本文選題：甲基苯丙胺 + 神經(jīng)損傷��； 參考：《南方醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 研究背景 甲基苯丙胺(Methamphetamine,METH,MA)屬于苯丙胺類神經(jīng)興奮劑(Amphetamine-Typed Stimulant,ATS),其鹽酸鹽為透明的結(jié)晶體,狀如冰,俗稱“冰毒”。METH具有多種藥理及毒理學(xué)特性,最初是作為減肥藥、抗疲勞劑使用,隨后發(fā)現(xiàn)其具有精神依賴性,因此被列入聯(lián)合國(guó)精神藥品公約管制的精神活性藥物,然而近年世界范圍內(nèi)對(duì)此藥的濫用呈上升趨勢(shì),國(guó)內(nèi)的濫用情況也十分嚴(yán)峻,所以對(duì)METH毒性機(jī)制的研究成為世界面臨的重大課題和研究熱點(diǎn)。動(dòng)物實(shí)驗(yàn)和臨床資料表明,METH濫用可導(dǎo)致顯著的精神和行為改變,如刻板運(yùn)動(dòng)、亢奮、易激惹等。研究顯示METH對(duì)心、肝、腎等組織器官均有毒性作用,但更主要的是對(duì)中樞神經(jīng)系統(tǒng)的毒性。METH可導(dǎo)致大腦紋狀體、海馬、皮質(zhì)等多部位損傷,包括神經(jīng)細(xì)胞凋亡、多巴胺(DA)耗竭、多巴胺轉(zhuǎn)運(yùn)體(DAT)減少和酪氨酸羥化酶活性(TH)下降。目前認(rèn)為,過(guò)量多巴胺的氧化作用、谷氨酸介導(dǎo)的興奮性毒性作用、線粒體功能紊亂以及大腦神經(jīng)元凋亡是METH導(dǎo)致中樞神經(jīng)損傷的可能機(jī)制,然而,當(dāng)前國(guó)內(nèi)外的研究結(jié)果尚不足以完全闡明METH確切的神經(jīng)毒性機(jī)制。 目的 大量研究支持氧化應(yīng)激是METH神經(jīng)毒性的重要機(jī)制。我們前一階段研究結(jié)果顯示METH引起大鼠紋狀體NOS活性升高,NO含量提高,產(chǎn)生大量的活性氧(ROS)和活性氮(RNS),這些物質(zhì)可引起神經(jīng)元SOD含量下降,導(dǎo)致中樞神經(jīng)系統(tǒng)損傷,而nNOS的抑制劑7-NI能減輕神經(jīng)毒性。研究表明不對(duì)稱性二甲基精氨酸(ADMA)是一氧化氮合酶(NOS)的內(nèi)源性競(jìng)爭(zhēng)性抑制劑,可抑制NO合成,而絕大部分的ADMA為二甲基精氨酸二甲基氨基水解酶(DDAH)降解。我們以往的研究發(fā)現(xiàn)METH中毒大鼠紋狀體區(qū)DDAH 1表達(dá)升高,此外各種研究表明ADMA與氧化應(yīng)激密切相關(guān)。然而METH是否通過(guò)干擾DDAH/ADMA系統(tǒng)影響NO的合成而產(chǎn)生神經(jīng)毒性,目前尚未見有相關(guān)報(bào)道。N—乙酰半胱氨酸(NAC)是一種強(qiáng)有力的抗氧化劑,其自身具有清除自由基功能,可促進(jìn)谷胱甘肽(GSH)的合成,增強(qiáng)組織抗氧化能力。 本研究假設(shè)METH誘導(dǎo)紋狀體氧化應(yīng)激,通過(guò)DDAH/ADMA系統(tǒng)介導(dǎo)神經(jīng)毒性損傷作用。為達(dá)此目的,建立METH中毒大鼠模型,運(yùn)用病理學(xué)技術(shù)、分子生物學(xué)、神經(jīng)化學(xué)等方法探討METH的神經(jīng)毒性,并且在此基礎(chǔ)上檢測(cè)NO上游調(diào)控系統(tǒng)DDAH的蛋白表達(dá)、ADMA含量及NOS活性等指標(biāo)的變化,此外還對(duì)NO下游作用產(chǎn)物過(guò)氧亞硝酸鹽(ONOO~-)進(jìn)行定量以及通過(guò)TUNEL方法檢測(cè)大腦神經(jīng)元的凋亡與氧化應(yīng)激的關(guān)系,最后研究抗氧化劑NAC對(duì)甲基苯丙胺神經(jīng)毒性的影響及其與DDAH/ADMA系統(tǒng)的關(guān)系,從一個(gè)新的角度探討METH神經(jīng)毒性機(jī)制。 方法 1 METH中毒模型的建立和毒性觀察 Wistar雄性大鼠,共40只,體重180g～220g,12 h光暗交替循環(huán),保持自由飲水及進(jìn)食。將動(dòng)物隨機(jī)分為4組,NS組腹腔注射生理鹽水,每天2次(8:00AM,6:00 PM),每次1mL,連續(xù)注射6天;METH組前兩天注射生理鹽水,第3～6天,腹腔注射METH(15mg/kg體重),注射方法同NS組;NAC組注射方法同NS組,用NAC替代生理鹽水,NAC的注射劑量為150mg/kg體重;NAC+METH組前兩天注射NAC,第3～6天,在METH注射前30min先腹腔注射NAC(150mg/kg體重),METH的注射方法同METH組。最后一次用藥72h后,處死大鼠取腦和其它器官。在實(shí)驗(yàn)過(guò)程中觀察動(dòng)物的體重、體溫和行為學(xué)改變;利用HE染色觀察動(dòng)物腦、心、肝、肺、腎組織病理學(xué)改變;高效液相色譜方法(HPLC)檢測(cè)紋狀體區(qū)DA及DOPAC含量的改變;利用ROS檢測(cè)試劑盒檢測(cè)紋狀體區(qū)ROS含量的變化。 2 NAC對(duì)METH神經(jīng)損傷的保護(hù)作用涉及DDAH/ADMA系統(tǒng) 利用熒光檢測(cè)方法,用ONOO~-氧化應(yīng)激試劑盒檢測(cè)紋狀體ONOO~-的變化;利用TUNEL方法觀察紋狀體神經(jīng)元的凋亡情況;Western Blotting檢測(cè)DDAH 1的蛋白表達(dá);HPLC檢測(cè)ADMA水平的改變;酶化學(xué)方法檢測(cè)NOS活性的變化。 結(jié)果 1.腹腔注射METH后,大鼠的體重顯著降低(P=0.000),體溫顯著性升高(P=0.000),并且出現(xiàn)顯著的行為學(xué)改變(P=0.000),主要表現(xiàn)為活動(dòng)增多、刻板樣運(yùn)動(dòng)活躍等。鏡下部分大鼠肝細(xì)胞水腫,肺廣泛充血、炎細(xì)胞浸潤(rùn),神經(jīng)元水腫,出現(xiàn)明顯的噬神經(jīng)現(xiàn)象。紋狀體的ROS水平顯著性升高(P=0.000),DA及DOPAC水平顯著降低(P=0.000;P=0.003),其差異有統(tǒng)計(jì)學(xué)意義。而使用NAC預(yù)處理的NAC+METH組與METH組相比,行為評(píng)分及ROS水平顯著性降低(P=0.000;P=0.000),而DA及DOPAC含量顯著性回升(P=0.000;P=0.040)。 2.與NS組相比,METH組紋狀體區(qū)DDAH 1蛋白表達(dá)水平明顯升高,ADMA含量顯著性降低(P=0.000),NOS活性、ONOO~-水平及神經(jīng)元凋亡數(shù)均顯著性增加(P=0.000,P=0.000,P=0.000)。而使用NAC預(yù)處理的NAC+METH組與METH組相比,紋狀體區(qū)DDAH 1蛋白表達(dá)水平呈一定程度的回落,ADMA含量顯著性回升(P=0.006),NOS活性、ONOO~-水平及神經(jīng)元凋亡數(shù)均顯著性降低(P=0.000,P=0.003,P=0.000)。且ADMA和ONOO~-含量之間存在負(fù)相關(guān)關(guān)系,ONOO~-含量與大腦神經(jīng)元凋亡數(shù)呈正相關(guān)關(guān)系。 結(jié)論 1.METH誘導(dǎo)紋狀體區(qū)組織內(nèi)ROS的產(chǎn)生和清除失衡,產(chǎn)生顯著的神經(jīng)毒性,引起大鼠神經(jīng)行為學(xué)改變,致使大腦神經(jīng)元損傷,導(dǎo)致DA和DOPAC含量下降及ROS水平的升高,而抗氧化劑NAC可減輕以上毒性改變。 2.METH誘導(dǎo)紋狀體區(qū)的NO上游調(diào)控DDAH/ADMA系統(tǒng)及下游產(chǎn)物的改變。主要表現(xiàn)為大鼠腦組織紋狀體區(qū)DDAH 1表達(dá)水平的升高,ADMA水平的降低,NOS活性、ONOO~-水平及神經(jīng)元凋亡數(shù)的增加,且ADMA和ONOO~-水平之間存在負(fù)相關(guān)關(guān)系,但是抗氧化劑NAC可一定程度逆轉(zhuǎn)以上改變,發(fā)揮神經(jīng)保護(hù)作用。 3.由此可見氧化應(yīng)激是METH神經(jīng)毒性的重要機(jī)制,而DDAH/ADMA系統(tǒng)可能是一個(gè)嶄新的神經(jīng)活性調(diào)控機(jī)制,在METH神經(jīng)損傷中發(fā)揮作用。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：D919

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