1,25-?OH?₂D₃ Improves Endoplasmic Reticulum Stress And Inhibits H₂O₂-induced Apoptosis In MIN6 Cells

Objective:To investigate the role of 1,25-dihydroxyvitamin D₃?1,25-?OH?₂D₃?in the prevention and treatment of diabetes mellitus?DM?.1,25-?OH?₂D₃ inhibits the expression of apoptosis-related molecules induced by hydrogen peroxide?H₂O₂?by improving endoplasmic reticulum stress?ERs?,and inhibited MIN6 cells apoptosis,which provides basis for the application of 1,25-?OH?₂D₃ in the prevention and treatment of Type 1 diabetes mellitus?T1DM?.Methods:Mouse insulinoma MIN6 cells were used as cell model.After pretreatment with different concentrations of 1,25-?OH?₂D₃ MIN6cells incubated with H₂O₂,CCK8 was used to detect the viability of MIN6 cells,and the optimal concentration and time for 1,25-?OH?₂D₃were obtained.Cells were stained with Hoechst 33258 dye and cell nucleus morphology was observed with fluorescence microscope.TUNEL staining and flow cytometry were used to detect the apoptotic levels of MIN6 cells.Western blot was used to detect the expression of VDR,ERs related molecules?GRP78,PERK,Phospho-PERK,ATF4,CHOP?and apoptosis related molecules?Bcl-2,Bax,MCL-1,Caspase-9,Cleaved caspase-9,Caspase-3,Cleaved caspase-3?expression.In addition,GSK2606414 is a highly effective and targeted PERK inhibitor.With GSK2606414 incubation,Western Blot was used to detect Phospho-PERK and ATF4 protein levels,and CCK8 to detect MIN6 cell viability.Results:?1?The results of CCK8 showed that the optimal concentration of 1,25-?OH?₂D₃ was 5 nmol/L,and the incubation time was 3 h.Compared with the H₂O₂ group,1,25-?OH?₂D₃ pretreatment can significantly increase the viability of MIN6 cells?P<0.05?.?2?Hoechst33258 staining showed that cells treated with H₂O₂ had an obvious apoptotic morphological change,including chromatin condensation and the formation of apoptotic bodies,while pretreatment with 1,25-?OH?₂D₃,cells showed almost normal nuclear with evenly distributed chromatin.Similarly,TUNEL staining showed that the apoptosis rate of MIN6 cells stimulated with H₂O₂ was markedly increased,while this effect was partially reversed by 1,25-?OH?₂D₃ pretreatment.?3?Flow cytometry was used to detect the percentage of apoptosis in MIN6 cells.The percentage of apoptosis in the 1,25-?OH?₂D₃ pretreatment group?9.32%?was significantly lower than the percentage of apoptosis in the H₂O₂ group?28.2%??P<0.05?.It was further confirmed that 1,25-?OH?₂D₃ can reduce the apoptosis of MIN6 cells induced by H₂O₂.?4?Western Blot results showed that compared with the H₂O₂ group,VDR expression increased significantly?P<0.05?,while Phospho-PERK,GRP78 and ATF4 levels decreased.In addition,as a downstream protein in the PERK pathway,the level of CHOP was found to be significantly downregulated with1,25-?OH?₂D₃ pretreatment compared with H₂O₂ group.Furthermore,results demonstrated that 1,25-?OH?₂D₃ pretreatment significantly decreased Bax expression and increased Bcl-2 expression compared with treatment of H₂O₂.Finally,results showed that Cleaved caspase-9 and Cleaved caspase-3 were highly expressed after H₂O₂ treatment,which were partially reversed by 1,25-?OH?₂D₃ pretreatment.?5?After incubation with the inhibitor of PERK,GSK2606414,the results showed that GSK2606414 strongly reduced the expression level of Phospho-PERK and ATF4 in H₂O₂-treated MIN6 cells.Subsequently,CCK8 results indicated 1,25-?OH?₂D₃ and GSK2606414 pretreatment effectively decreased the percentage of apoptotic cells compared with H₂O₂ treatment group.Conclusion:?1?1,25-?OH?₂D₃ improves ERs by inhibiting the PERK-ATF4-CHOP signaling pathway.?2?1,25-?OH?₂D₃ inhibits H₂O₂-induced apoptosis in MIN6 cells by regulating the expression of apoptosis-related proteins.