Effects Of HUC-MSCs-CM And 3-MA On Autophagy And Apoptosis Of Microglia After Radiation

Objectives To study the effects of human umbilical cord mesenchymal stem cells conditioned medium(hUC-MSCs-CM)and 3-MA on autophagy,apoptosis and inflammatory response of microglia after 10 Gy radiation,and to provide new ideas for the prevention and treatment of radiation-induced brain injury.Methods 1 hUC-MSCs were isolated and cultured by enzyme digestion.Cell morphology was observed under inverted microscope,and cell phenotype was identified by flow cytometry.The third to fourth generation of hUC-MSCs with good logarithmic growth were cultured in serum-free IMEM medium for 48 h,and the supernatant was collected for preparation of hUC-MSCs-CM.2 Using 6MV-XRay linear accelerator 10 Gy irradiation to stimulate BV2 microglia to establish a radiation cell model,and the intervention of hUC-MSCs-CM and 3-MA were carried out.3 The experiment was divided into five groups: A blank control group(Control group),B Simple 10 Gy ionizing radiation group(RT group),C 10Gy+hUC-MSCs-CM co-stimulation group(MSCs group),D 10 Gy + 3-MA co-stimulation group(3-MA group),E 10 Gy + 3-MA + hUC-MSCs-CM costimulation group(MSCs + 3-MA group).4 The proliferation of BV2 microglia was detected by CCK-8.Annexin V / PI double staining flow cytometry was used to detect the apoptosis rate of each group,TUNEL combined with immunohistochemistry was used to detect the apoptosis of BV2 microglia,Western blot was used to detect the expression of Cleavedcaspase-3 protein.Collect the culture supernatant,extract the total protein and RNA,detect the expression of Beclin1 by real-time fluorescence quantitative PCR(RTPCR),detect the expression of autophagy related protein LC3 by Western blot,detect the concentration of TNF-? and IL-1? in the supernatant by ELISA.Results 1 CCK-8 results: compared with Control group,the proliferation of BV2 cells in RT group increased(P<0.05),compared with RT group,the proliferation of BV2 cells in MSCs group,3-MA group and MSCs + 3-MA group decreased(P<0.05).2 The results of flow cytometry,TUNEL and Cleavedcaspase-3 showed that compared with Control group,the apoptosis of BV2 cells in RT group increased,the expression of Cleavedcaspase-3 increased(P<0.05),compared with RT group,the apoptosis of BV2 cells in MSCs and 3-MA groups decreased and the expression of Cleavedcaspase-3 decreased(P<0.05),compared with MSCs and 3-MA groups,the apoptosis of BV2 cells in MSCs + 3-MA group decreased and the expression of Cleavedcaspase-3 decreased(P<0.05).3 The results of autophagy related protein and autophagy activity showed that the expression of Beclin1 and LC3 II / LC3 I in BV2 cells of RT group was higher than that of Control group(P<0.05),compared with RT group,the expression of Beclin1 and LC3 II / LC3 I in BV2 cells of MSCs and 3-MA groups were lower(P<0.05),compared with MSCs and 3-MA groups,the expression of Beclin1 and LC3 II / LC3 I in BV2 cells of MSCs + 3-MA group was lower(P<0.05).4 Compared with Control group,TNF-? and IL-1? secretion of BV2 cells in RT group increased(P<0.05),compared with RT group,TNF-? and IL-1? secretion of BV2 cells in MSCs and 3-MA groups decreased(P<0.05),compared with MSC and 3-MA groups,TNF-? and IL-1? secretion of BV2 cells in MSCs + 3-MA group decreased(P<0.05).Conclusions 1 10 Gy ionizing radiation can promote the proliferation and apoptosis of microglia,hUC-MSCs-CM and / or 3-MA can inhibit the proliferation and apoptosis of microglia.2 hUC-MSCs-CM and(or)3-MA can inhibit the over autophagy of microglia after ionizing radiation and reduce the secretion of inflammatory factors,and there is a conbine effect between hUC-MSCs-CM and 3-MA.Figure 8;Table 9;Reference 134...