摘要
目的:探讨温肺降浊方通过 Aβ25-35诱导PC12细胞损伤对线粒体自噬的影响.方法:通过重组大鼠β-NGF分化PC12细胞,Aβ25-35干预其成为阿尔茨海默病细胞模型,确定Aβ25-35造模的最佳浓度和时间点,设立温肺降浊方含药血清低(2％)、中(4％)、高(8％)浓度组.MDC染色和JC-1染色法观察自噬小体和线粒体膜电位,免疫荧光化学染色、qPCR和Western Blot检测各组细胞中PINK1、PARKIN、Ubl、Ub、p-Ub、LC3B mRNA和(或)蛋白表达.结果:β-NGF分化PC12细胞在第6天后成为海马神经元细胞形态结构,AD细胞造模条件以5 μmol/L终浓度的Aβ25-35干预48 h最佳.与正常对照组比较,模型组自噬小体显著增多,线粒体膜电位明显降低,PINK1、PAR-KIN、Ubl、Ub、p-Ub、LC3B mRNA和(或)蛋白表达显著升高(P＜0.05).与模型组比较,温肺降浊方含药血清可使模型细胞自噬小体减少,线粒体膜电位明显升高,PINK 1、PARKIN、Ubl、Ub、p-Ub、LC3B mRNA和(或)蛋白表达显著降低(P＜0.05).结论:温肺降浊方可减轻 Aβ25-35诱导的PC12细胞损伤,其机制可能与PINK1/PARKIN介导的线粒体自噬通路有关.
Abstract
Objective:To explore the effect of Wenfei jiangzhuo formula on mitochondrial autophagy through Aβ25-35 induced PC 12 cell injury.Methods:PC12 cells were differentiated by recombinant ratβ-NGF,Aβ25-35 intervened to become the AD cell model,the opti-mal concentration and time point of Aβ25-35 model was determined.The low(2％),medium(4％)and high(8％)concentration of Wen-fei jiangzhuo formula drug-containing serum groups were set up.MDC staining and JC-1 staining methods were used to observe the auto-phagosome and mitochondrial membrane potential.Immunofluorescence chemical staining,qPCR and Western Blot were used to detect the mRNA and/or protein expressions of PINK1,PARKIN,Ubl,Ub,p-Ub and LC3B in the cells of each group.Results:The PC 12 cells differentiated byβ-NGF became the morphological structure of hippocampal neurons after six days,and the optimal molding condition of AD cell was 5 μmol/L Aβ25-35 intervention for 48 hours.Compared with the normal control group,the model group had a significant increase in autophagosomes,a significant reduction in mitochondrial membrane potential,and significant increases in mRNA and/or protein expressions of PINK 1,PARKIN,Ubl,Ub,p-Ub,LC3B(P＜0.05).Compared with the model group,the Wenfei jiangzhuo formula drug-con-taining serum had a significant reduction in autophagosomes of model cell,a significant increase in mitochondrial membrane potential,and significant reductions in mRNA and/or protein expressions of PINK1,PARKIN,Ubl,Ub,p-Ub,LC3B(P＜0.05).Conclusion:Wenfei jian-gzhuo formula can attenuate Aβ25-35-induced PC12 cell injury possibly through PINK1/PARKIN mediated mitophagy pathway.
基金项目
国家自然科学基金(82260904)
广西中医脑病临床研究中心项目(桂科AD20238028)
广西壮族自治区高等学校高水平创新团队及卓越学者计划(桂教人才202006)
广西中医药大学博士研究生创新项目(YCBXJ2023010)
国家科技期刊平台
NSTL
万方数据