Effects of Orail overexpression activating calcineurin/TFEB pathway on autophagy and cell activity in Parkinson's disease cell models
Objective To investigate the effects of calcineurin(CaN)/transcription factor EB(TFEB)pathway activated by calcium release-activated calcium modulator 1(Orai1)protein overexpression on autophagy level and cell activity in Parkinson's disease(PD)cell models.Methods The cultured SH-SY5Y cells were treated with 1-methyl-4-phenyl-pyridinium(MPP+)to establish the PD cell model.SH-SY5Y cells were divided into the control group,MPP+group,MPP++oe-NC group,MPP++oe-Orai1 group,and MPP++oe-Orai1+FK506(CaN inhibitor)group.CCK-8 was used to detect the cell activity.A permeable calcium fluorescence probe was used to detect the levels of intracellular calcium.ELISA was used to detect the content of CaN.RT-qPCR was used to detect the mRNA expression levels of Orai1,Microtubule-associated protein 1 light chain 3(LC3),and P62.Western blot was used to detect the expression levels of Orai1,stromal interaction molecule 1(STIM1),CaN,TFEB,LC3 Ⅱ/LC3 Ⅰ,P62,and TH.Results(1)Compared with the MPP++oe-NC group,the intracellular calcium level,the LC3 mRNA expression level(P＜0.01 or P＜0.05),and the LC3Ⅱ/LC3Ⅰ protein ratio(P＜0.05)in the MPP++oe-Orai1 group was increased,and the P62 protein expression level was decreased(P＜0.01).The effects of Orai1 overexpression on LC3 mRNA expression level,LC3 Ⅱ/LC3 Ⅰ protein ratio,and P62 protein expression level(P＜0.01 or P＜0.05)were inhibited by CaN inhibitor FK506 intervention.(2)Compared with the MPP++oe-NC group,CaN expression level and TFEB nuclear translocation in the MPP++oe-Orai1 group were increased(all P＜0.05).Compared with the MPP++oe-Orai1 group,TFEB nuclear translocation in the MPP++oe-Orai1+FK506 group was reduced(P＜0.01).(3)Compared with the MPP-+oe-NC group,the TH expression level and cell activity in the MPP++oe-Orai1 group were increased(all P＜0.01).Compared with the MPP++oe-Orai1+FK506 group,the TH expression level and cell activity of the MPP-+oe-Orai1 group were decreased(all P＜0.01).Conclusions Orai1 protein overexpression can enhance the autophagy level and cell activity in PD model cells,and the mechanism is related to the activation of the CaN/TFEB pathway by Orai1 protein overexpression.
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维普
