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

doi:10.11958/20221595

天津医药 ›› 2023, Vol. 51 ›› Issue (5): 449-453.doi: 10.11958/20221595

• 细胞与分子生物学 • 下一篇

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

王飞¹^,²(), 张小蕾², 李含章², 李亚楠², 胡梦妮², 马骏²^,^△()

1.武汉市第一医院中医部（邮编430022）
2.湖北中医药大学针灸骨伤学院

收稿日期:2022-09-29 修回日期:2022-11-21 出版日期:2023-05-15 发布日期:2023-05-05
通讯作者: △E-mail:376201631@qq.com
作者简介:王飞（1982），男，主治医师，主要从事针灸治疗脑病及神经系统疾病方面研究。E-mail：wangfei202@163.com
基金资助:
国家自然科学基金项目(81473788)

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

WANG Fei¹^,²(), ZHANG Xiaolei², LI Hanzhang², LI Yanan², HU Mengni², MA Jun²^,^△()

1. Department of Traditional Chinese Medicine, Wuhan First Hospital, Wuhan 430022, China
2. School of Acupuncture and Bone Injury, Hubei University of Chinese Medicine

Received:2022-09-29 Revised:2022-11-21 Published:2023-05-15 Online:2023-05-05
Contact: △E-mail:376201631@qq.com

王飞, 张小蕾, 李含章, 李亚楠, 胡梦妮, 马骏. 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[J]. 天津医药, 2023, 51(5): 449-453.

WANG Fei, ZHANG Xiaolei, LI Hanzhang, LI Yanan, HU Mengni, MA Jun. Effects of inhibiting PI3K/Akt/mTOR signaling pathway on autophagy, apoptosis and PD characteristic expression proteins in MPP⁺-treated SH-SY5Y cells[J]. Tianjin Medical Journal, 2023, 51(5): 449-453.

摘要/Abstract

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。

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

Abstract:

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 protein α-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 of α-syn, TH, p62, microtubule-associated protein 1 light chain 3B (LC3B), p-mTOR, mTOR, p-PI3K, 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/Ⅰ decreased, and the level of p62 protein increased (P<0.05). The level of TH protein decreased and the level of α-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.

Key words: Parkinson disease, phosphatidylinositol 3-kinases, proto-oncogene proteins c-akt, TOR serine-threonine kinases, autophagy, apoptosis, 1-methyl-4-phenylpyridinium, SH-SY5Y cells

中图分类号:

R742

图/表 8

图1 流式细胞仪检测SH-SY5Y细胞凋亡情况

Fig.1 Apoptosis of SH-SY5Y cells detected by flow cytometry

表1 各组SH-SY5Y细胞存活率、凋亡率比较 (n=6,%,$\bar{x}\pm s$)

Tab.1 Comparison of survival rate and apoptosis rate of SH-SY5Y cells between the five groups

组别	细胞存活率	细胞凋亡率
Control组	100.00±0.00	9.36±0.98
MPP⁺组	52.64±3.25^a	23.48±3.07^a
IGF-1组	58.69±4.15^a	19.67±3.24^a
LY294002组	136.29±8.34^ab	4.19±0.45^ab
MPP⁺+LY294002组	78.24±7.52^bc	16.35±2.18^bc
F	226.163^＊＊	70.666^＊＊

表2 各组SH-SY5Y细胞自噬空泡比例及p62、LC3B蛋白表达水平比较 (n=6,$\bar{x}\pm s$)

Tab.2 Comparison of autophagic vacuoles ratio, expression levels of p62 and LC3B proteins between the five groups

组别	自噬空泡比例（%）	p62	LC3BⅡ/Ⅰ
Control组	75.36±9.36	0.45±0.03	0.89±0.09
MPP⁺组	24.36±2.58^a	1.28±0.09^a	0.24±0.03^a
IGF-1组	26.47±2.64^a	1.15±0.10^a	0.17±0.02^a
LY294002组	89.89±9.08^ab	0.24±0.02^ab	1.28±0.11^ab
MPP⁺+LY294002组	61.51±5.59^bc	0.79±0.06^bc	0.57±0.05^bc
F	119.538^＊＊	256.787^＊＊	267.938^＊＊

图2 抑制PI3K/Akt/mTOR信号通路对MPP+处理的SH-SY5Y细胞自噬的影响 A：荧光显微镜下观察AO对SH-SY5Y细胞自噬空泡的染色情况（比例尺=50 μm）；B：Western blot检测SH-SY5Y细胞中p62和LC3B的表达

Fig.2 Effects of inhibiting PI3K/Akt/mTOR signaling pathway on autophagy of SH-SY5Y cells treated with MPP+

图3 Western blot检测各组SH-SY5Y细胞中TH和α-syn蛋白表达

Fig.3 TH and α-syn protein expression in SH-SY5Y cells of each group detected by Western blot assay

表3 各组SH-SY5Y细胞中TH和α-syn蛋白水平比较 (n=6,$\bar{x}\pm s$)

Tab.3 Comparison of TH and α-syn protein levels in SH-SY5Y cells of each group

组别	TH	α-syn
Control组	0.73±0.07	0.34±0.03
MPP⁺组	0.22±0.03^a	0.78±0.08^a
IGF-1组	0.24±0.04^a	0.75±0.06^a
LY294002组	0.98±0.10^ab	0.16±0.02^ab
MPP⁺+LY294002组	0.48±0.04^bc	0.56±0.05^bc
F	167.842^＊＊	154.391^＊＊

表4 各组SH-SY5Y细胞中p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR比较 (n=6,$\bar{x}\pm s$)

Tab.4 Analysis of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR ratios in SH-SY5Y cells in each group

组别	p-PI3K/PI3K	p-Akt/Akt	p-mTOR/mTOR
Control组	0.44±0.05	0.31±0.04	0.35±0.04
MPP⁺组	0.81±0.07^a	0.79±0.08^a	0.76±0.06^a
IGF-1组	0.85±0.06^a	0.84±0.06^a	0.79±0.05^a
LY294002组	0.26±0.04^ab	0.14±0.01^ab	0.11±0.02^ab
MPP⁺+LY294002组	0.60±0.06^bc	0.53±0.05^bc	0.59±0.05^bc
F	114.574^＊＊	192.190^＊＊	235.189^＊＊

图4 Western blot检测各组SH-SY5Y细胞中p-PI3K、PI3K、p-Akt、Akt、p-mTOR、mTOR蛋白表达

Fig.4 The p-PI3K, PI3K, p-Akt, Akt, p-mTOR and mTOR protein expression in SH-SY5Y cells of each group detected by Western blot assay

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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

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