Orai1过表达激活Calcineurin/TFEB通路对帕金森病细胞模型自噬及细胞活性的影响  

Effects of Orai1 overexpression activating calcineurin/TFEB pathway on autophagy and cell activity in Parkinson's disease cell models

作　　者：侯萌 王昭君[2] 张皓洁 樊泽新 王荔[1] HOU Meng;WANG Zhaojun;ZHANG Haojie;FAN Zexin;WANG Li(不详;Department of Neurology,Second Hospital of Shanxi Medical University,Taiyuan Shanxi 030000,China)

机构地区：[1]山西医科大学第二医院神经内科,030000 [2]山西医科大学生理学系,030000

出　　处：《中国神经免疫学和神经病学杂志》2024年第5期343-351,共9页Chinese Journal of Neuroimmunology and Neurology

基　　金：山西省自然科学基金面上项目(20210302123288);山西省卫生健康委科研课题面上项目(2021064)。

摘　　要：目的研究钙释放激活钙通道调节分子1(calcium release-activated calcium modulator 1,Orai1)蛋白过表达激活钙调神经磷酸酶(Calcineurin,CaN)/转录因子EB(transcription factor EB,TFEB)通路对帕金森病(Parkinson disease,PD)细胞模型自噬水平与细胞活性的影响。方法将培养的SH-SY5Y细胞给予1-甲基-4-苯基-吡啶离子(1-methyl-4-phenyl-pyridinium,MPP^(+))处理建立PD细胞模型,并分为对照组、MPP^(+)组、MPP^(+)+oe-NC组、MPP^(+)+oe-Orai1组与MPP^(+)+oe-Orai1+FK506(CaN抑制剂)组。采用CCK-8检测各组SH-SY5Y细胞的存活率;采用细胞可渗透钙离子荧光探针检测各组细胞胞内钙离子水平;采用ELISA法检测各组细胞CaN含量;采用RT-qPCR检测各组细胞Orai1、微管关联蛋白1A/1B轻链3(microtubule-associated protein 1 light chain 3,LC3)、P62 mRNA相对表达水平;采用Western blot检测各组细胞Orai1、基质相互作用分子1(stromal interaction molecule 1,STIM1)、CaN、TFEB、LC3Ⅱ/LC3Ⅰ、P62以及酪氨酸羟化酶(tyrosine hydroxylase,TH)蛋白表达水平。结果(1)与MPP^(+)+oe-NC组比较,MPP^(+)+oe-Orai1组胞内钙离子水平、LC3 mRNA表达水平升高(P<0.01或P<0.05),LC3Ⅱ/LC3Ⅰ蛋白比值增高(P<0.05),P62蛋白表达水平减少(P<0.01),而CaN抑制剂FK506干预可抑制Orai1过表达对细胞LC3 mRNA表达水平、LC3Ⅱ/LC3Ⅰ蛋白比值及P62蛋白表达水平的影响(P<0.01或P<0.05)。(2)与MPP^(+)+oe-NC组比较,MPP^(+)+oe-Orai1组细胞CaN表达水平增加,TFEB核移位增加(均P<0.05),而MPP^(+)+oe-Orai1+FK506组细胞TFEB核移位较MPP^(+)+oe-Orai1组减少(P<0.01)。(3)与MPP^(+)+oe-NC组比较,MPP^(+)+oe-Orai1组细胞TH表达水平和细胞活性增加(均P<0.01),而MPP^(+)+oe-Orai1+FK506组细胞TH表达水平和细胞活性较MPP^(+)+oe-Orai1组降低(均P<0.01)。结论Orai1蛋白过表达可增强PD模型细胞自噬水平与细胞活性,其机制与Orai1蛋白过表达激活CaN/TFEB通路有关。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,LC3Ⅱ/LC3Ⅰ,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/LC3Ⅰprotein 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,LC3Ⅱ/LC3Ⅰprotein 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

关 键 词：Orai1 Calcineurin/TFEB 自噬 帕金森病 多巴胺能神经元 

分 类 号：R742.5[医药卫生—神经病学与精神病学]