甲卡西酮神经毒性损伤中NO相关氧化应激通路的初步研究

New psychoactive substance would be about to the global epidemic third generation drug shocked and replaced the first classic and the second synthetic drug, the toxicity and perniciousness of which was even more serious. Methcathinone, a typical new psychoactive substance, induced psychological dependence and drug abuse. The activity of antioxidant enzymes was decreased dose-dependently; nNOS protein expression level was elevated obviously; mitochondrial membrane potential declined; caspase3 activation enhanced; which indicated increase of apoptosis while autophagy expression decreased in previous work. DDAH1/ADMA/NOS system played a critical role in the oxidative stress by increasing NO and ROS, which enhanced the nitrification level of glutathione-S-transferase pi (GSTP1), as a result of combination of GSTP1-P35/P25-CDK5 with CDK5 and cytoskeleton damage. It would be proposed the pathway was involved with toxic mechanism of methcathinone. In this research, culture in vitro and in vivo with different dose methcathinone, interference with inhibitors will be used by step to investigate the changes and the regulation of key factors, and to reveal the pathway on the apoptosis and autophagy by behavioristics, pathology, molecular biology, neurobiology and so on. This study is designed to definitude the mechanism of oxidative stress and provides basis for a comprehensive explanation of the neurotoxicity and new targets for drug therapy.

新精神活性物质或将成为全球流行的第三代毒品强力冲击、替代传统和合成毒品，其毒性、危害性较之传统毒品有过之而无不及。典型代表甲卡西酮摄入后易致精神依赖和药物滥用。我们前期研究显示，甲卡西酮染毒大鼠体内抗氧化酶活性降低，nNOS蛋白表达水平明显升高，线粒体膜电位下降、caspase3活化增强，凋亡增加，自噬表达下调。结合氧化应激通路DDAH1/ADMA/NOS系统诱使NO含量升高、ROS改变；NO促进GSTP1-P35/P25-CDK5复合物形成、破坏细胞骨架。我们推测甲卡西酮可能通过上述通路产生神经毒性作用，具体机制有待探索。本项目拟应用行为学、病理学、分子生物学、神经生物学等技术，建立不同剂量甲卡西酮染毒体内、外模型，经多种抑制剂分步骤干预、观察染毒后信号通路中主要要素表达变化、调控及细胞凋亡、自噬，明确氧化应激作用效应，为阐明甲卡西酮神经毒性损伤机制及寻找药物治疗靶点提供基础。

新精神活性物质已然成为全球流行的第三代毒品强力冲击、替代传统和合成毒品，其毒性、危害性较之传统毒品有过之而无不及。典型代表甲卡西酮与苯丙胺类药物结构相似，具有中枢神经兴奋作用，近年来被大肆滥用，致残、致死案例在国内外均有报道；其摄入后，主要中毒症状为心动加速、高温、焦虑、抑郁等，易致精神依赖和药物滥用。.为证实甲卡西酮产生神经毒性作用的通路，我们进行了如下实验：在行为学实验中，发现甲卡西酮染毒可降低大鼠的认知功能，使其空间位置记忆能力显著下降；对心肌产生明显毒性；TUNEL法观察到部分凋亡神经细胞显亮黄色，其形态呈细胞核固缩、变小、边缘化，海马区凋亡的神经元多集中在齿状回颗粒细胞层、锥体细胞层CA3区；甲卡西酮染毒大鼠体内抗氧化酶活性降低，nNOS蛋白表达水平明显升高，线粒体膜电位下降、caspase3活化增强，凋亡增加，自噬表达下调。结合氧化应激通路DDAH1/ADMA/NOS系统诱使NO含量升高、ROS改变；NO促进GSTP1-P35/P25-CDK5复合物形成、破坏细胞骨架。.利用已分化的PC12细胞株，通过DDAH1抑制剂L-257、NOS抑制剂L-NAME干预DDAH1/ADMA/NOS、通过7-NI、CDK5抑制剂Roscovitine干预GSTP1硝基化及抑制GSTP1-P35/P25-CDK5下游通路，对比染毒组PC12细胞和多种抑制剂分步骤干预的各组细胞，发现染毒组细胞活力均下降，细胞角状突起变短、断裂、消失，体积缩小，表面有毛刺状突起，贴壁少且不伸展，逐渐呈圆形、漂浮，胞浆内可见环状区域，通透、明亮，细胞间边界模糊，细胞数量减少。抑制剂组细胞呈多角型，贴壁较松，部分连接紧密、成团生长。染毒组细胞同样抗氧化酶活性降低，nNOS蛋白表达水平明显升高，线粒体膜电位下降、caspase3活化增强，凋亡增加，自噬表达下调。.通过上述实验，我们证实甲卡西酮致损伤机制系通过介导氧化应激作用，进而引起神经细胞凋亡和自噬，以调节神经毒性，在明确前述效应的基础上，本项目有望为阐明及寻找药物治疗靶点提供基础。