Regulation Of MCU By Orai1 In Amyotrophic Lateral Sclerosis (ALS) SOD1 G93A NSC34 Cells And Its Mechanism

Objective:SOD1 gene mutation is a common mutation in amyotrophic lateral sclerosis(amyotrophic lateral sclerosis,ALS).It is confirmed that Orai1 regulates MCU to participate in the pathogenesis of ALS through CREB in stai-transfected SOD1 G93 A NSC34 cells.Methods:1.Stably transfected SOD1 G93 A NSC34 cells were obtained by lentivirus transfection technology,and transfection efficiency was detected by immunofluorescence and QRT-PCR.2.Group without drug intervention: Western blot and immunofluorescence staining were used to detect the expression of Orai1,P-CREb,MCU and CREB proteins in NSC34 cells and SOD1 G93 A NSC34 cells.3.Grouping after drug intervention: In stably transfected SOD1 G93 A NSC34 cells,Orai1 protein inhibitor(MRS1845,MRS)and MCU protein excitant(Spermine,SPE)were used for Western blot and immunofluorescence staining.The expressions of Orai1,P-CREB,MCU,CREB and other proteins in each group were detected,and the experimental groups were as follows: SOD1 G93 A NSC34 cell group,SOD1G93 A NSC34+MRS group,SOD1 G93 A NSC34+SPE group,SOD1 G93 A NSC34+MRS+SPE group.4.Effect of cell activity: THE cell activity of SOD1 G93 A NSC34 group,SOD1 G93 A NSC34+MRS group and SOD1 G93 A NSC34+SPE group was detected by CCK8 method.Results:1.Grouping results without drug intervention: Western blot results showed that the expression of Orai1 protein,P-CREB protein and MCU protein in SOD1 G93 A NSC34 cell group was higher than that in NSC34 cell group(P<0.05);Immunofluorescence staining showed that the fluorescence intensity of Orai1 protein,P-CREB protein and MCU protein was consistent with Western blot results(P<0.05);2.Grouping results after drug intervention: Compared with SOD1 G93 A NSC34 cell group,the expression levels of Orai1 protein,P-CREB protein and MCU protein in SOD1 G93 A NSC34+MRS group were significantly decreased(P<0.05);There were no significant differences in Orai1 and P-CREB protein in SOD1 G93 A NSC34+SPE group,while the expression of MCU protein showed an increasing trend(P<0.05);In SOD1 G93 A NSC34+MRS+SPE group,the expression of Orai1 protein and p-CREB protein decreased(P<0.05),and there was no significant difference in MCU expression.Immunofluorescence staining showed that the fluorescence intensity of Orai1 protein,P-CREB protein and MCU protein was consistent with Western blot results(P<0.05);3.Results of cell activity experiment: Compared with SOD1 G93 A NSC34 cell group,the cell activity of SOD1 G93 A NSC34+MRS group was enhanced;The cell activity of SOD1 G93 A NSC34+SPE group decreased(P < 0.05).Conclusion:The high expression of Orai1,P-CREb and MCU proteins was related to the pathogenesis of SOD1 G93 A NSC34 cell model.Orai1 regulates the expression of MCU protein by regulating CREB phosphorylation and participates in the pathogenesis of SOD1 G93 A NSC34 cell model.Silencing Orai1 protein protected cell activity in SOD1G93 A NSC34 cell model.