Effects And Mechanism Of Conditioned Medium Derived From Bone Marrow Mesenchymal Stem Cell With Inflammatory Activation On Regeneration Of Radiation-induced Intestinal Injury In Vitro

BACKGROUNDRadiation-induced intestinal injury often occurs after radiotherapy of abdominal and pelvic malignancies,with clinical manifestations of abdominal pain,diarrhea,bloody stools,malnutrition,and life-threatening complications such as sepsis and septic shock,which seriously affect the quality of life of tumor patients.However,there is no ideal treatment for radiation intestinal injury.Mesenchymal stem cells(MSCs)have strong self-differentiation and paracrine ability and are widely used in tissue damage repair.Conditioned medium derived from mesenchymal stem cells(MSC-CM)is rich in a variety of paracrine factors secreted from mesenchymal stem cells(MSCs)and has no risk of stem cell transplantation,making it an ideal alternative to stem cell transplantation.Our previous study has demonstrated that MSC-CM with inflammatory activation improves the structural and functional restoration of the small intestine after radiation-induced intestinal injury and improves the survival status of rats with acute radiation injury,but its potential cellular mechanism has not been further explored.OBJECTIVETo observe the effect of MSC-CM with inflammatory activation on the proliferation and apoptosis of intestinal epithelial cells(IEC-6)after radiation injury and to investigate the paracrine change of pre-activated MSC-CM;,thus provides theoretical basis and laboratory basis for the optimization therapeutic effect of MSC-CM.METHODS1.IEC-6 cells were divided into four groups:control group,radiation injury group,normal MSC-CM(MSC-CMIEC-6(NOR))group and inflammatory pre-activated MSC-CM(MSC-CMIIEC-6(IR))group.IEC-6 cells in the latter three groups were exposed to 10 Gy X-ray irradiation and cultured in DMEM-F12 medium,MSC-CMIEC-6(IR)and MSC-CMIEC-6(NOR)respectively.Cells in the control group were only cultured in DMEM-12 medium.The number of survival of 10 Gy-irradiated IEC-6 in all groups were evaluated at 1,3,5,7 days after radiation.Cultured cells were measured at collected at 3 days after radiation to observe the proliferation of IEC-6 cells by using proliferating cell nuclear antigen immunofluorescence staining,and to observe the apoptosis by using TUNEL apoptosis staining and western blot assay.2.Using ELISA Kit to identified the level of paracrine facotors in pre-activated MSC-CM.RESULTS AND CONCLUSION1.Compared with the radiation injury group and MSC-CMIEC-6(NOR)group,the number of PCNA positive cells in MSC-CMIEC-6(IR)group increased significantly(P<0.05),the number of TUNEL positive cells decreased(P<0.05),and the expression of apoptosis-related protein Caspases-3 decreased significantly(P<0.05),which indicates that the conditioned medium from MSCs under radiation-induced inflammatory condition(MSC-CMIEC6(IR),but not non-activated MSC-CM,improves survival of radiation-injured IEC-6 cell by regulating cellular homeostasis via effects on the apoptosis and proliferation of intestinal epithelium cell.2.Using cytokine array,we identified some molecules such as VEGF,bFGF,IGF-1 and IL-10 significantly up-regulated in MSC-CMIEC-(IR).These factors are associated with angiogenesis,apoptosis inhibition and cell regeneration,which suggests that the upregulation of the above factors may facilitation in the repair of radiation intestinal injury.