The Experimental Study On The Intervention Of Human Umbilical Cord Mesenchymal Stem Cells In Rat Ileal Pouchitis

Objective:Pouchitis is a major complication after total colorectal resection and ileal pouch-anal anastomosis?IPAA?,the incidence of which in patients with ulcerative colitis is high.Severe cases may even result in waste pouches,permanent ileostomy,and significantly reduce postoperative quality of life.However,the pathogenesis still remains unclear.Mesenchymal stem cells?MSCs?are non-hematopoietic stem cells with multi-directional differentiation potential.Their self-renewal and differentiation potential can exert inflammatory inhibition through various mechanisms,and promote tissue regeneration and repair,for which they have been widely used in the treatment of many diseases and show a significant effect.However,there is not any relevant research about MSCs in the area of pouchitis.This study aims to study the effect of intraperitoneal injection of human umbilical cord mesenchymal stem cells on pouchitis by establishing a stable dextran surfate sodium?DSS?-induced ileal pouchitis rat model and explore the mechanism.Methods:1.18 clean male SD rats weighing 300-340 g were randomly divided into DSS group,IPAA group and Sham group?n=6?.Rats in DSS group and IPAA group underwent IPAA procedure and rats in DSS group received 4%DSS ad libitum for 4consecutive days from the 31^st days after surgery.Rats in Sham group underwent switch abdominal surgery.Body weight,food intake,water intake,fecal score,bloody stool,and disease activity index?DAI?were recorded postoperatively.At the 35^th day after operation,the ileal specimens from Sham group and the pouch specimens from DSS group and IPAA group were collected for HE staining to evaluate histopathological scores,immunohistochemistry to evaluate the expression of myeloperoxidase?MPO?,PCR and ELISA to detecte the mRNA and protein expression levels of the inflammatory factors IL-1?,IL-6,IL-10,and TNF-?.2.The MSCs were derived from human umbilical cord,stably cultured in vitro,and transfected with the lentivirus expressing the fluorescent protein to trace the dynamic distribution of the stem cells.Rat models of pouchitis were established and randomly divided into MSC group,PBS group,and DSS group?n=12 per group?.Intraperitoneal injection of 2×10⁶ MSCs once,1 ml of PBS once and no special intervention were respectively performed to rats in each group.The changes in body weight and fecal score,bloody stools,and DAI were recorded.6 rats were sacrificed in each group at the 4^th day after the administration,and the remaining 6 rats were sacrificed at the 7^th day.The pouch specimens were obtained for histopathological score,MPO expression level,IL-1?,IL-6,IL-10 and TNF-?mRNA and protein expression levels.Results:1.The body weight of rats began to decline immediately after IPAA procedure,gradually increased after reaching the minimum on the 11^th day and reached the preoperative weight at about the 22^nd day after the operation.Meanwhile,the food intake and water intake returned to normal levels.There was no significant difference in fecal scores between IPAA group and Sham group on the 30^thh postoperatively?P=0.718?.After induced by DSS,the experimental rats showed apathetic,weight loss,bloody stools,and significantly increased fecal score?P=0.002?.Compared with IPAA group and Sham group,DAI in DSS group was significantly higher?P=0.003?,mucosal ulceration,erosion,and villous structure destruction were observed in the intestinal mucosa,and histopathological scores increased?P=0.004,P=0.003?,the expression of MPO protein increased?P<0.001?,the mRNA and protein expression of IL-1?,IL-6,TNF-?increased?P<0.01?,and the mRNA and protein levels of IL-10 were reduced?P=0.004?.2.After intraperitoneal injection of MSCs,the accumulation towards the pouch was evident,and the fluorescence imaging disappeared after 4 days of injection.The mental status of rats in MSCs group was improved,body weight gradually increased,bloody stools disappeared,and fecal scores were significantly reduced?D4:P=0.023,D7:P=0.020?.Besides,congestive erosions on intestinal wall disappeared,mucosal was smooth and intact,and villus grew.Whereas the body weight of rats in PBS group and DSS group increased slowly after 5 days of withdrawal of DSS solution,the fecal score decreased at the 7^th day after discontinuation?P=0.020,P=0.023?,and the villus destruction was improved.At the 4^th day after MSCs treatment,DAI was lower than that of the other two groups?P=0.003,P=0.002?,the histopathology score was decreased?P=0.002,P=0.003?,the expression of MPO protein was decreased?P=0.004,P=0.004?,the mRNA and protein levels of IL-1?,IL-6,TNF-?decreased?P<0.01?,and the mRNA and protein levels of IL-10 increased significantly?P=0.004?.At the 7^th day after MSCs treatment,DAI was lower than that of the other two groups?P=0.018,P=0.018?,the histopathology score was decreased?P=0.002,P=0.003?,the expression of MPO protein was decreased?P=0.004?,the mRNA and protein expression of IL-6 decreased?P<0.01?and the mRNA and protein levels of IL-10 significantly increased?P=0.004?.Conclusions:1.The experimental ileal pouchitis model after IPAA procedure in rats was stably established,which can be used for subsequent experimental studies.2.Intraperitoneal injection of MSCs has a therapeutic effect on DSS-induced ileal pouchitis.3.The therapeutic effect of MSCs on rat pouchitis may be through reducing the expression of pro-inflammatory factors and up-regulating the expression level of anti-inflammatory factors so as to reduce the inflammatory damage.