Ginsenoside RG1 Enhances The Paracrine Effects Of Bone Marrow-derived Mesenchymal Stem Cells On Radiation Induced Intestinal Injury And The Mechanism

Background and ObjectivesPatients with malignant abdominal tumors usually suffer from radiation-induced intestinal injury due to exposure to radiation.It is reported that 3%-10% of patients undergoing abdominal radiotherapy may have intestinal fistulas and even enterogenous sepsis due to radiotherapy.At present,the pathogenesis of radiation-induced intestinal injury is not clear,and clinical treatments such as drugs and surgery are not effective.Regenerative medicine oriented by mesenchymal stem cells has broad prospects in the treatment of myocardial infarction,Alzheimer’s disease,hepatitis B cirrhosis,radiation encephalopathy,radiation-induced intestinal injury and other diseases.In recent years,studies have shown that stem cells secrete many cytokines or nutrient factors to target cells or organs mainly through paracrine effect,playing an important role in regeneration and repair.Compared with unstable stem cell transplantation,the conditioned medium(CM)derived from bone marrow mesenchymal stem cells(BM-MSCs)has the characteristics of low tumorigenicity and low immunogenicity.Previous studies have suggested that the effect of CM is equivalent to or even better than stem cell transplantation.However,the therapeutic effect of stem cell-derived conditioned medium is closely related to the concentration of paracrine factors,and it is particularly critical to find ways to enhance the paracrine effect of stem cells.Here we examined if and how RG1 enhances the paracrine effects of bone marrow-derived mesenchymal stem cells(BM-MSCs)on radiation induced intestinal injury(RⅢ).MethodsIrradiated IEC-6 were co-cultured with CM from inactivated BM-MSCs(MSC-CM)or BM-MSCs pre-activated by RG-1(RG1-MSC-CM)or DMEM-F12 to screen the optimal RG1 concentration.The cellular proliferation,apoptosis,p53 expression,and inflammatory factor level were also assessed.Irradiated rats randomly received intraperitoneal injection of conditioned medium(CM)derived from non-activated BM-MSCs(MSC-CM)or BM-MSCs pre-activated by RG-1(RG1-MSC-CM).Intestinal samples were collected,followed by the evaluation of histological and functional change,apoptosis,proliferation,inflammation,angiogenesis and stem cell regeneration.Results(1)RG1 enhanced the paracrine effect of BM-MSCs on irradiated IEC-6 and1μM was the optimal dose.Compared with the control group,the survival rate,PCNA and p53 expression,apoptosis,and inflammatory factor levels in the RG1-MSC-CM group were significantly ameliorated.(2)RG1-MSC-CM improved the survival rate of rats by improving intestinal functiolen and histological changes.Compared with the control group,RG1-MSC-CM instead of MSC-CM can significantly improve the barrier and absorption function and structural damage of the intestinal tract of radiation-damaged rats,thereby improving the survival rate of rats.Among them,the average survival time of RG1-MSC-CM group,MSC-CM group and DMEM-F12 group was 12.4±3.9,6.3±4.7,5.5±4.3 days respectively.(3)RG1-MSC-CM significantly improved intestinal proliferation/apoptosis,inflammation,angiogenesis and stem cell regeneration in rats injured by radiation.Compared with the control group,RG1-MSC-CM but not MSC-CM can significantly improve the intestinal PCNA expression,TUNEL expression,inflammatory factor level,CD31+ expression,and Lgr5 expression in radiation-damaged rats.(4)The mechanism of the superior paracrine efficacy of RG1-MSC-CM is related to the high release of two key cytokines VEGF and IL-6.After RG1pre-stimulated MSC,three factors in the conditioned medium were significantly increased.The concentrations of VEGF,IL-6,and IL-10 were 2484.2±200.6 pg/ml,1525.6±212.5 pg/ml and 130.0±10.7 pg/ml respectively,which are 6,5,and 1.5 times higher than MSC-CM,respectively.the therapeutic effect of RG1-MSC-CM on apoptosis,proliferation,inflammation response and survival of irradiated rats was partially inhibited by antibodies against VEGF or IL-6 individually and further blocked by combined neutralization of VEGF and IL-6.In addition,combined injection of IL-6 and VEGF could exhibit a similar therapeutic effect as treated with RG1-MSC-CM.Conclusion1)RG1-MSC-CM,but not MSC-CM,improved survival and promoted the structural and functional restoration of RⅢ by regulating intestinal regeneration,inflammation and angiogenesis.2)RG1 enhanced the BM-MSCs secretion of VEGF and IL-6 therefore providing superior therapeutic efficacy on RⅢ.