中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (2): 319-327.doi: 10.4103/1673-5374.244795

• 原著:退行性病与再生 • 上一篇    下一篇

甲基苯丙胺诱导α-突触核蛋白聚集产生了神经毒性

  

  • 出版日期:2019-02-15 发布日期:2019-02-15
  • 基金资助:

    中国国家自然科学基金项目（81373240，81671865）

     

Implications of alpha-synuclein nitration at tyrosine 39 in methamphetamine-induced neurotoxicity in vitro and in vivo

Hong-Hua Qiao 1 , Lin-Nan Zhu 1 , Yue Wang 2 , Jia-Liang Hui 3 , Wei-Bing Xie 1 , Chao Liu 4 , Ling Chen 1 , Ping-Ming Qiu 1   

  1. 1 School of Forensic Medicine, Southern Medical University, Guangzhou, Guangdong Province, China
    2 Kingmed Institute for Forensic Science, Guangzhou, Guangdong Province, China
    3 First Clinical Medicine College, Southern Medical University, Guangzhou, Guangdong Province, China
    4 Guangzhou Forensic Science Institute, Guangdong Province Key Laboratory of Forensic Genetics, Guangzhou, Guangdong Province, China
  • Online:2019-02-15 Published:2019-02-15
  • Contact: Ping-Ming Qiu, PhD, qiupm@163.com; Ling Chen, PhD, lingpzy@163.com.
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81373240 and 81671865.

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摘要：

甲基苯丙胺是一种苯丙胺类精神兴奋剂，能损伤多巴胺能神经元，并导致与帕金森病等神经退行性疾病相似的特征性病理学改变。但其具体作用机制尚不明确。为此，实验设计以暴露于甲基苯丙胺的SH-SY5Y细胞和C57BL/6J小鼠作为体内外帕金森病病理模型。(1)体外实验以0，0.5，1.0，1.5，2.0，2.5mmol/L甲基苯丙胺干预24h或以2.0mmol/L甲基苯丙胺干预0，2，4，8，16，24h培养SH-SY5Y细胞，另以0.1 mmol/L 一氧化氮抑制剂N-硝基-L-精氨酸干预以暴露于2.0mmol/L METH 24h的SH-SY5Y细胞；(2)体内实验以C57BL/6J小鼠腹腔注射N-硝基-L-精氨酸(8mg/kg)，共注射8次，间隔12h，每次注射0.5h后腹腔注射甲基苯丙胺15mg/kg；(3)以Western blot分析检测细胞和小鼠脑组织中的一氧化氮合酶、α-突触核蛋白、5G4、酪氨酸39位点的硝基化修饰的α-突触核蛋白、裂解的caspase-3和裂解的聚腺苷酸二磷酸核糖转移酶的表达，以免疫荧光染色检测NeuN、酪氨酸39位点的硝基化修饰的α-突触核蛋白和5G4阳性反应，以ELISA测量小鼠脑组织中多巴胺水平；(4)甲基苯丙胺暴露后，SH-SY5Y细胞和C57BL/6J小鼠脑组织中α-Syn的表达增加，α-Syn 5G4聚集增多，酪氨酸39位点的硝基化修饰的α-突触核蛋白、一氧化氮合酶、裂解的caspase-3和裂解的聚腺苷酸二磷酸核糖转移酶表达增加，且小鼠脑组织中多巴胺含量降低。而当以N-硝基-L-精氨酸联合干预SH-SY5Y细胞和C57BL/6J小鼠后，上述变化明显改善；(5)实验结果表明，酪氨酸39位点硝基化修饰的α-突触核蛋白聚集参与了甲基苯丙胺的神经毒性作用。

orcid: 0000-0002-5579-1124 (Ping-Ming Qiu)

关键词: &alpha, -突触核蛋白, 硝基化&alpha, -突触核蛋白, 帕金森病, 甲基苯丙胺, N-硝基-L-精氨酸, &alpha, -突触核蛋白聚集, 细胞凋亡, 神经毒性, 神经再生

Abstract:

Methamphetamine is an amphetamine-type psychostimulant that can damage dopaminergic neurons and cause characteristic pathological changes similar to neurodegenerative diseases such as Parkinson’s disease. However, its specific mechanism of action is still unclear. In the present study, we established a Parkinson’s disease pathology model by exposing SH-SY5Y cells and C57BL/6J mice to methamphetamine.  In vitro experiments were performed with 0, 0.5, 1.0, 1.5, 2.0 or 2.5 mM methamphetamine for 24 hours or 2.0 mM methamphetamine for 0-, 2-, 4-, 8-, 16-, and 24-hour culture of SH-SY5Y cells. Additional experimental groups of SH-SY5Y cells were administered a nitric oxide inhibitor, 0.1 mM N-nitro-L-arginine, 1 hour before exposure to 2.0 mM methamphetamine for 24 hours. In vivo experiments: C57BL/6J mice were intraperitoneally injected with N-nitro-L-arginine (8 mg/kg), eight times, at intervals of 12 hours. Methamphetamine 15 mg/kg was intraperitoneally injected eight times, at intervals of 12 hours, but 0.5-hour after each N-nitro-L-arginine injection in the combined group. Western blot assay was used to determine the expression of nitric oxide synthase, α-synuclein (α-Syn), 5G4, nitrated α-synuclein at the residue Tyr39 (nT39 α-Syn), cleaved caspase-3, and cleaved poly ADP-ribose polymerase (PARP) in cells and mouse brain tissue. Immunofluorescence staining was conducted to measure the positive reaction of NeuN, nT39 α-Syn and 5G4. Enzyme linked immunosorbent assay was performed to determine the dopamine levels in the mouse brain. After methamphetamine exposure, α-Syn expression increased; the aggregation of α-Syn 5G4 increased; nT39 α-Syn, nitric oxide synthase, cleaved caspase-3, and cleaved PARP expression increased in the cultures of SH-SY5Y cells and in the brains of C57BL/6J mice; and dopamine levels were reduced in the mouse brain. These changes were markedly reduced when N-nitro-L-arginine was administered with methamphetamine in both SH-SY5Y cells and C57BL/6J mice. These results suggest that nT39 α-Syn aggregation is involved in methamphetamine neurotoxicity.

Key words: nerve regeneration, alpha-synuclein, nitrated α-synuclein, Parkinson’s disease, methamphetamine, N-nitro-L-arginine, alpha-synuclein aggregation, apoptosis, neurotoxicity, neural regeneration