中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (5): 865-874.doi: 10.4103/1673-5374.268902

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

39 ℃条件下甲基苯丙胺可诱导RIP3/MLKL介导的皮质神经元程序性坏死

  

  • 出版日期:2020-05-15 发布日期:2020-06-01

RIP3/MLKL-mediated neuronal necroptosis induced by methamphetamine at 39°C

Li-Min Guo1, Zhen Wang1, 2, Shi-Ping Li3, Mi Wang1, Wei-Tao Yan1, Feng-Xia Liu4, Chu-Dong Wang5, Xu-Dong Zhang6, Dan Chen1, Jie Yan5, Kun Xiong1#br#   

  1. 1 Department of Neurobiology and Human Anatomy, School of Basic Medical Science, Central South University, Changsha, Hunan Province, China
    2 Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu Province, China
    3 Department of Neurology, People’s Hospital of Lianhua, Pingxiang, Jiangxi Province, China
    4 Department of Human Anatomy, School of Basic Medical Science, Xinjiang Medical University, Urumqi, Xinjiang Uygur Autonomous Region, China
    5 Department of Forensic Science, School of Basic Medical Science, Central South University, Changsha, Hunan Province, China
    6 Narcotics Division, Municipal Security Bureau, Changsha, Hunan Province, China
  • Online:2020-05-15 Published:2020-06-01
  • Contact: Kun Xiong, MD,xiongkun2001@163.com; Jie Yan, wills212156@csu.edu.cn.
  • Supported by:
    This study was funded by the National Natural Science Foundation of China, No. 81971891 (to KX), 81571939 (to KX), 81772134 (to KX), 81772024 (to JY), and 81860781 (to FXL); the Key Research and Development Program of Hunan Province of China, No. 2018SK2091 (to KX); the Natural Science Foundation of Hunan Province of China, No. 2017JJ2339 (to JY); the Wu Jie-Ping Medical Foundation of the Minister of Health of China, No. 320.6750.14118 (to KX).

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摘要：

甲基苯丙胺(Methamphetamine, METH)又叫冰毒，其滥用者通常会出现神经毒性作用，同时伴随出现高热（39℃），幻觉和其他一些精神症状，但详细机制仍不清楚。为此，实验探讨了甲基苯丙胺对胚胎大鼠皮质神经元的影响。(1)实验将SD大鼠原代皮质神经元采用1 mM的甲基苯丙胺在39 ℃条件下干预培养，采用碘化丙啶(PI)染色和乳酸脱氢酶释放法(LDH)检测到明显的细胞坏死样死亡，而用受体相互作用蛋白1(RIP1)抑制剂nec-1预处理可抑制此现象；(2)Western blot结果显示：大鼠原代皮质神经元用1 mM甲基苯丙胺处理后放置在39 ℃培养箱培养3 h后，受体相互作用蛋白3(RIP3)和混合系激酶区域样蛋白(MLKL)表达增加，用RIP3抑制剂GSK'872干预处理后，PI染色，LDH检测和CCK8检测显示，大鼠原代皮质神经元坏死率降低，RIP3及MLKL蛋白表达显著降低；(3)同时实验采用免疫组织化学染色进一步观察到甲基苯丙胺滥用的人尸体大脑组织RIP3和MLKL的表达均上调。(4)上述结果表明，甲基苯丙胺+39 ℃可诱导RIP3/MLKL介导的皮质神经元程序性坏死，从而导致神经毒性。研究方案经中南大学湘雅三医院医学伦理委员会批准(批准号：2017-S026，批准时间：2017-03-07；批准号：2017-S033，批准时间：2017-03-07)。

orcid: 0000-0002-3103-6028 (Kun Xiong) 

         0000-0001-6877-5193 (Jie Yan)

关键词: 甲基苯丙胺, 高热, 程序性坏死, 受体相互作用蛋白3(RIP3), 混合系激酶区域样蛋白(MLKL), 皮质神经元, 成人脑组织, Nec-1, GSK’872, 协同作用

Abstract: Methamphetamine is one of the most prevalent drugs abused in the world. Methamphetamine abusers usually present with hyperpyrexia (39°C), hallucination and other psychiatric symptoms. However, the detailed mechanism underlying its neurotoxic action remains elusive. This study investigated the effects of methamphetamine + 39°C on primary cortical neurons from the cortex of embryonic Sprague-Dawley rats. Primary cortex neurons were exposed to 1 mM methamphetamine + 39°C. Propidium iodide staining and lactate dehydrogenase release detection showed that methamphetamine + 39°C triggered obvious necrosis-like death in cultured primary cortical neurons, which could be partially inhibited by receptor-interacting protein-1 (RIP1) inhibitor Necrostatin-1 partially. Western blot assay results showed that there were increases in the expressions of receptor-interacting protein-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) in the primary cortical neurons treated with 1 mM methamphetamine + 39°C for 3 hours. After pre-treatment with RIP3 inhibitor GSK’872, propidium iodide staining and lactate dehydrogenase release detection showed that neuronal necrosis rate was significantly decreased; RIP3 and MLKL protein expression significantly decreased. Immunohistochemistry staining results also showed that the expressions of RIP3 and MLKL were up-regulated in brain specimens from humans who had died of methamphetamine abuse. Taken together, the above results suggest that methamphetamine + 39°C can induce RIP3/MLKL regulated necroptosis, thereby resulting in neurotoxicity. The study protocol was approved by the Medical Ethics Committee of the Third Xiangya Hospital of Central South University, China (approval numbers: 2017-S026 and 2017-S033) on March 7, 2017.

Key words: GSK’872, human brain tissue, hyperpyrexia, methamphetamine, mixed lineage kinase domain-like protein, necrostatin-1, necroptosis, nerve regeneration, neural regeneration, rat cortical neurons, receptor-interacting protein-3, synergistic effect