Study On The Mechanism Of The Protective Effect Of Intestinal Trefoil Factor On Inflammatory Bowel Disease In Mice Model

IntroductionInflammatory bowel disease(IBD),includes Crohn's disease(CD) and ulcerative colitis(UC),represents a group of chronic disorders characterized by inflammation of the gastrointestinal tract,typically with a relapsing and remitting clinical course.Based on recent studies,the pathogenesis of IBD seems to involve a complex interplay between certain genetic,environmental,and immunological factors.The epidemiological studies show that the incidence of IBD is high in the adolescence,who can take 25%-30%of patients diagnosed of IBD.In China,we can see the incidence of IBD increases gradually based on recent 10 years studies.So now IBD is a hackneyed kind of digestive system diseases.The aetiology and pathogenesis of inflammatory bowel disease(IBD) remain unknown,but increasing evidence suggests that the dysregulation of mucosal inflammation response may play an important role in the pathogenesis of IBD.The human intestinal tract mucosa is exposed to an enormous microbial flora.An unbalanced activation of the mucosal immune system driven by the commensal flora in a genetically susceptible host appears to cause intestinal inflammation in IBD patients. There is growing evidence that several mediators,adhesion molecule,cytokines, chemokines and inflammatory cell play key role in the pathogenesis of IBD.The chronic inflammatory process leads to disruption of the epithelial barrier,and the formation of epithelial ulceration.As unknown etiology,the current therapy remains largely symptomatic.The development of novel therapeutic agents for IBDs is an active field;some new strategies for cytokine antagonist therapy have been developed.Intestinal epithelial cells(IEC) recognition of microorganisms is based on recognition of conserved signature molecules in microorganisms called pathogen(or microbe)-associated molecular patterns(PAMP).There are two main types Pattern recognition receptor(PRR)families mediate the response of immune cells to PAMP to date.The first are the Toll-like receptors(TLR)family that share homology with the antifungal extracellular receptor Toll.The TLR4 of the IEC of the patients of IBD is upregulated,whith result in NF-κB p65 activation,leading to the production of the antimicrobial factors cytokines(including TNF-α)and chemokines,then initiate innate and adaptive immunity.The key pathology of the IBD is intestinal tract mucosal erosion and ulceration, epithelium extensive deletion,lamina propria inflammatory cell infiltration.Although the pathomechanism of the IBD is unknown,increasing evidence suggests that epithelium apoptosis excess and inflammatory cell apoptosis insufficiency may play an important role.The disproportion of the apoptosis of cell can induce autoimmune disease;autoimmune disorder.The family of trefoil factor(TFF)peptides,with its three members TFF1(pS2), TFF2(spasmolytic polypeptide),and TFF3(intestinal trefoil factor/ITF)represents typical secretory products of mucin producing cells.These small molecules are predominantly expressed in the digestive tract of mammals.Mice over expressing human TFF1 or rat TFF3 in the intestine displayed increased resistance to intestinal damage and ulceration.It has also been shown that mice lacking TFF3 are more susceptible to DSS colitis.Some reports show that TFF3 participate in the immune response and apoptosis regulation.Although evidence from in vitro and in vivo experiments suggests TFF as therapeutic agents in inflammatory bowel disease,the molecular function of TFF peptides in the gut is not completely understood.Materials and Methods1.Animal modelFemale,BALB/C mouse(22-26 g,6-8 weeks old;China Medical University Experimental Animal Research Center,Shenyang,China).Mice were randomly divided into 3 groups.Colitis was induced by a single intracolonic administration of 0.2 ml of 50%ethanol(vol/vol)containing TNBS(Sigma)at 25 mg/ml,based on a method described previously.Group A were treated by intraperitoneal injection of 0.1 ml of normal saline once a day as the positive control;group B had intraperitoneal injection with 0.1 ml(5mg/ml)of TFF3 once a day;group C,selected as the empty control, received 50%ethanol intracolonic instillation on day 0 and normal saline intraperitoneal injection for following days.The treatment was consecutive for 5 days and mice(eight per group)were killed on day 7 and day 14 after induction of colitis.2.Tissue preparation and processWeight of mice as well as diarrhoea was recorded daily after induction of colitis. Stool occult blood was surveyed to evaluate disease activity index(DAI).Each group of mice was anesthetize by intra-peritoneal injection of 10%chloral hydrate at the day 7 and 14,and were killed.Colon tissues were collected and stored respectively according to the following protocols.(1)0.5cm specimens were taken from the area of the treated colon segment. Samples were fixed in 4%paraformaldehyde and 2.5%glutaral for hematoxylin-eosin (HE)stain,immunohistochemistry,terminal deoxynucleotidyl transferase-mediated dUTP(TUNEL)and transmission scanning electron microscope.(2)Colon specimens was homogenated for measurement of myeloperoxidase (MPO)after removing water with filter and weighing.(3)The other colon tissues were collected,then put it in liquid nitrigen immdiately and stored at -70℃for measurement of mRNA for Real time quantitative RT-PCR and protein for Western Blotting.(4)Splenocytes and mesenteric lymph node(MLN)cells were isolated for flow cytometry.3.Experimental methods and analysis index(1)The appearance,weight of mice as well as diarrhoea was recorded daily.(2)The pathology change of the colon.①Assessment of macroscopic damage.②Assessment of microscopic damage by the light microscope.③Ultramicrostructure changes and villi of colon tissues were examined by transmission electron microscope(TEM).(3)The content of myeloperoxidase(MPO)in colon tissues was measured by histomedical method.(4)The expression of TNF-α,TLR4,NF-κBp65 protein were evaluated by immunohistochemistry. (5)Apoptosis of epithelial cells by TUNEL.(6)The expession of TLR4,NF-κBP65,Bcl-2 and Bax mRNA were determined respectively by Real time quantitative RT-PCR.(7)Western Blotting analysis for TLR4,NF-κBP65.(8)Apoptosis of Splenocytes and mesenteric lymph node(MLN)cells and expression of Fas,Bcl-2 were monitoring by flow cytometry.4.Statistical analysisSPSS version 13.0 was used to perform statistical analysis,with all data were presented as means±SD.One way analysis of variance and Independent t-test was used for statistical analysis of the differences between the groups.A value of P＜0.05 was considered statistically significant.ResultsClinical manifestation of miceAfter TNBS/ethanol intracolonic administrated,the mice in TNBS group appeared lazy,dispirited,anorexia,diarrhea,bloody stool and abdominal distention.The symptoms above were obvious on the 3 days later,and such symptom as tachypnea, cyanosis in lip,less activity,mess body hairs without gloss appeared.Stool occult blood is +～++++.The seriously sick mice showed spasm,cyanosis in limbs,chilly body and even dead.The body weight of survivor descend most on the second day, with the average descent was 9.87%in TNBS group.On the 7th day,the average body weight descent was 8.19%,then it to approach the normal body weight without induction of colitis on the 14th day.Whereas in ITF group,the clinical manifestation lessened compared with TNBS group.In ethanol group,the mice were survived well.The pathological findings of colonic tissues1.Macroscopic presentationMacroscopic examination of the colon after TNBS induction showed colonic mucosal injury,hyperaemia,oedema,erosion,and ulceration.The symptoms on the 7th day were severe,then improved on the 14th day.The macroscopic score in TNBS group was much higher than that in ethanol group.Compared with TNBS group, Striking differences were observed in animals treated with TFF3.Macroscopic scores for ITF group,was lower than those for TNBS group. 2.Histological evaluationAlmost no colonic damage was found in ethanol group,but the histological manifestation in mice with TNBS induced colitis in TNBS group showed frank ulceration was apparent with a total loss of epithelium in some cases.The remaining epithelium was often vacuolated and necrotic,and gross intramural inflammatory infiltrate was apparent in all animals.A large number of neutrophil,monocyte,and eosinophil infiltration in mucosa and submucosa,ulceration,and mucosal damage. Compared with TNBS group,after TFF3 treatment,the epithelium in ITF group was mainly intact,with only occasional small lesions.Branching glands characteristic of epithelial rebuilding were common,and the extent of inflammatory cell penetration (mainly neutrophils and macrophages)was reduced.3.The change of colonic tissues in TEMEpithelial cells are pillar-like,cell nuclear was orbicular-ovate.The colon villi of common enterocyte lined up in order,tight junction integrity,endoplasmic reticulum and mitochondria clearly in the controls.In TNBS group,it could be seen cell nucleus condense,chromoplasm margin,break and depletion of part if microvilli,widen and disrupted tight junction.The change of colonic tissues achieve the peak on the 7th day, while the injury improved on the 14th day.The changes of colon in ITF group lightened compared with TNBS group.4.The DAI scoring of miceThe DAI scoring in TNBS group was higher than that in ethanol group on the 7th, 14th day(p＜0.01).The DAI scoring in ITF group was lower than that in TNBS group on the 7th,14th day(p＜0.01).Tissue MPO activityA pronounced inflammatory response after TNBS induction was confirmed in TNBS group by the significant rise in MPO activity.But striking differences were seen in ITF group,which significantly decreased(p＜0.05).Tissue MPO activities in ethanol group were lowest.The apoptosis of colonic epitheliumThe colonic epithelium in the TNBS group contains more apoptotic cell than that in the ITF group.Apoptotic activity in the TNBS group was higher than that of ITF group(p＜0.01).The colonic epithelium in the ethanol group contains few apoptotic cells.The apoptosis of splenocytes and mesenteric lymph node(MLN) cells and the regulating factorThe number of apoptotic splenocytes and mesenteric lymph node(MLN)cells in TNBS and ITF groups were lower than those in control group(P＜0.01).There were no significant difference between two groups.The number of cell expressing Fas in splenocytes and MLN cells in the TNBS group was lower than that in the control group (P＜0.01).For Bcl-2,there were no statistics difference among three groups.The expression of TNF-α,TLR4,and NF-κB p651.Immunohistochemical assessment of TNF-α,TLR4,and NF-κB p65 expressionThe expression of TNF-α,TLR4,and NF-κB p65 protein were all upregulated in colitis mice compared with the normal animals.The expression in ITF group was decreased compared with TNBS group.2.Western blotting assay for TLR-4,NF-κB p65 protein expressionWestern blotting analysis showed that there was weak expression of TLR4 and NF-κBp65 protein in ethanol group(p＜0.01).TLR4 and NF-κBp65 protein was significantly increased in TNBS group,but obviously decreased in ITF group compared with TNBS group(p＜0.01).TLR-4,NF-κBp65,Bcl-2 and Bax mRNA expression in colonic tissueTLR4,NF-κBp65 and Bax genes were all upregulated in colitis mice compared with the normal animals.Transcriptional levels of TLR4,NF-κB p65 in ITF groups were downregulated compared with TNBS group(p＜0.01).Bcl-2 gene was downregulated in colitis mice compared with the normal animals.Transcriptional levels of Bcl-2 in ITF groups were upregulated compared with TNBS group(p＜0.01).Conclusions1.ITF can relieve histopathological damage of colonic tissue in trinitrobenzene sulphonic acid induced inflammatory bowel disease in mice,and promote the reparation of damaged tissue.2.Intraperitoneal injection of ITF is an convenience and effective research method to trinitrobenzene sulphonic acid induced inflammatory bowel disease in mice.3.TLR4/NF-κBp65 mRNA and proteins expression was up-regulated and cytokine TNF-αwas high-expression of colonic tissue in trinitrobenzene sulphonic acid induced inflammatory bowel disease in mice.4.One of the mechanism of Intraperitoneal injection of ITF was down-regulated TLR4/NF-κBp65 mRNA and proteins expression and inhibit cytokine TNF-αactivation resulting in protection for colitis in murine models of IBD.5.The colonic epithelium mucosa of trinitrobenzene sulphonic acid induced inflammatory bowel disease in mice consists excess apoptosis,which may concern with the ratio ofBcl-2/Bax mRNA.6.ITF can down-regulate colonic epithelium mucosae apoptosis of trinitrobenzene sulphonic acid induced inflammatory bowel disease in mice,and the mechanism was in part due to regulate the ratio of Bcl-2/Bax mRNA.7.The asplenocytes and mesenteric lymph node(MLN)cells of trinitrobenzene sulphonic acid induced inflammatory bowel disease in mice consists insufficient apoptosis,which may concerned with the Fas/FasL signal transduction.8.ITF may have no regulation to apoptosis of the asplenocytes and mesenteric lymph node(MLN)cells from colitis in murine models of IBD.