骆驼蓬总碱对Tau蛋白的降解作用

Degradation of Tau by total alkaloid of harmaline

目的研究骆驼蓬总碱(total alkaloid of harmaline,TAH)通过调节神经细胞自噬促进Tau蛋白的降解作用。方法完全培养基培养稳定表达绿色荧光蛋白-微管相关蛋白1轻链3(green fluorescent protein-microtubule associated protein 1 light chain 3,GFP-LC3)的大鼠肾(normal rat kidney,NRK)细胞、人神经母细胞瘤细胞(SH-SY5Y),分成对照组(CT组)、雷帕霉素(rapamycin,Rapa)组和TAH组,利用激光共聚焦显微镜观察自噬体数量的变化,蛋白免疫印迹技术检测自噬底物蛋白(p62/sequestosome-1,P62/SQSTM1)和自噬体标志性蛋白微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)LC3-II蛋白表达水平的变化;完全培养基培养表达Tau-绿色荧光蛋白(tau-green fluorescent protein,Tau-GFP)的人胚肾-293(human embryonic kidney-293,HEK-293)细胞,分成对照组(CT组)、模型组(DOX组)和药物组(DOX+TAH组),结合溶酶体标记探针Lyso-tracker Red,利用激光共聚焦显微镜观察Tau-GFP的荧光变化及溶酶体活性的变化,蛋白免疫印迹技术检测Tau-GFP和LC3-II蛋白表达水平的变化。结果与CT组比较,TAH组细胞中自噬体数目增多,P62蛋白表达水平降低,LC3-II蛋白表达水平升高,差异有统计学意义(P<0.05);DOX诱导Tau蛋白表达后,与DOX组比较,DOX+TAH组Tau-GFP荧光强度减弱,蛋白表达水平降低,LC3-II蛋白表达水平升高,溶酶体活性增强,差异有统计学意义(P<0.05)。结论TAH可以降解Tau蛋白,并通过诱导神经细胞自噬发挥此作用。

Objective To investigate the role of total alkaloid of harmaline(TAH)in promoting the degradation of Tau by regulating autophagy in neuronal cells.Methods Normal rat kidney(NRK)cells stably expressing green fluorescent protein-microtubule associated protein 1 light chain 3(GFP-LC3)and human neuroblastoma cells(SH-SY5Y)were cultured in complete medium,then cells were grouped into control(CT group),rapamycin(Rapa group)and TAH,the number of autophagic puncta was counted in GFP-LC3 NRK cells,the expression of sequestosome-1 and microtubule associated protein 1 light chain 3(LC3)proteins in NRK and SH-SY5Y cells were detected by protein immunoblotting.Human embryonic kidney-293(HEK-293)cells expressing tau-green fluorescent protein(Tau-GFP)were cultured in complete medium and grouped into CT group,DOX group and DOX+TAH group.The fluorescence changes of Tau-GFP and lysosomal activity were observed by laser confocal microscopy,Tau-GFP and LC3-II protein expression levels were detected by protein immunoblotting in combination with the lysosomal labeling probe Lyso-tracker Red.Results Compared with the CT group,the number of LC3 puncta was increased,the expression level of P62 was decreased,while the expression level of LC3-II was dramatically increased in the TAH group(P<0.05).After DOX induced Tau protein expression,compared with the DOX group,the fluorescence intensity of Tau-GFP was weakened,the expression level of Tau-GFP was decreased,and the expression level of LC3-II was significantly increased,lysosomal activity was enhanced in the DOX+TAH group(P<0.05).Conclusion TAH could degrade Tau protein,playing a protective role on neurodegenerative disease cells by inducing autophagy.