N-乙酰半胱氨酸对甲基苯丙胺神经损伤保护作用的研究

发布时间：2018-06-25 21:12

本文选题：甲基苯丙胺 + 神经损伤　；参考：《南方医科大学》2009年硕士论文

【摘要】： 研究背景甲基苯丙胺(Methamphetamine,METH,MA)属于苯丙胺类神经兴奋剂(Amphetamine-Typed Stimulant,ATS),其盐酸盐为透明的结晶体,状如冰,俗称“冰毒”。METH具有多种药理及毒理学特性,最初是作为减肥药、抗疲劳剂使用,随后发现其具有精神依赖性,因此被列入联合国精神药品公约管制的精神活性药物,然而近年世界范围内对此药的滥用呈上升趋势,国内的滥用情况也十分严峻,所以对METH毒性机制的研究成为世界面临的重大课题和研究热点。动物实验和临床资料表明,METH滥用可导致显著的精神和行为改变,如刻板运动、亢奋、易激惹等。研究显示METH对心、肝、肾等组织器官均有毒性作用,但更主要的是对中枢神经系统的毒性。METH可导致大脑纹状体、海马、皮质等多部位损伤,包括神经细胞凋亡、多巴胺(DA)耗竭、多巴胺转运体(DAT)减少和酪氨酸羟化酶活性(TH)下降。目前认为,过量多巴胺的氧化作用、谷氨酸介导的兴奋性毒性作用、线粒体功能紊乱以及大脑神经元凋亡是METH导致中枢神经损伤的可能机制,然而,当前国内外的研究结果尚不足以完全阐明METH确切的神经毒性机制。目的大量研究支持氧化应激是METH神经毒性的重要机制。我们前一阶段研究结果显示METH引起大鼠纹状体NOS活性升高,NO含量提高,产生大量的活性氧(ROS)和活性氮(RNS),这些物质可引起神经元SOD含量下降,导致中枢神经系统损伤,而nNOS的抑制剂7-NI能减轻神经毒性。研究表明不对称性二甲基精氨酸(ADMA)是一氧化氮合酶(NOS)的内源性竞争性抑制剂,可抑制NO合成,而绝大部分的ADMA为二甲基精氨酸二甲基氨基水解酶(DDAH)降解。我们以往的研究发现METH中毒大鼠纹状体区DDAH 1表达升高,此外各种研究表明ADMA与氧化应激密切相关。然而METH是否通过干扰DDAH/ADMA系统影响NO的合成而产生神经毒性,目前尚未见有相关报道。N—乙酰半胱氨酸(NAC)是一种强有力的抗氧化剂,其自身具有清除自由基功能,可促进谷胱甘肽(GSH)的合成,增强组织抗氧化能力。本研究假设METH诱导纹状体氧化应激,通过DDAH/ADMA系统介导神经毒性损伤作用。为达此目的,建立METH中毒大鼠模型,运用病理学技术、分子生物学、神经化学等方法探讨METH的神经毒性,并且在此基础上检测NO上游调控系统DDAH的蛋白表达、ADMA含量及NOS活性等指标的变化,此外还对NO下游作用产物过氧亚硝酸盐(ONOO~-)进行定量以及通过TUNEL方法检测大脑神经元的凋亡与氧化应激的关系,最后研究抗氧化剂NAC对甲基苯丙胺神经毒性的影响及其与DDAH/ADMA系统的关系,从一个新的角度探讨METH神经毒性机制。方法 1 METH中毒模型的建立和毒性观察 Wistar雄性大鼠,共40只,体重180g～220g,12 h光暗交替循环,保持自由饮水及进食。将动物随机分为4组,NS组腹腔注射生理盐水,每天2次(8:00AM,6:00 PM),每次1mL,连续注射6天;METH组前两天注射生理盐水,第3～6天,腹腔注射METH(15mg/kg体重),注射方法同NS组;NAC组注射方法同NS组,用NAC替代生理盐水,NAC的注射剂量为150mg/kg体重;NAC+METH组前两天注射NAC,第3～6天,在METH注射前30min先腹腔注射NAC(150mg/kg体重),METH的注射方法同METH组。最后一次用药72h后,处死大鼠取脑和其它器官。在实验过程中观察动物的体重、体温和行为学改变;利用HE染色观察动物脑、心、肝、肺、肾组织病理学改变;高效液相色谱方法(HPLC)检测纹状体区DA及DOPAC含量的改变;利用ROS检测试剂盒检测纹状体区ROS含量的变化。 2 NAC对METH神经损伤的保护作用涉及DDAH/ADMA系统利用荧光检测方法,用ONOO~-氧化应激试剂盒检测纹状体ONOO~-的变化;利用TUNEL方法观察纹状体神经元的凋亡情况;Western Blotting检测DDAH 1的蛋白表达;HPLC检测ADMA水平的改变;酶化学方法检测NOS活性的变化。结果 1.腹腔注射METH后,大鼠的体重显著降低(P=0.000),体温显著性升高(P=0.000),并且出现显著的行为学改变(P=0.000),主要表现为活动增多、刻板样运动活跃等。镜下部分大鼠肝细胞水肿,肺广泛充血、炎细胞浸润,神经元水肿,出现明显的噬神经现象。纹状体的ROS水平显著性升高(P=0.000),DA及DOPAC水平显著降低(P=0.000;P=0.003),其差异有统计学意义。而使用NAC预处理的NAC+METH组与METH组相比,行为评分及ROS水平显著性降低(P=0.000;P=0.000),而DA及DOPAC含量显著性回升(P=0.000;P=0.040)。 2.与NS组相比,METH组纹状体区DDAH 1蛋白表达水平明显升高,ADMA含量显著性降低(P=0.000),NOS活性、ONOO~-水平及神经元凋亡数均显著性增加(P=0.000,P=0.000,P=0.000)。而使用NAC预处理的NAC+METH组与METH组相比,纹状体区DDAH 1蛋白表达水平呈一定程度的回落,ADMA含量显著性回升(P=0.006),NOS活性、ONOO~-水平及神经元凋亡数均显著性降低(P=0.000,P=0.003,P=0.000)。且ADMA和ONOO~-含量之间存在负相关关系,ONOO~-含量与大脑神经元凋亡数呈正相关关系。结论 1.METH诱导纹状体区组织内ROS的产生和清除失衡,产生显著的神经毒性,引起大鼠神经行为学改变,致使大脑神经元损伤,导致DA和DOPAC含量下降及ROS水平的升高,而抗氧化剂NAC可减轻以上毒性改变。 2.METH诱导纹状体区的NO上游调控DDAH/ADMA系统及下游产物的改变。主要表现为大鼠脑组织纹状体区DDAH 1表达水平的升高,ADMA水平的降低,NOS活性、ONOO~-水平及神经元凋亡数的增加,且ADMA和ONOO~-水平之间存在负相关关系,但是抗氧化剂NAC可一定程度逆转以上改变,发挥神经保护作用。 3.由此可见氧化应激是METH神经毒性的重要机制,而DDAH/ADMA系统可能是一个崭新的神经活性调控机制,在METH神经损伤中发挥作用。
[Abstract]:Research background
Methamphetamine (Methamphetamine, METH, MA) belongs to the amphetamine type Stimulant (Amphetamine-Typed Stimulant, ATS). Its hydrochloride is a transparent crystal like ice. It is commonly known as "ice poison".METH has a variety of pharmacological and toxicological characteristics. It was originally used as a weight loss drug, antifatigue agent, and subsequently found to have a mental dependence. Therefore, it is found to have mental dependence. As a psychoactive drug controlled by the United Nations Convention on psychotropic drugs, the drug abuse in the world has increased in recent years, and the abuse in China is also very severe. So the study of the toxic mechanism of METH has become a major topic and research hotspot in the world. Animal experiment and clinical data show that the abuse of METH can lead to the abuse of the drug. Significant mental and behavioral changes, such as stereotyped movement, hyperactivity, irritability, etc., have shown that METH has toxic effects on the tissues and organs of the heart, liver and kidney, but mainly the toxicity of.METH to the central nervous system can lead to multiple lesions in the brain striatum, hippocampus, and cortex, including neuron apoptosis, dopamine (DA) depletion and dopamine transfer. The decrease of transporter (DAT) and tyrosine hydroxylase activity (TH) decrease. At present, it is believed that excessive dopamine oxidation, glutamic acid mediated excitotoxicity, mitochondrial dysfunction and brain neuron apoptosis are the possible mechanisms of METH leading to central nerve injury. However, the current research results at home and abroad are not enough to fully elucidate METH The exact mechanism of neurotoxicity.
objective
A large number of studies support oxidative stress as an important mechanism for METH neurotoxicity. Our previous study showed that METH induced increased NOS activity in the striatum and increased NO content in rat striatum, producing a large number of reactive oxygen species (ROS) and active nitrogen (RNS). These substances could cause the decrease of SOD content in neurons, leading to the damage of the central nervous system and the inhibition of nNOS. 7-NI can reduce neurotoxicity. Studies have shown that asymmetric two methyl arginine (ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS), which inhibits NO synthesis, and the overwhelming majority of ADMA is two methyl arginine two methylamino hydrolase (DDAH) degradation. Our previous study found the expression of DDAH 1 in the striatum of METH poisoned rats In addition, various studies have shown that ADMA is closely related to oxidative stress. However, whether METH produces neurotoxicity by interfering with the influence of DDAH/ADMA system on the synthesis of NO, it has not yet been reported that.N - acetylcysteine (NAC) is a powerful antioxidant, and its self has the function of scavenging free radicals and can promote glutathione (GSH). ) synthesis to enhance the antioxidant capacity of the tissue.
This study assumes that METH induces oxidative stress in the striatum and mediates neurotoxicity through the DDAH/ADMA system. In order to achieve this purpose, a rat model of METH poisoning is established. The neurotoxicity of METH is explored by means of pathological techniques, molecular biology and neurochemistry, and on this basis, the protein expression of DDAH in the upstream NO system of NO is detected, ADM The changes in the content of A and the activity of NOS, as well as the quantitative of peroxy nitrite (ONOO~-) in the downstream of NO, and the relationship between the apoptosis of brain neurons and oxidative stress by TUNEL method. Finally, the effects of antioxidant NAC on methamphetamine neurotoxicity and the relationship with the DDAH/ADMA system are studied. The mechanism of METH neurotoxicity is discussed.
Method
Establishment and toxicity observation of 1 METH poisoning model
Wistar male rats, 40 rats, body weight 180g to 220g, 12 h light dark alternate circulation, keep free drinking water and eat. The animals were randomly divided into 4 groups. Group NS was injected with saline, 2 times a day (8:00AM, 6:00 PM), each 1mL, for 6 days; the METH group was injected with saline, third to 6 days, and intraperitoneal injection of METH (15mg/kg weight), injection method Group NS, group NAC, group NS, group NS, NAC instead of normal saline, NAC injection amount of 150mg/kg weight; NAC+METH group injected NAC in the first two days, third to 6 days, before METH injection NAC (150mg/kg weight), and the injection method was in the same group. The brain and other organs were executed after the last drug use. In the experiment process, the experiment process was carried out. To observe the body weight, body temperature and behavioral changes of the animals, observe the pathological changes of the brain, heart, liver, lung and kidney of animals by HE staining, the changes of the content of DA and DOPAC in the striatum by high performance liquid chromatography (HPLC), and the changes of the ROS content in striatum by ROS detection kit.
2 the protective effect of NAC on METH nerve injury involves DDAH/ADMA system.
The changes of ONOO~- in striatum were detected by ONOO~- oxidation stress kit and TUNEL method was used to observe the apoptosis of neurons in striatum; Western Blotting was used to detect the protein expression of DDAH 1; HPLC was used to detect the change of ADMA level, and the enzyme chemical method was used to detect the change of NOS viability.
Result
1. after intraperitoneal injection of METH, the body weight of the rats decreased significantly (P=0.000), the body temperature was significantly increased (P=0.000), and a significant behavioral change (P=0.000) was found. The main manifestations were the increase of activity and the activity of stereotyped exercise. The hepatocytes were edema, extensive hyperemia of the lungs, infiltration of inflammatory cells, edema of neurons, and obvious neurophagocytosis in some rats. The ROS level of the striatum was significantly higher (P=0.000), and the level of DA and DOPAC decreased significantly (P=0.000; P=0.003), and the difference was statistically significant. While the NAC pretreated NAC+METH group was significantly lower in the behavior score and ROS level than the METH group (P=0.000; P=0.000).
2. compared with the NS group, the expression level of DDAH 1 protein in the striatum of METH group was significantly increased, the content of ADMA decreased significantly (P=0.000), NOS activity, ONOO~- level and the number of neuron apoptosis increased significantly (P=0.000, P=0.000, P=0.000). The expression level of the 1 protein in the striatum was to a certain extent compared with the NAC pretreated NAC+METH group. The ADMA content increased significantly (P=0.006), NOS activity, ONOO~- level and neuron apoptosis decreased significantly (P=0.000, P=0.003, P=0.000), and there was a negative correlation between ADMA and ONOO~- content, and ONOO~- content was positively correlated with the number of neuronal apoptosis.
conclusion
1.METH induced the imbalance in the production and clearance of ROS in the striatum tissue, resulting in significant neurotoxicity, causing neurobehavioral changes in rats, causing neuronal damage in the brain, resulting in a decrease in the content of DA and DOPAC and the increase of ROS levels, and the antioxidant NAC can reduce the above toxicity.
2.METH induced the changes in the NO upstream of the striatum to regulate the DDAH/ADMA system and the downstream products. The main expression is the elevation of the expression level of DDAH 1 in the striatum, the decrease of ADMA level, the activity of NOS, the increase of ONOO~- level and the number of neuron apoptosis, and the negative correlation between the level of ADMA and ONOO~-, but the antioxidant NAC can be found. The above changes can be reversed to some extent and play a neuroprotective role.
3. it can be seen that oxidative stress is an important mechanism of METH neurotoxicity, and the DDAH/ADMA system may be a new regulatory mechanism of neuroactivity, which plays a role in the METH nerve injury.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919

【参考文献】