Paracrine Effect And Mechanism Of Bone Marrow Mesenchymal Stem Cells With Inflammatory Activation On Inflammatory Response Of Radiation-induced Intestinal Injury

BACKGROUNDAcute radiation-induced intestinal injury(RⅢ)is common in the abdominal cavity,pelvic malignant tumor radiotherapy patients.As high as 90-95%of the patients could have varying degrees of acute radiation-induced intestinal injury.Symptoms such as frequent vomiting,diarrhea and hematochezia may occur clinically,and severe patients may present sepsis,anemia or emaciation,which could seriously affect the quality of life of patients and the further application of radiotherapy.At present,the mechanism of acute radiation-induced intestinal injury is not completely clear,and there is no effective treatment in clinic.Therefore,it is urgent to explore the pathogenesis of acute radiation-induced intestinal injury in order to find more effective treatment Previous studies have shown that the pathogenesis of acute radiation-induced intestinal injury is essentially inflammation.Recent studies have shown that bone marrow mesenchymal stem cells(BMSCs)have powerful anti-inflammatory and immunomodulatory properties.Our previous experiments have demonstrated that conditioned medium from bone marrow MSCs with inflammatory activation(MSC-CMIEC-6(IR)could improve the structural and functional repair of the small intestine after acute radiation-induced intestinal injury,but its effect and mechanism on inflammatory response to acute radiation-induced intestinal injury have not been defined.In this experiment,we investigated the effect and mechanism of conditioned medium derived from bone marrow mesenchymal stem cells with inflammatory activation on inflammatory response to acute radiation-induced intestinal injury.METHODS1.Isolation,Culture and Identification of bone marrow mesenchymal stem cells.The phenot)ypic CD29,CD34,CD44,CD45 was identified by flow cytometry after primary isolation,culture and passage.2.Preparation of bone marrow mesenchymal stem cells conditioned medium.Preparation of bone marrow mesenchymal stem cells conditioned medium with Nflammatory activation(MSC-CMIEC-6(IR)and normal bone marrow mesenchymal stem cells conditioned medium(MSC-CMIEC-6(nor),and then all of them were stored in refrigerator.3.Seventy Sprague-Dawley rats were randomly divided into four groups:control group(n=10),irradiation group(IR+DMEM-F12 group)(n=20),irradiation+MSC-CMIEC-6(NOR)group(IR+MSC-CMIEC-6(NOR)group)(n=20)and irradiation+MSC-CMIEC-6(IR)group(IR+MSC-CMIEc-6(IR)group)(n=20),followed by continuous administration via the tail vein and abdominal cavity.Intestinal tissue and serum samples were collected 3 days after radiation for hematoxylin-eosin staining and analysis of the concentratons of inflammatory factors via ELISA(n=10/group).The Kaplan-Meier method was used for analyzing the survival rate of irradiated rats(n=10/group).4.The concentrations of tumor necrosis factor activator gene 6(TSG-6)protein,Interleukin 10(IL-10),transforminggrowth factor β1(TGF-β1)and indoleamine 2,3-dioxygenase(IDO)in MSC-CMIEC-6(IR)and MSC-CMIEC-6(NOR)were analysed via ELISA respectively.5.Mesenteric lymph nodes were collected 3 days after radiation for analysis of the percentage of CD4 Foxp3+Treg cells via flow cytometry(n=10/group).RESULTS1.Compared with the IR+DMEM-F12 group and IR+MSC-CMIEC-6(NOR)group,the intestinal structure(small intestine epithelial villi and crypt)was significantly improved in the IR+MSC-CMIEC-6(IR)group,and survived longer(p<0.05).2.The changes in the levels of inflammatory factors were most significant 3 days after irradiation.Compared with the IR+DMEM-F12 group and IR+MSC-CMIEC-6(NOR)group,the pro-inflammatory factors such as interleukin-1β,interleukin-6 and tumor necrosis factor-α in the small intestine were significantly decreased whereas the level of anti-inflammatory factor interleukin-10 was significantly increased in the IR+MSC-CMIEC-6(IR)group(P<0.05).Similarly,the levels of pro-inflammatory fectors such as Activin A,interleukin-1β,interleukin-6 and tumor necrosis factor-a in the serum samples were significantly decreased and the level of anti-inflammatory factor IL-10 was significantly increased(P<0.05).3.Compared with the MSC-CMIEC-(NOR)group,the concentrations of tumor necrosis factor activator gene 6 protein,Interleukin 10,transforming growth factor β1 and indoleamine 2,3-dioxygenase(IDO)in MSC-CMIEC-6(IR)were significantly increased in the MSC-CMIEC-6(IR)group(P<0.05).4.The percentage of CD4+Foxp3+Treg cells in mesenteric lymph nodes in the IR+MSC-CMIEC-6(IR)group was significantly higher than any other group(P<0.05).CONCLUSION1.These results suggest that MSC-CMIEC-6(IR)can inhibit the whole body level and intestinal mucosal inflammation in rats with acute radiation-induced intestinal injury,and regulate the immune response in inflammatory process,improve the repair of intestinal mucosal injury,and meanwhile prolong the survival time of the rats.2.The mechanism of MSC-CMIEC-6(IR)regulating intestinal inflammatory response in rats with acute radiation-induced intestinal injury may include:(1)MSCs with inflammatory activation may secrete anti-inflammatory factors to regulate the balance of proinflammatory factor/anti-inflammatory factor.(2)MSC-CMIEC-6(IR)may promote the transformation of t lymphocytes into CD4+Foxp3+ Treg cells in the mesenteric lymph nODes of the intestine,and increase the percentage of CD4+Foxp3+ Treg cells,thus inhibiting the inflammatory response at systemic and mucosal levels in acute radiation-induced intestinal injury.