The Therapeutical Effect And Mechanism Of Triptolide In Ameliorating Anastomosis Inflammation And Fibrosis Of IL-10^-/-Mice Receiving Ileocaecal Resection

Crohn's disease(CD),which is one category of inflammatory bowel disease(IBD),represents a group of chronic intestine inflammation,damage and distortion of tissue architecture.Based on studies in human and in animal colitis model it was considered that acquired immune as well as activated innate immunity play an important role in CD,although the definite pathogens are still unknown.Surgery is not a curative method for CD and recurrences after surgery are common,but surgeries are still required by 70%-75%of CD patients if drug treatment fails or to correct complications such as stenosis and bowel obstruction.ICR is most common in CD among many surgery interventions as ileum and caecum are the commonly onset regions of CD.Reoperatipons were often needed owing to the frequent anastomotic recurrences in patients underwent surgical intervention.To prevent the relapse of CD in anastomosis is very important as the high rate of recurrence of anastomostic inflammation and fibrosis.Numerous animal models were developed to explicate the pathogenesis of CD.The model of interleukin-10 deficient(IL-10-/-)mice reported to spontaneously develop a Thl-mediated chronic colitis has many similarities to CD except the onset of small intestine(SI).A model of ICR in IL-10-/-mice provides us a completely new method to research the postsurgical inflammation and fibrosis in the SI and anastomosis and it may be useful for preclinical testing during postsurgical inflammation as it was reported recently.As an active component isolated from Chinese herb Tripterygium wilfordii Hook F(TWHF),triptolide has a potent anti-inflammatory and immunosuppressive properties and has beenused widely to treat autoimmune diseases for many years in China.Numerous reports show that triptolide inhibits the expression of RPB1(the RNA polymerase II main subunit),XPB(a subunit of TFIIH)and NF-?B-mediated transcription.Recent studies have shown that triptolide inhibited the induction of miR-155 in LPS-stimulated macrophages.MicroRNAs(miRNAs)comprise a novel class of nucleotide between 19 and 24 bases in length,which are noncoding and post-transcriptionally regulate expression of 30%of the genes in eukaryotic organisms.Encoded within a region known as B cell integration cluster(Bic)gene,miR-155 has a powerful ability in regulating immune cells such as T cells,B cells and dendritic cells(DCs).Src homology 2-containing inositol phosphatase-1(SHIP-1),a target of miR-155,is a potent inhibitor of many inflammatory pathways.Recent studies have discovered the key role for SHIP-1 in controlling Thl/Th2 bias and regulating Tregs as well as Th17 cells.As the balance of T cell subset plays a significant role in CD,we speculated that the miR-155/SHIP-1 pathway may be an important pathway in the pathogenesis of CD.Intestinal fibrosis,defined as an excessive accumulation of scar tissue in the intestinal wall,is a common complication of CD which is associated with long-lasting,debilitating consequences that negatively affect patients' quality of life.TNF-? is one of the most important factors which are associated with intestinal fibrosis.Up-regulation of miR-155 which has a powerful ability in regulating immune cells such as T cells,B cells and dendritic cells(DCs)may contribute to increased production of TNF-?.The relationship of these two factors was called TNF-?/miR-155 axis.So we hypothesis that triptolide may ameliorate the fibrosis of intestinal tract by attenuating TNF-a/miR-155 axis.The purpose of this study is to investigate the therapeutic effect of triptolide in the new model of IL-10-/-mice receiving ICR.Our objective was twofold:first,to establishe the model of IL-10-/-mice performed ICR and evaluation of this model was performed;second,to investigate the therapeutic effect of triptolide on ameliorating the inflammation and fibrosis of anastomosis by attenuating miR-155/SHIP-1 pathway and TNF-a/miR-155 axis separately.PART1The development and evaluation of the model of ileocaecal resection in IL-10-/-miceObjective:To investigate the feasibility of developing the model of ileocaecal resection(ICR)in IL-10-/-mice and to observe the severity of inflammation of fibrosis of anastomosis.Methods:Wild type mice were assigned to negative surgery(NS)group.IL-10-/-mice were randomized to positive surgery(PS)group and control group.All mice of NS group and PS group were performed ICR,the disease activity index(DAI)was evaluated every week.Mice were weighed and sacrificed under anesthesia with pentobarbital 8 weeks after the start of experiment.The intestinal samples were paraffin-embedded,sectioned,and stained with hematoxylin and eosin,CD4 monoclonal antibody and Sirius red respectively.The histologic score,analyses of the number of CD4 antibody stained cells per area and fibrosis score were also been demonstrated.The levels of myeloperoxidase(MPO),IL-1?,transforming growth factor-?(TGF-?)in intestinal tissue and calprotectin in feces were measured by enzyme linked immunosorbent assay(ELISA).Results:The severity of inflammation and fibrosis of anastomosis in PS group were significantly higher than NS group.The histologic score,CD4 antibody stained area and fibosis score of PS group were also higher than NS group.The levels of MPO,IL-1p,TGF-? in intestinal tissue and calprotectin in feces of PS group were increased compared with NS group.Conclusion:The inflammation and fibrosis of anastomosis of IL-10-/-mice performed ICR were more severe than WT mice performed ICR.The model of ICR in IL-10-/-mice provides us a new way to investigate the postsurgical inflammation and fibrosis of Crohn's disease patients' anastomosis.PART 2-1The therapeutical effect and mechanism of triptolide in ameliorating anastomosis inflammation of IL-10-/-mice performed ileocaecal resectionObjective:To investigate the therapeutical effect of triptolide in ameliorating anastomosis inflammation of IL-10-/-performed ICR and its effect on miR-155/SHIP-1 signaling pathway.Methods:IL-10-/-mice were randomized to Saline-treated ICR(ST-ICR)group and Triptolide-treated ICR(TT-ICR)group.All mice of these two groups were performed ICR,the disease activity index(DAI)was evaluated every week.All mice of ST-ICR group wereintraperitoneally injected saline for 8weeks,and mice of ST-ICR group were intraperitoneally injected triptolide for 8weeks.All mice were weighed and sacrificed under anesthesia with pentobarbital 8 weeks after the start of experiment.The intestinal samples were paraffin-embedded,sectioned,and stained with hematoxylin and eosin,CD4 monoclonal antibody.The histologic score,analyses of the number of CD4 antibody stained cells per area were also been demonstrated.The levels of MPO,IFN-y,IL-6,IL-17,and IL-4 in intestinal tissue and calprotectin in feces were measured by ELISA.The level of miR-155 was measured by quantitative real time polymerase chain reaction(qRT-PCR)and the level of SHIP-1 was determined by Western blot.Results:The severity of inflammation of anastomosis in TT-ICR group was significantly lower than ST-ICR group.The histologic score,CD4 antibody stained area were also lower than ST-ICR group.The levels of MPO,IFN-y in intestinal tissue and calprotectin in feces of TT-ICR group were decreased compared with ST-ICR group,but no differences of the levels of IL-6,IL-17,and IL-4 in intestinal tissue were seen between two groups.The level of miR-155 in TT-ICR group was lower than ST-ICR group,however the level of SHIP-1 was increased compared with ST-ICR group.Conclusion:Our results suggested that triptolide attenuate the anastomosis inflammation of IL-10-/-mice performed ICR by the mechanism involving downregulation of miR-155/SHIP-1 signaling pathway.PART 2-2The therapeutical effect and mechanism of triptolide in ameliorating anastomosis fibrosis of IL-10-/-mice performed ileocaecal resectionObjective:To investigate the therapeutical effect of triptolide in ameliorating anastomosis fibrosis of IL-10-/-performed ICR and its effect on TNF-?/miR-155 axis.Methods:IL-10-/-mice were randomized to Saline-treated ICR(ST-ICR)group and Triptolide-treated ICR(TT-ICR)group.All mice of these two groups were performed ICR,the disease activity index(DAI)was evaluated every week.All mice of ST-ICR group were intraperitoneally injected saline for 8weeks,and mice of ST-ICR group were intraperitoneally injected triptolide for 8weeks.All mice were weighed and sacrificed under anesthesia with pentobarbital 8 weeks after the start of experiment.The intestinal samples were paraffin-embedded,sectioned,and stained with Sirius red,the fibrosis score were also been demonstrated.The levels of IL-1?,TGF-? were measured by ELISA.Procollagen al were measured by qRT-PCR,the level of TNF-? was determined by ELISA and qRT-PCR.Results:The fibrosis score and level of procollagen al of anastomosis in TT-ICR group were significantly lower than ST-ICR group.The levels of IL-1?,TGF-? were also decreased compared with ST-ICR group.The levels of TNF-? measured by ELISA and qRT-PCR were significantly lower than ST-ICR group.Conclusion:Our results suggested that triptolide attenuate the anastomosis fibrosis of IL-10-/-mice performed ICR by the mechanism involving downregulation of TNF-a/miR-155 axis.