1 and IL-6)in IBDpatients(Lee et al.2009).Various pre-clinical and clinicalstudies confirmed the higher expression levels of TLR-2 andTLR-4inIBDsubjects.ItwasshownthatTLR-4expression

23、isprofoundly upregulated in the initial inflammatory processinvolved in IBD(Kordjazy et al.2018;Szebeni et al.2008).With such assumption,many investigations have focusedon anti-inflammatory substances,preferably natural products,with minimum undesired adverse effects to improve IBD.Low efficacy and

24、high risk of current chemical medicationssuchasSulfasalazine,arealsoadditionalreasons topursuit fornew drugs(Saaga et al.2014).Herbal medicines withestablished therapeutic effects and lower adverse effects,could be appropriate alternative treatments for IBD.Herbalmedicines can affect IBD through sev

25、eral mechanisms,eitherthrough their antioxidant and anti-inflammatory properties orby modification of the immune system(Rahimi et al.2009).Cinnamaldehyde(CA)is an,-unsaturated aromatic alde-hyde.Thetrans-isomerofCAisthemainactiveingredientoftheessential oil of Cinnamomum spp.Bark,which is presented

26、as apale yellow oily liquid(Deng et al.2020).The compound isfound in considerable quantities in the stem bark of C.cassia(He et al.2005).CA is the most abundant compound in cinna-mon oil,accounting for 65%to 75%of all compounds(Chianget al.2019).Cinnamomum species have been extensively ex-plored for

27、 their healing effects on gastrointestinal disorders suchas IBD(Hawrelak and Myers 2010;Kwon et al.2011;Muhammad et al.2015;Hagenlocher et al.2016).CA has abroad spectrum of pharmacological properties such as anti-in-flammatory,anti-tumor,anti-bacterial,antidiabetic,anti-obesity,andneuro-andcardio-p

28、rotectiveeffects(Hwaetal.2012;Zhanget al.2015).The anti-inflammatory activity of CA is well justi-fied,which is derived from several mechanisms including sup-pressionofmacrophageactivity,reductioninILsandtheTNF-activities,and also decrease in oxidative reactions(Chao et al.2008;Qu et al.2019).Respec

29、ting the anti-inflammatory andantioxidant activities of CA and the inflammatory mechanismsinvolved in IBD,it is believed that this compound may havesome preventive effects on IBD progression.Hence,the objec-tive of the current study was to evaluate the possible anti-inflammatoryactivityofCAinacetica

30、cid-inducedcolitisinrats.Materials and methodsChemicalsAll chemicals used in present study were of analytical grade.Chemicals and CA were purchased from Sigma-AldrichBiologia(Munich,Germany).Rat-specific TNF-,and IL-6 enzyme-linked immunosorbent assay ELISA kits were prepared byEnzo Life Sciences,Lo

31、rrach/Germany.AnimalsMale Wistar albino rats were provided by the animal labora-tory of Medicinal Plants Research Center,Institute ofMedicinal Plants.Thirty-six rats,weighing 180-200 g,werehoused under standard conditions of diet,temperature(232 C),humidity(557%)and 12/12 h light/dark cycle(6 ratsin

32、 each cage).Current animal study was performed accordingto the“Principles of Laboratory Animal Care”(NIH publica-tion 8223,revised in 1985 and 1996)and based onGuidelines and Protocols of the Research Ethics Committeeof Islamic Azad Tehran Medical Sciences University-Pharmacy and Pharmaceutical Bran

33、ch Faculty,confirmed byMinistry of Health and Medical Education.Colitis inductionAnimals were fasted for 24 h,with free access to water.Following anesthesia with an intramuscular injection of keta-mine(4 mg/100 g)and xylazine(1 mg/100 g)mixture,amedical-grade polyurethane canal inserted into the col

34、on rec-tally(approximately 8 cm).Two mL of a dilute solution ofacetic acid(4%)was administrated to all animals,except insham group(Fakhraei et al.2014).There was no mortality inanimals,either following colitis induction or after samplesadministration.Since the animal model of acute colitis wasinduce

35、d with acid solution,welfare of animals has been mon-itored carefully.Following colitis induction,some extent ofstress,anorexia,and diarrhea was observed.Experimental designTwenty-fourhoursaftercolitisinduction,animalsweretreatedwith different concentration of CA and Dexamethasone.CA(purity 95%)was

36、dissolved in water,using short timesonification.Dosage of CA was selected based on our pilotstudy.Based on randomclassification,ratswere distributed to6 groups(n=6);1.Control group:Colitis induced by acetic acid(2 mL,4%),rats were treated by saline,once a day,for 2 days.2.Dexamethasone/Dexagroup:Col

37、itisinducedbyaceticacid(2 mL,4%),rats were treated by 1 mL Dexamethasone1 mg/kg intraperitoneally,once a day,for 2 days.3.CA-2 group:Colitis induced by acetic acid(2 mL,4%),1mLCAwasadministratedat2mg/kgviagavage,onceaday,for 2 days.4.CA-4 group:Colitis induced by acetic acid(2 mL,4%),1mLCAwasadminis

38、tratedat4mg/kgviagavage,onceaday,for 2 days.5.CA-8 group:Colitis induced by acetic acid(2 mL,4%),1mLCAwasadministratedat8mg/kgviagavage,onceaday,for 2 days.6.Shamgroup:Ratsweretreatedwith1mLsaline(gavage),once a day,for 2 days.Animals were euthanized after 48 h.The protruding part ofcolon(the last 8

39、 cm of the colon)was cut open on ice andcleansed leniently with normal saline.Colonic segments weredivided into two sections;one for histopathologic evaluation(kept in 10%formalin),and the other part kept at 80 C forbiochemical assessments.Macroscopic evaluation of colonic damageThe cleanedand isola

40、tedcolonal instance was monitoredbymagnifying lens,and scored via Morris criteria as;0 for anuninjured zone,1 for localized hyperemia without any ul-cers,2 for linear ulcer without any significant inflamma-tion,3 for linear ulcer with inflammation which only ob-served in one site,4 for existence ulc

41、er and inflammation intwo or more sites,and 5 for ulcer and inflammation in twoor more sites with a width of more than 1 cm(Morris et al.1989).Microscopic evaluation of colonic damageMicroscopic histological evaluation was performed using alight microscopy.Colon sections were collected and fixedwith

42、 neutral formalin 10%.Formalin fixed colon tissueswere embedded in paraffin,and then sectioned with a mi-crotome(RM2235 Rotary Microtome)to obtain 5 m-thickparaffin sections.These sections were stained withHematoxylin and Eosin and the rate of tissue damageswas evaluated.The presence and severity of

43、 acute inflam-mation in colonic sections was examined by a histopathol-ogist that was blinded to treatment allocation during theanalysis(Stumhofer et al.2006).Biochemical assaysDetermination of inflammatory mediatorsColonal sections were mixed with an ice cold potassium phos-phate buffer(pH 6.0)cont

44、aining hexadecyltrimethylammoniumbromide(HTAB,5%)and homogenized.The lying liquid wascollected and cooled at 80 C for later analysis.According tothe guidelines,enzyme linked immunosorbent assay ELISA kitswereusedforcalculatingTNF-,andIL-6levelsat620nm.TheBiologiatissue protein level was indicated in

45、 pg/mg based on Bradfordmethod(0.0061x+0.0195,R2=0.987)(Farzaei et al.2015).Myeloperoxidase(MPO)activity assayMPO is an indicator of polymorphonuclear leukocyte accu-mulation and colonic inflammation.Colonic homogenate wasprepared as described in previous section and the homogenatewas sonicated inan

46、 ice bathfor 10 s,then itwassubjected toasequence of freezing and thawing for 3 times,sonicated againfor 10 s and centrifuged for 15 min at 15,000 rpm at 4 C.0.1 mL of supernatant was added to 2.9 mL phosphate buffer(50mM,pH=6)containing0.0005%H2O2and0.167mg/mLO-dianisidine and the absorption was ca

47、lculated at 460 nm.The MPO activity was defined as the quantity of enzymedegrading 1 mM of peroxide per min at 25 C and wasexpressed in units per gram(U/g)of protein(Krawisz et al.1984).Real time-reverse transcription polymerase chain reaction(RT-PCR)Total RNA was extracted from colon tissues with T

48、RIzol re-agent kit and kept in RNAlater storage solution.2.5 g ofRNA was reverse-transcribed using iScript cDNA SynthesisKit and Oligo dT specific primers.The primer sequences thatwere used for RT-PCR in terms of genes as follows:Tlr-4-F:5-GCAGGTCGAATTGTATCGCC-3R:5-GGGTTTTAGGCGCAGAGTTT-3Gapdh-F:5-AC

49、TGAGCAAGAGAGGCCCTA-3R:5-TATGGGGGTCTGGGATGGAA-3Real-timePCRwasperformedonLightCycler96usingthecomparative Ctquantitation method.GAPDH was amplifiedasinternalcontrol.CtvaluesofGAPDH were subtractedfromCtvalues of the target genes(Ct).Ct values of treated ratswere compared with Ct values of untreated a

50、nimals.Relative changes in gene expression were determined usingthe 2-Ctmethod compared with endogenous referenceGAPDH mRNA level(Momtaz et al.2019).Statistical analysisResults are shown as means standard error.Data were ana-lyzed using one-way analysis of variance(ANOVA)for mul-tiple comparisons be