抑制PI3K/Akt/mTOR信号通路对MPP+处理的SH-SY5Y细胞自噬、凋亡及PD特征蛋白表达的影响  

Effects of inhibiting PI3K/Akt/mTOR signaling pathway on autophagy,apoptosis and PD characteristic expression proteins in MPP^(+)-treated SH-SY5Y cells

作　　者：王飞[1,2] 张小蕾 李含章 李亚楠 胡梦妮 马骏 WANG Fei;ZHANG Xiaolei;LI Hanzhang;LI Yanan;HU Mengni;MA Jun(Department of Traditional Chinese Medicine,Wuhan First Hospital,Wuhan 430022,China;School of Acupuncture and Bone Injury,Hubei University of Chinese Medicine)

机构地区：[1]武汉市第一医院中医部,430022 [2]湖北中医药大学针灸骨伤学院

出　　处：《天津医药》2023年第5期449-454,共6页Tianjin Medical Journal

基　　金：国家自然科学基金项目(81473788)。

摘　　要：目的探讨抑制磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对1-甲基-4-苯基吡啶离子(MPP+)处理的SH-SY5Y细胞自噬、凋亡及帕金森病(PD)特征蛋白α-突触核蛋白(α-syn)、酪氨酸羟化酶(TH)表达的影响。方法以MPP+、PI3K/Akt激活剂胰岛素样生长因子-1(IGF-1)、PI3K/Akt抑制剂LY294002作用于人神经母细胞瘤SH-SY5Y细胞,CCK-8检测细胞存活率;流式细胞术检测细胞凋亡率;吖啶橙(AO)染色检测自噬空泡;Western blot检测α-syn、TH、p62、微管相关蛋白1轻链3B(LC3B)、p-mTOR、mTOR、p-PI3K、PI3K、p-Akt、Akt蛋白水平。结果MPP+干预显著降低SH-SY5Y细胞存活率,且呈现剂量依赖性(P<0.05)。MPP+和IGF-1处理后SH-SY5Y细胞存活率降低,凋亡率增高(P<0.05);细胞中自噬空泡减少,LC3BⅡ/Ⅰ降低,p62蛋白水平增高(P<0.05);TH蛋白水平降低,α-syn蛋白水平增高(P<0.05);p-PI3K/PI3K、p-Akt/Akt与p-mTOR/mTOR增高(P<0.05)。LY294002对SH-SY5Y细胞的影响与MPP+和IGF-1相反,且LY294002可在一定程度上逆转MPP+对SH-SY5Y细胞的影响(P<0.05)。结论抑制PI3K/Akt/mTOR信号通路可能对MPP+诱导的SH-SY5Y细胞毒性具有保护作用。Objective To investigate effects of inhibiting phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway on autophagy,apoptosis,the expression of Parkinson's disease(PD)characteristic proteinα-synuclein(α-syn)and tyrosine hydroxylase(TH)in SH-SY5Y cells treated with 1-methyl-4-phenylpyridine(MPP+).Methods SH-SY5Y cells were treated with MPP+,PI3K/Akt activator insulin-like growth factor-1(IGF-1)and PI3K/Akt inhibitor LY294002.CCK-8 was performed to measure the cell viability.Flow cytometry was used to detect cell apoptosis rate.Acridine orange(AO)staining was used to detect autophagic vacuoles.Western blot assay was used to measure protein levels ofα-syn,TH,p62,microtubule-associated protein 1 light chain 3B(LC3B),p-mTOR,mTOR,pPI3K,PI3K,p-Akt and Akt.Results MPP+intervention significantly reduced the survival rate of SH-SY5Y cells in a dose-dependent manner(P<0.05).The survival rate of SH-SY5Y cells decreased and the apoptosis rate increased after MPP+and IGF-1 treatment(P<0.05).The autophagic vacuoles in cells decreased,the ratio of LC3BⅡ/Ⅰdecreased,and the level of p62 protein increased(P<0.05).The level of TH protein decreased and the level ofα-syn protein increased(P<0.05).The ratios of p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR were increased(P<0.05).The effect of LY294002 on SH-SY5Y cells was opposite to that of MPP+and IGF-1,and LY294002 was able to reverse the effect of MPP+on SH-SY5Y cells to a certain extent(P<0.05).Conclusion Inhibition of PI3K/Akt/mTOR signaling pathway has a protective effect on MPP+-induced SH-SY5Y cytotoxicity,which provides a new theoretical basis for the treatment of PD.

关 键 词：帕金森病 磷酸肌醇3-激酶类 原癌基因蛋白质c-akt TOR丝氨酸-苏氨酸激酶 自噬 细胞凋亡 1-甲基-4-苯基吡啶离子 SH-SY5Y细胞 

分 类 号：R742[医药卫生—神经病学与精神病学]