S-亚硝基谷胱甘肽还原酶在吗啡成瘾中的功能和机制探讨

Drug addiction is a serious social problem and continues to cause an enormous financial burden on society. It is characterized by drug dependence sensitization and craving. The exact mechanism of drug addiction remains unclear. Morphine, a typical of opiate, is widely used for pain relief. However, the clinical usage of morphine is limited by side effects including addiction, analgesic tolerance and opioid-induced hyperalgesia. We have found that autophagy plays an active role in morphine-induced effect. Morphine could induce mitochondrial dysfunction and led to autophagy (Autophagy 2013). The Atg5- and Atg7-dependent autophagy in dopaminergic neurons participated in the development of addictive behaviors (Autophagy 2017). Most recently, we found that the mRNA expression levels of the S-nitrosoglutathione reductase (GSNOR) was significantly decreased in the spinal cord and cortex tissues of mice with morphine injection by using RNA-seq. GSNOR regulates oxidative stress and autophagy. However, the physiological function of GSNOR in morphine addiction has not been studied so far. In this proposal, we aimed to analyze the expression change of GSNOR and its regulation, and to investigate the molecular mechanism of GSNOR in cellular and animal models with morphine addiction. We will define the role of GSNOR in morphine addiction through knockdown and overexpression of the GSNOR gene at the cellular level and knockout of this gene in mouse neural cells. By using the mouse models with knockout or overexpression of the Gsnor gene, we will further explore the effects of GSNOR on synaptic plasticity, neurotransmitter release, and addictive behaviors (such as reward effect, behavioral sensitization and withdrawal symptom). Our results will uncover the role of GSNOR in the pathogenesis of morphine addiction and may provide helpful information for opiate withdrawal and pain therapy.

毒品成瘾严重危害人类健康，表现为难以控制的渴求和依赖行为，其机制至今未明。吗啡是最常用的镇痛药物，长期使用会带来耐受和成瘾等副作用，限制了其临床应用。我们发现，慢性吗啡诱导线粒体功能异常诱发自噬（Autophagy 2013），进而调控成瘾行为（Autophagy 2017）。近期通过转录组测序，我们发现S-亚硝基谷胱甘肽还原酶（GSNOR）表达在吗啡成瘾过程中发生显著变化。GSNOR可调控氧化应激和自噬，其是否调控吗啡成瘾还不清楚。本项目拟从细胞系、原代神经元、小鼠模型等层面，利用基因过表达、敲降及敲除技术，研究GSNOR对神经元功能的影响；利用该基因敲除和过表达小鼠，研究GSNOR在自噬、突触可塑性和吗啡成瘾行为（如奖赏效应、行为敏感化和戒断症状）中的作用，探索其参与吗啡成瘾的机制。研究结果有望解析GSNOR在吗啡成瘾中的作用机制，为成瘾的治疗和临床镇痛提供思路与科学依据。

毒品成瘾严重危害人类健康，表现为难以控制的渴求和依赖行为，其机制至今未明。吗啡是最常用的镇痛药物，长期使用会带来耐受和成瘾等副作用，限制了其临床应用。本项目从动物模型出发，发现慢性吗啡注射诱导小鼠皮层的S-蛋白亚硝基化显著升高，同时伴随着S-亚硝基谷胱甘肽还原酶（GSNOR）的表达显著降低。随后在Gsnor基因敲除小鼠和神经元过表达GSNOR的小鼠中发现，GSNOR参与吗啡成瘾/镇痛耐受的形成。进一步通过亚硝基化组学、蛋白活性检测以及抑制剂注射小鼠实验结果表明，GSNOR调控PKCα蛋白的S-亚硝基化修饰参与吗啡镇痛耐受。另一方面，我们还发现，发现在帕金森（PD）动物模中，GSNOR通过抑制CDK5蛋白亚硝基化修饰，激活CDK5的活性，进而诱导CDK5介导的自噬，从而PD的发生发展；GSNOR通过调控TBK-1蛋白的S-亚硝基化在抗病毒天然免疫中扮演重要的作用。目前项目任务书的研究工作内容已经全部完成，同时深入研究了GSNOR在PD和抗病毒先天免疫过程中的作用机制。项目执行期内培养毕业博士研究生3人，在影响因子>10的领域权威SCI期刊发表标署本项目资助的论文3篇（Redox Biology 2020, 34: 101560；Redox Biology 2021, 2021. 47: 102172；Science China Life Sciences 2022; 65: 648-650），影响因子>5的两篇（Molecular Neurobiology, 2021;58:4628-4638；Free Radical Biology and Medicine, 2022, 189: 111-121），申请国家发明专利2项。团队成员2人次入选中国科学院青年创新促进会。我们的研究结果阐释GSNOR在吗啡镇痛耐受中的作用机制，为成瘾的治疗和临床镇痛提供思路与科学依据。