The Mechanisms Of Triptolide In Relieving The Anastomotic Fibrosis In Interleukin-10 Knock Out Mice Undergoing Ileocecal Resection And Anastomosis

Part ?:Triptolide exerts protective effects against postoperative ileocolonic anastomosis inflammation and fibrosis in IL-10deficient miceObjective:To establish an animal model which can simulates postoperative condition of Crohn's disease?CD?patients by performing the ileocecal resection and anastomosis?ICA?in interleukin-10 knock out?IL-10KO?mice,and observe the pathological changes in ileocolonic anastomosis in the models which received different treatments including triptolide?TPL?.Methods:A model of IL-10KO mice undergoing ICA was used to study postsurgical inflammation and fibrosis of anastomosis.18 IL-10KO mice aged 8 weeks were randomly divided into 3 groups on average as follows:IL-10KO group?normal control,in which the mice had no surgery?,IL-10KO-ICA group?the saline-treated group subjected to ICA?and TT-IL-10KO-ICA group?TPL-treated group subjected to ICA?.12 Wild-type?WT?mice of matching ages were assigned to the WT group?normal control,in which the mice had no surgery?and WT-ICA group?the saline-treated group subjected to ICA?on average.The effects of TPL treatment on ileocolonic anastomosis were determined by histopathological evaluation,western blot analysis and ELISA.Disease activity index?DAI?was conducted according to the reports discribed previously,HE staining and inflammation score were used to evaluate the inflammation degree;Immunohistochemical?IHC?staining with CD₄expression was used to measure the area of CD₄⁺cell infliltration;Masson staining and fibrosis score were employed to determine the severity of intestinal or anastomotic fibrosis.Western blot was performed to determine the Collagen 1?Col-1?and Collagen?Col-3?level.Enzyme linked immunosorbent assay?Elisa?was used to measure the level of transforming growth factor-??TGF-??,Tumor Necrosis Factor-??TNF-??,interferon-??IFN-??,IL-4,IL-6and IL-17.Results:?1?All mice were sacrificed by decapitation 8 weeks after surgery,obvious swelling and thickening in intestinal wall as well as luminal stenosis at the anastomosis site can be observed following ileocecal resection and anastomosis in IL-10KO-ICA group,mean colonic general score in IL-10KO-ICA group was significantly higher than other groups;While after treatment of TPL,all symptoms were all significantly relieved and the mean colonic general score in TT-IL-10KO-ICA was also significantly reduced compared with IL-10KO-ICA group.?2?DAI and CDAI score of IL-10KO group were both significantly higher than WT and WT-ICA group,while the two scores in IL-10KO-ICA group were significantly higher than IL-10KO group,and after treatment of TPL,DAI and CDAI score in TT-IL-10KO-ICA group were effectively reduced and significantly lower than IL-10KO-ICA group.?3?HE staining shows that intestinal wall of mice in IL-10KO-ICA group was obviously thickened with a large number of inflammatory cells infiltration,and the mean inflammation score of IL-10KO-ICA group was significantly higher than other groups;After intraperitoneal injection of TPL,the inflammation score in TT-IL-10KO-ICA group was significantly reduced than IL-10KO-ICA group.?4?IHC staining with CD4 expression shows that there are abundant CD₄⁺cells infiltration in the intestinal wall especially around anastomosis site,and the area of CD4+cell infiltration of IL-10KO-ICA group was significantly higher than other groups;After treatment of TPL,area of CD4+cell infiltration was obviously narrowed.Small amounts of CD4+cells infiltration in intestinal wall can also be observed in IL-10KO group;?5?Masson staining suggests that no obvious collagen deposition can be seen in WT group,While compared with WT group,collagen deposition in anastomosis was much more obvious in WT-ICA group.Collagen deposition in all layers especially submucosa of intestinal wall around anastomosis can be observed in IL-10KO-ICA group,and TPL can significantly inhibits collagen deposition of TT-IL-10KO-ICA group.?6?Fibrosis score of WT-ICA group was significantly higher than WT group,and compared with other groups,fibrosis score in IL-10KO-ICA group was also significantly higher.TPL treatment can effectively reduce fibrosis score of TT-IL-10KO-ICA group.?7?Western blot indicates that Col-1,3 level in WT-ICA group were significantly higher than WT group,besides,Col-1,3 level in IL-10KO-ICA group were significantly higher than other groups including WT,WT-ICA and IL-10KO group;After treatment of TPL,Col-1,3 were significantly inhibited in TT-IL-10KO-ICA group.?8?Results of Elisa suggested that along with the rise of inflammatory severity,the level of IL-4?IL-6?IL-17?TNF-??TGF-?and IFN-?ascended simultaneously.After the treatment of triptolide,the anastomotic concentration of all cytokines were decreased especially IL-6?TNF-??TGF-?and IFN-?.Conclusion:IL-10KO mice develop enterocolitis spontaneously;IL-10KO mice with ileocecal resection and anastomosis is a satisfactory animal model which effectively simulates the postoperative state of CD patients,including postoperative anastomotic inflammation and fibrosis;TPL is valuable in the prevention of postsurgical anastomotic inflammation and fibrosis,besides,TPL can attenuated expression of multiple cytokines that closely related to inflammation and fibrosis in IL-10KO mice performed ICAPart ?: TPL inhibits functions of fibroblasts which derived from anastomosis of IL-10 KO mice by mechanisms involving mi R-16-1/HSP70 signaling pathwayObjective: To elucidate the relationship between TPL and mi R-16-1/HSP70 pathway and identify if TPL influences functions of fibroblasts which derived from anastomosis of IL-10 KO mice by mechanism involving mi R-16-1/HSP70 signaling pathway.Methods: Fibroblasts were derived form anastomosis of IL-10 KO mice which underwent ileocecal resection and anastomosis.Immunofluorescence staining of vimentin was applied for identification of fibroblasts;RT-q PCR was used to determine the level of mi R-16-1 and HSP70,antagomir-16-1 and agomir-16-1 were employed to down and up-regulate mi R-16-1 respectively,then functional and morphological information of fibroblasts in different groups would be recorded.MTT assays were used to test proliferative ability and scratch-wound experiments were used to determine motility of fibroblasts;Western blot was performed to determine the level of HSP70,Col-1 and Col-3.Results:?1?IHC staining proved that cells derived from anastomosis of IL-10 KO mice which underwent ileocecal resection are vimentin positive;?2?TPL can significantly inhibited the expression of mi R-16-1 in fibroblasts;?3?Luciferase reporter assay indicated that HSP70 m RNA is direct target of mi R-16-1;?4?HSP70 level in fibroblasts can be significantly elevated by treatment of TPL;?5?MTT assay showed that proliferative ablity of fibroblasts can be significantly inhibited by either treatment of TPL or merely down-regulation of mi R-16-1;While if we up-regulate mi R-16-1 by agomir-16-1 in fibroblasts which pretreated with TPL,the proliferative ability of fibroblasts can be significantly enhanced compared with those only treated with TPL.?6?Likewise,scratch-wound experiments and wound healing rate both indicated that motility of fibroblasts can be significantly inhibited by either treatment of TPL or merely down-regulation of mi R-16-1;But after up-regulation of mi R-16-1 by agomir-16-1 in fibroblasts which pretreated with TPL,the motility ability of fibroblasts can be significantly enhanced compared with those only treated with TPL.?7?Results of western blot showed that the level of both Col-1 and Col-3 can be significantly decreased by either treatment of TPL or merely down-regulation of mi R-16-1;While after up-regulate mi R-16-1 in fibroblasts which pretreated with TPL,the level of Col-1 and Col-3 of fibroblasts can be significantly increased compared with those only treated with TPL.Conclusion: TPL can inhibits proliferative ability,motility as well as collagen synthesis ability of fibroblasts derived from anastomosis of IL-10 KO mice which underwent ileocecal resection and anastomosis,and mi R-16-1/HSP70 pathway plays an important role in this process.Part ?: mi R-16-1/HSP70 pathway takes part in the process of TPL ameliorating postoperative ileocolonic anastomosis fibrosis in IL-10 deficient miceObjective: To clarify the role of mi R-16-1/HSP70 pathway in the process of TPL ameliorating postoperative ileocolonic anastomosis fibrosis in IL-10 deficient mice which underwent ileocecal resection and anastomosis.Methods: Agomir-16-1 and antagomir-16-1 were used to up and down regulate mi R-16-1 respectively.General conditions of animals and severity of anastomosis fibrosis were recorded,besides,DAI,CDAI and fibrosis scores were used to determine the severity of disease in mice with different treatments.Western blot and RT-q PCR were used to determine the HSP70 level,and mi R-16-1 level was determined by RT-q PCR,Col-1,3 level were detected by Western blot.Results:?1?Antagomir-16-1 significantly suppressed the level of mi R-16-1 in ileoccolic anastomosis of IL-10 KO mice.?2?No obvious change of DAI and CDAI scores can be detected through down-regulation of mi R-16-1 for mice underwent ileocecal resection and anastomosis.?3?There was no significant change of fibrosis severity for IL-10 KO mice which treated by antagomir-16-1 than before,likewise,fibrosis scores were also showed no remarkable change before and after intraperitoneal injection of antagomir-16-1.What' more,down-regulation of mi R-16-1 failed to inhibit synthesis rate of Col-1 and Col-3.?4?TPL significantly inhibited expression of mi R-16-1 in anastomosis of IL-10 KO mice.?5?Intraperitoneal injection of agomir-16-1 reversed the suppressive effects of TPL on mi R-16-1 synthesis and significantly up-regulate the level of mi R-16-1 in ileocolic anastomosis of IL-10 KO mice.?6?DAI and CDAI scores were significantly raised in TPL-treated IL-10 KO mice by intraperitoneal injection of agomir-16-1.?7?Besides,overexpression of mi R-16-1 also significantly reversed the effects of TPL ameliorating postoperative anastomotic fibrosis in IL-10 KO mice.For mice which treated by TPL,overexpression of mi R-16-1 significantly up-regulated fibrosis scores in anastomosis.?8?Results of western blot also showed that the level of Col-1 and Col-3 can be significantly elevated by agomir-16-1 for mice which treated by TPL before.Conclusion: Mi R-16-1/HSP70 pathway takes part in the process of TPL ameliorating postoperative ileocolonic anastomosis fibrosis in IL-10 deficient mice which underwent ileocecal resection and anastomosis.