Roles Of RIP1 In Regulation Of Intestinal Epithelial Repair After Injury

Background and Objective:Inflammatory bowel disease(IBD)is a chronic disease with nonspecific inflammation occurs in the gut.The incidence of IBD in China is increasing year by year with the improvement of living standards.The occurrence and development of IBD are affected by many factors,including diet,environment,genetics and so on.Therefore,it is important to understand the mechanism of occurrence and development of IBD.Receptor-interacting protein kinase 1(RIP1)is a serine/threonine protein kinase that is abundantly expressed in intestinal epithelial cells.RIP1 is involved in regulating many physiological processes such as cell survival,apoptosis,and necroptosis.It has reported that epithelial barrier dysfunction is often detected in IBD patients.The integrity of epithelium is important to maintain the gut homeostasis.In the physiological state,the gut epithelium is quickly renewed every 5-7 days.Therefore,the proliferation and death of epithelial cells must be strictly regulated to maintain the integrity of the epithelium.In this study,we aim to answer two questions.1)to determine whether RIP1 affects the proliferation and differentiation of intestinal epithelial cells;2)to determine whether RIP1 knockdown affects the epithelial repair after injury.Our study will help to understand the mechanism how RIP1 promotes epithelial barrier repair after colitis,and this will be beneficial to IBD treatment.Methods:1.RIP1 haplodeficiency(RIP1+/-)mice were generated using the knockout first strategy.The epithelial morphology of small intestine and colon was assessed by paraffin sectioning and H&E staining.To determine the effect of RIP1 on epithelial cell proliferation and differentiation,immunohistochemistry staining and qPCR were used to detect the expression of active stem cells marker Lgr5 and quiescent stem cells marker Bmi1.2.To establish the experimental colitis mouse model,RIP+/-and wildtype mice were caged with drinking water containing 3%DSS for seven days and followed by fresh water for additional two days.After cervical dislocation,the length of the colon was measured,and the colon tissue and colonic epithelial cells were collected and stored in liquid nitrogen.Tissue and cell samples were used to extract mRNA for detection of proliferation related genes by qPCR.The colon tissue was collected,fixed with 4%formaldehyde,embedded in paraffin.Tissue sections were stained with H&E and immunohistochemistry to analyze the expression and location of markers for cell proliferation and diferentiation.3.Crypts were isolated from both RIP1+/-mice and wildtype mice and cultured as intestinal organoids in vitro.Organoids were collected and frozen with liquid nitrogen.Samples were used to extract mRNA for detection the expression of genes related to proliferation,differentiation,and NF-κB signaling pathway by qPCR.4.RIP1 knockdown L929 cell lines were generated by lentivirus infection.Cells were stained with PI/Hoechst to detect the effect of RIP1 knockdown on cell survival.To induce cell death,cells were treated with zVAD,or necroptosis inhibitor Necrostain-1(Nec-1).Cell survival and cell death were monitored by time-lapse microscopy.The expression of cell death proteins were determined by Western blot.Results:Our study used the knockout first technique to generate mouse model with global knockdown of RIP1.Compared to wildtype mice,the expression of RIP1 protein in intestinal and colon epithelial cells reduced to 46.98±9.79%and 60.32±8.10%,respectively,in RIP1+/-mice.H&E staining showed that RIP1+/-mice had a normal epithelial structure without infiltration of immune cells of the small intestine and colon.The number of ChgA+ enteroendocrine and Muc2+ goblet cells in the villus and the number of Lgr5+ and Bmi1+ stem cells in the crypt were comparable between RIP1+/-and wildtype mice.However,the length of the villus and crypt was shorter in RIP1+/-mice than wildtype mice.In addition,the number of Ki67+ cells in the crypt was significantly reduced in RIP1+/-mice.RIP1+/-mice and wildtype mice were treated with DSS to induce acute colitis.Compared with wildtype mice,RIP1+/-mice loss weight quickly and had a short colon after DSS treatment.The number of Ki67+ cells in the crypts was nearly undetectable,and the expression of proliferation-related genes and stem cell marker genes was significantly reduced in RIP1+/-mice treated with DSS.Interestingly,the number of ChgA+ cells increased but the expression of Muc2 decreased in RIP1+/-mice treated with DSS.Similar results were observed in intestinal organoids.Compared with organoid cultured from wildtype mice,organoids from RIP1+/-showed reduced expression of proliferation-related genes,stem cell marker genes,and different mature cell marker genes.Next,the expression of molecules of NF-κB signaling pathway was analyzed.The results showed that in RIP1+/organoids,the expression of some of the NF-κB signal-related genes including IKKβ and P65 reduced significantly.L929 cells are a an ideal cell line to investigate cell apoptosis and necroptosis.We generated RIP1 knockdown(KD)cell lines(RIP1KDL929(P1)).PI-positive cells increased significantly in RIP1KDL929(P1)cells,indicating that RIP1 is necessary to maintain the survival of L929 cells.To induce cell death,RIP1KD L929(P4)cells were treated with zVAD.Nec-1 completely suppressed cell death of the control cells but only partially inhibit cell death of RIP1KD L929(P4)cells.This result indicates that RIP1 knockdown might induce some other kind of cell death other than necroptosis.Conclusions:Our study had successfully generated a RIP1 haplodeficiency(RIP 1+/-)mice model with reduced RIP 1 expression in the gut epithelium.Compared with RIP1-/-mice and RIP 1 intestinal epithelial specific knockout(RIP1ΔIEC)mice,RIP1+/-mice were survival,fertile,and apparently normal in the gut.In the physiological condition,RIP1 did not affect epithelial cell stemness and differentiation.Although RIP1+/-intestine showed low level of proliferative capability,it was still morphologically normal and showed no signs of immune cell infiltration.However,when treated with DSS,the proliferative capacity and number of stem cells in the colon were further reduced,and these may lead to unsuccessful healing of epithelium in RIP+/-mice.Surprisingly,the differentiation of mature secretory cells was also aberrant in RIP1+/-mice.Finally,our results suggested that RIP1 may regulate intestinal epithelial proliferation through the NF-κB pathway.RIP1 is required for the survival of L929 cells and RIP1 knockdown may lead to the switch of necroptosis to other kind of cell death in L929 cells.