Effects Of HUC-MSCs-CM On Apoptosis And Perk Pathway Of Endoplasmic Reticulum Stress Induced By Radiation In HT22 Cells

Objectives To investigate the effects of human umbilical cord mesenchymal stem cell conditioned mediu(hUC-MSCs-CM)intervention on apoptosis and endoplasmic reticulum stress PERK-ATF4-CHOP pathways stimulated by radiation in hippocampus neuronal cell of mice(HT22).To elucidate its related mechanisms and to provide new ideas for the prevention and treatment of radiation-induced brain injury(RBI).Methods 1 hUC-MSCs were isolated and cultured by enzyme digestion.Cellular morphology was observed by inverted microscope and cellular phenotype was identified by flow cytometry.The third generation hUC-MSCs with good growth log were selected and cultured in serum-free IMEM medium for 48 hours,and the supernatant was collected to hUC-MSCs-CM.2 HT22 cells were irradiated by 6MV-XRay linear accelerator to RBI preparation,then pretreated by hUC-MSCs-CM and PERK inhibitor GSK2606414 respectively.The experiment was divided into five groups:blank control group(Control group),single irradiation group(RT group),irradiation hUC-MSCs-CM co-stimulation group(RT+MSCs-CM group),single hUC-MSCs-CM stimulation group(MSCs-CM group),irradiation GSK2606414 co-stimulation group(RT+GSK2606414 group).Each group was cultured for 24 hours after the above stimulation,the morphological changes of each group were observed by inverted microscope,the apoptosis rate of each group was detected by Annexin V/PI double staining flow cytometry,and the expression level of GRP78,p-PERK,ATF4,CHOP protein in each group was detected by Western Blot method.Results 1 The inverted microscope showed that the morphology of the third generation hUC-MSCs was similar to fibroblasts and showed the long fusiform and vortex growth.hUC-MSCs cell phenotypes detected by flow cytometry:hUC-MSCs expression of stromal cell-related surface antigen CD73,CD90 and CD 105;do not express hematopoietic stem cell markers CDllb and CD34,do not express human leukocyte coantigen CD45 and MHC-? class of HLA-DR.2 Compared with the Control group,the number of cells decreased significantly in RT group,and the cells crumpled,rounded and floating cells increased.After radiation-induced HT22 cells were intervened by MSCs-CM and GSK2606414 respectively,the number of cells increased,cell adherent growth,complete morphology,no cell shrinkage and few floating cells.Compared with Control group,the apoptosis rate of RT group was significantly higher(P<0.05).The apoptosis rate of radiation-induced HT22 cells decreased after RT+MSCs-CM co-stimulation and RT+GSK2606414 co-stimulation respectively(P<0.05).GRP78,p-PERK,ATF4 and CHOP protein expression levels increased in RT group compared with Control group(P<0.05).This result was consistent with the down-regulation of GRP78,p-PERK,ATF4 and CHOP protein expression of radiation-induced HT22 cells which intervented by the PERK pathway inhibitor GSK2606414(P<0.05).Conclusions 1 hUC-MSCs-CM intervention reduces radiation-induced apoptosis in HT22 cells.2 hUC-MSCs-CM intervention attenuates radiation-induced endoplasmic reticulum stress in HT22 cells and may be associated with PERK-ATF4-CHOP pathway.Figure 10;Table 5;Reference 159...