甲基苯丙胺对PC12细胞的毒性损伤、蛋白质硝基化、L-257抗损伤等作用的机制研究
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摘要
研究背景：
甲基苯丙胺(Methamphetamine, METH)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,外观状如冰,俗称“冰毒”,属于一种新型毒品,也是目前危害最为严重和广泛的毒品之一。冰毒具有中枢神经兴奋性、致幻、食欲抑制和拟交感能效应等药理学、毒理学特性。过去曾作为抗疲劳剂、减肥药广泛使用,但随后发现其具有严重的精神依赖性,已被列为联合国精神药品公约管制的精神活性药物。由于METH具有见效快、兴奋作用持续时间长,价格低廉,化学合成技术简单,多途径摄取等特点,导致快速地滥用及蔓延。冰毒号称“毒品之王”,其最大的特点是致瘾性强,戒断困难。近年国际和国内大中城市的滥用情况十分严峻,吸毒不仅造成吸毒者机体和精神损害,而且造成艾滋病等多种疾病传播,严重影响人体身心健康。毒品相关的刑事犯罪和社会治安等问题,对社会的稳定造成极大危害。因此,METH的神经毒性损伤、成瘾机制的研究,是全世界面临的重大课题和研究热点。
METH具有多种药理、毒理学特性,尤其是具有很强的中枢兴奋作用和强烈的药物依赖性,长期滥用可以引起中枢神经系统神经化学、神经病理学和行为学的改变。对METH成瘾者而言,毒品影响的不仅是精神,还包括神经结构和功能的损伤性改变。近年来的研究显示,METH有较强的神经毒性,可导致大脑出现与阿尔茨海默病(Alzheimer's disease, AD)和帕金森病(Parkinson's disease, PD)等退行性神经病变类似的病理学改变。现阶段的研究结果显示,其可能的机制主要包括：氧化应激损伤作用、体温平衡失调、离子稳态破坏以及细胞凋亡相关通路的激活等。但是,现有的研究结果仍不足以充分阐释METH的神经毒性机制,对其毒性作用尚缺乏有效的防治手段。
在我们前一阶段的研究中,对METH处理大鼠的额叶皮质、纹状体、海马等部位的差异表达蛋白质进行鉴定和分析,发现氧化应激、能量代谢障碍、蛋白酶体功能失调以及凋亡等可能是参与METH神经毒性的主要机制。其中,一种新型的一氧化氮(nitric oxide, NO)生成调节酶二甲基精氨酸二甲基氨基水解酶1(Nω, Nω-dimethyl-L-arginine dimethylaminohydrolase, DDAH1)在纹状体和额叶皮质的高表达尤其令人瞩目。DDAH是近年发现的一种蛋白酶,在体内存在生物学活性相似的两种亚型DDAH1和DDAH2,其中DDAH1主要分布于中枢神经系统。DDAH主要参与不对称二甲基精氨酸(asymmetric dimethylated L-arginine, ADMA)的分解代谢,体内95%以上的ADMA均由DDAH分解代谢,调控正常生理情况下不同的组织器官内的ADMA浓度维持在相应的水平。研究发现,UDMA是含有甲基化精氨酸蛋白质的分解产物,是内源性的一氧化氮合酶(nitric oxide synthase, NOS)的抑制剂,对三种亚型的NOS均有抑制作用。蛋白质甲基化是生物体内普遍存在的一种蛋白质翻译后修饰现象,蛋白质的甲基化和甲基化蛋白质的降解,是机体内存在的正常生理过程。某些病理情况下,体内ADMA浓度下降可导致其对NOS的抑制作用减弱,引起组织内NO合成增加。可见,DDAH可以通过调节组织内ADMA的降解率,从而调节组织中NOS的活性和NO的合成。正常生理条下,中枢神经系统中的NO主要通过神经型NOS (nitric oxide synthase, nNOS)产生,作为神经递质发挥生理作用并可调节神经轴突的可塑性。而在氧化应激等病理条件下,产生的过量NO,则有可能对组织造成损害。研究证明,氧化应激可在损伤过程中产生大量的NO,NO和超氧阴离子的产物可造成脑组织DNA、蛋白质和脂质氧化损伤而诱导神经细胞损伤、功能丧失和死亡,在衰老和AD等多种神经退行性疾病的发病机制中起重要作用。
我们前一阶段的体内实验研究结果显示METH可引起大鼠脑组织多部位神经毒性损伤,多巴胺及代谢产物的耗竭,纹状体NOS活性升高,NO含量提高,小胶质细胞激活等。运用蛋白质组学的方法,对METH注射后大鼠纹状体、皮质、海马等部位部位的差异表达蛋白质进行鉴定和分析,寻找和鉴定出30个差异表达蛋白点,3个NO相关蛋白即硝化蛋白。同时,我们采用PC12细胞的体外急性METH中毒模型的研究表明,氧化应激、能量代谢障碍、蛋白酶体功能失调、凋亡及重要结构蛋白的硝化等可能是METH神经毒性的主要机制。
蛋白质硝基化作用是一种特殊的蛋白质翻译后修饰作用,属于氧化应激反应的通路之一。在大量NO介导下,组织内产生大量的活性氮类物质(reactive nitrogen species, RNS)可对相关蛋白质的3位酪氨酸产生硝基化作用,生成3-硝基酪氨酸(3-NT),从而导致蛋白功能的失活。研究表明,许多典型的神经退行性疾病中可发生大量的蛋白质硝基化水平升高现象。因此我们首次提出,DDAH1/ADMA/NOS通路介导的蛋白质硝基化作用可能是METH神经毒性机制的重要组成部分。
为了更深入的研究蛋白质硝基化作用与METH的神经毒性机制之间的关系,本次研究采用多巴胺能神经元体外模型PC12细胞系进行体外实验。PC12细胞在神经生长因子等诱导后,形态学及功能方面向交感神经元分化。由于PC12细胞的神经元分化特性,目前已做为一个广泛使用的神经元体外模型,用于神经生物、中枢神经系统疾病、神经毒素等方面的研究,也是一个可行的METH神经蛋白组学研究模型。因此,我们运用PC12细胞系进行METH体外损伤实验,探讨METH对PC12细胞的毒性损伤作用,并在此基础上运用分子生物学、蛋白质组学及质谱分析技术,对METH作用PC12后的硝基化蛋白质进行定性、定量分析,进一步探讨DDAH1/ADMA/NOS通路介导的蛋白质硝基化作用对靶蛋白的功能影响。同时,我们采用DDAH1的经典抑制剂L-257对该通路进行有效抑制,通过与阴性对照组进行比较,分析DDAH1/ADMA/NOS通路与蛋白质硝基化作用间的因果关系,并对硝基化蛋白进行验证。
目的：
建立单纯METH急性中毒和经L-257预处理的METH急性中毒PC12细胞模型,运用细胞生物学、蛋白质组学、稳定同位素标记氨基酸细胞培养技术(stable isotope labeling with amino acids in cell culture, SILAC)等方法,了解METH的神经毒性作用及L-257预处理对METH神经毒性的保护作用,检测PC12细胞内与METH毒性作用密切相关的硝基化因素NOS、NO含量的表达变化,对硝基化水平升高的蛋白进行定性、定量分析,探讨DDAH1/ADMA/NOS通路介导的蛋白质硝基化作用与METH神经毒性之间的关系。
方法：
1.METH对PC12细胞毒性损伤及L-257的保护作用
采用已分化的PC12细胞,用高糖DMEM培养基(含10%胎牛血清,1：100双抗)进行培养,将细胞置于37℃,5%C02的培养箱中。实验分为空白对照组(阴性对照)和实验组,实验组根据L-257浓度的不同而分为4组,L-257含量分别为0μmol/L(阳性对照)、10μmol/L、50μmol/L、100μmol/L,各组METH含量均为2.0mmol/L。细胞生长至对数生长期,达80%汇合后各实验组分别用上述不同浓度METH和L-257处理,对照组加入等体积的DMEM培养液,继续培养24h。倒置显微镜下观察PC12细胞形态改变,MTT法检测细胞存活率,nnexin V-FITC/PI双染法结合流式细胞术检测细胞凋亡率,NO检测试剂盒硝酸还原酶法检测细胞上清液NO含量,NOS检测试剂盒检测NO上游NOS活力,Western blot技术检测NO下游产物总3-硝基酪氨酸表达水平。
2.应用SILAC技术对PC12细胞内METH诱导的硝基化蛋白的鉴定
采用同位素对PC12细胞进行标记,以使各实验组内总蛋白分子量分别增多4-6和8-10,通过不同分子量对各组间的蛋白质进行区分。已分化的PC12细胞分为3组：(1)轻链组(对照组,L),采用轻链培养液(高糖DMEM培养基+10%胎牛血清)进行培养；(2)中链组(METH+L-257, M),采用中链培养液(缺乏L-精氨酸-L赖氨酸的高糖DMEM培养基+10%的富含2H4-L-赖氨酸和13C6-L-精氨酸的胎牛血清)进行培养,分子量偏移增加4至6；(3)重链组(METH, H),采用重链培养液(缺乏L-赖氨酸-L-赖氨酸的高糖DMEM培养基+10%的富含13C6-’N2-L-赖氨酸和13C6-15N4-L-精氨酸的胎牛血清)进行培养,分子量偏移增加8至10。
将3组PC12细胞接种于6孔板中,给予对应的同位素培养液进行培养,稳定传代6代以上,再次接种于10cm2培养皿中,每组2个培养皿。当细胞生长至对数生长期,达80%汇合时弃去上层培养液,并进行以下处理：轻链组加入高糖DMEM培养基；中链组加入含2.0mmol/L METH和50μmol/L抑制剂L-257的中链DMEM;重链组加入含2.0mmol/L METH的重链DMEM。以上三组细胞继续培养24小时,分别提取各组总蛋白。然后将3组蛋白混合,采用免疫共沉淀法提取总硝基化蛋白。SDS电泳分离总硝基化蛋白,对各个条带进行切胶、酶切和肽段抽提。采用LC-MS/MS质谱对肽段进行扫描,对各组内的硝基化蛋白进行定性、定量分析。
3. METH作用下L-257对GSTP1蛋白表达的影响
实验分为空白对照组(阴性对照)和实验组,实验组根据L-257浓度的不同而分为4组,L-257含量分别为Oμmol/L (阳性对照)、10μml/L、50μmol/L、100μmo/L各实验组均采用2.0mmol/LMETH处理。采用Western-blot技术对各组GSTP1蛋白表达进行定量分析。
结果：
1.1METH对PC12细胞形态学影响及L-257的保护作用
METH+L-257处理组PC12细胞胞体逐渐萎缩变圆,变圆细胞的胞质透亮度增加,胞浆内可见环形透亮区,细胞突起变短、断裂、消失,细胞间神经网络结构逐渐消失,因细胞突起萎缩可在细胞表面形成毛刺状,细胞边界不清晰,并可见细胞脱壁漂浮现象。随着L-257浓度降低,细胞形态损伤呈增强趋势,100μmol/L及50μmol/L处理组主要以胞体变圆及胞浆透亮增加为主,而降低至25μmol/L时突起的改变开始变明显,并有毛刺状结构形成,此现象在10μmol/L及Oμmol/L组尤为明显,细胞突起及网络结构基本消失。
1.2METH作用下L-257对PC12细胞存活率的保护作用
结果显示,随着L-257浓度逐渐增加,MTT法测得经METH(2.0mmol/L)处理的PC12细胞OD值逐渐升高,细胞存活率逐渐升高,各实验组与对照组相比有显著性差异(P<0.001,F=816.687)。
1.3METH作用下L-257对PC12细胞凋亡的保护作用
结果显示,随着L-257浓度的不断升高,经METH (2.0mmol/L)处理的PC12细胞凋亡率逐渐下降,各实验组与对照组相比有显著性差异(P<0.001,F=119.436)。
1.4METH作用下L-257对PC12细胞培养液NO浓度的影响
结果显示,随着L-257浓度逐渐增加,经METH (2.0mmol/L)处理的PC12细胞培养上清液中NO含量逐渐减少。各实验组与对照组相比,除100剂量组与对照组比较无显著性差异(P=0.170),其余各组均有显著性差异(P<0.001,F=50.030)。
1.5METH作用下L-257对PC12细胞NOS活性的影响
结果显示随着L-257浓度逐渐增加,经METH(2.0mmol/L)处理的PC12细胞总NOS活力逐渐减少。各实验组与对照组相比,除100剂量组与对照组比较无显著性差异(P=0.246),其余实验组与对照组相比均有显著性差异(P<0.008,F=29.043)。
1.6METH作用下L-257对PC12细胞总硝基化蛋白表达的影响
结果显示,阴性对照组与阳性对照组相比总硝基化蛋白表达在15-170KD范围内普遍升高。L-257对硝基化作用可产生明显的抑制效果：随着L-257浓度的升高,硝基化蛋白的总体表达水平逐渐降低。
2.1SILAC方法对硝基化蛋白的鉴定和比较
SILAC方法共鉴定出蛋白42个,其中硝基化水平升高的共27个：升高0-10%的为6个,升高10%-20%的为12个,升高20%-30%的为6个,30%以上的为1个,L-257对蛋白质的硝基化作用具有不同程度的抑制效果。其中共有15种核糖体蛋白硝基化水平不同程度地升高,71kDa热休克同源蛋白、辅肌动蛋白α1和谷胱甘肽S转移酶P1的硝基化水平明显升高。
2.2METH作用下GSTP1蛋白表达变化
结果显示,阴性对照组与阳性对照组(单纯METH)相比,GSPT1蛋白表达显著降低。L-257对GSPT1蛋白的下降趋势可起到明显的抑制作用：随着L-257浓度的升高,GSTP1蛋白的表达逐渐上升。
结论：
1. METH对PC12细胞具有神经毒性作用,能导致细胞形态学改变,L-257对METH的神经毒性具有明显的保护作用。
2. METH可通过DDAH1/ADMA/NOS通路使PC12细胞NOS活力和NO含量增加,通过氧化应激引起靶蛋白硝基化水平升高,造成PC12细胞活力下降,诱导细胞凋亡。
3.L-257可通过抑制DDAH1/ADMA/NOS通路而有效抑制靶蛋白的硝基化作用,从而降低PC12细胞的凋亡率和死亡率,发挥显著的神经毒性保护作用。
4. METH诱导的蛋白质硝基化作用参与了氧化应激、凋亡和细胞骨架损伤等神经损伤过程,可能是METH神经毒性的重要机制。
BACKGROUND
Methamphetamine (METH), also known as "ice" for its ice-like and crystalline appearance, belongs to amphetamine-type stimulants (ATS). METH is one of the most popular drugs which have brought about great hazard to the society. METH is characterized by the toxicological and pharmacological features of central excitability, hallucinations, inhibited appetite and sympatheticomimetic effects. It was first used as antifatigue and anti-obesity agents. But soon it was put onto the list of controlled psychoactive drugs by the United Nations Convention on Psychotropic Drugs because of its psychological dependence. In recent years, the use of METH spreads quickly and wildly because it is easy to synthesis with little cost. METH is called "the king of drugs" because it is impossible to get rid of METH addiction once someone has used it. Nowadays the problem of METH addiction has been getting serious in big cities all around the world. METH not only results in damages to the body and the spirit, but also gives rise to many diseases such as AIDS. The METH related crimes also impair the order and the stability of the entire society. Thus, the mechanism study of METH induced neurotoxicity and addiction has become one of the most important topics for researchers.
METH has many pharmacological and toxicological features among which the significant central excitability is the most important one. Long-term abuse of METH can lead to pathological, praxiological and neurochemistry changes to the central nerve system. For those who abuse METH, it is not only the spirit but also the structures and the functions of the CNS that have been affected. Previous studies show that METH induced neurotoxicity can cause disease recessive neuropathies such as Alzheimer's (AD) and Parkinson's disease (PD) in the brain. In recent years it has been revealed that the possible mechanism of METH induced neurotoxicity could be consist of oxidative stress, disturbance of energy metabolism, disorder of proteasomes and apoptosis. It has been reported that the up-regulation of Nω, Nω-dimethyl-L-arginine dimethylaminohydrolase (DDAH1), a newfound regulatory enzyme of NO, in corpus striatum and cortex of frontal lobe has caused the attentions from researchers all over the world. DDAH is a protease which has two isoforms with similar biologic activity in vivo, DDAH1and DDAH2. DDAH1is mainly seen in the CNS. DDAH is chiefly in charge of the metabolism of asymmetric dimethylated L-arginine (ADMA). About95%ADMA are catabolized by DDAH in order to maintain its balance in vivo. ADMA is the decomposed products of methylated Arginine which is common Protein Post-translational Modification. Protein methylation and decomposition of methylated proteins are normal physiological processes in human and animals. ADMA is the inhibitor of endogenous nitric oxide synthase (NOS) and has dramatic inhibitory effect to all the three isoforms. In certain pathological conditions, with the decrease of ADMA and its inhibitory effect the production of NO increases significantly. While in normal conditions, the production of NO, which serves as neurotransmitters performing physiological effects and modulating the plasticity of neuritis, is mainly induced by the neurologic nitric oxide synthase (nNOS) in the CNS. It has been indicated that the production of NO induced by oxidative stress plays a key role in process of aging and some degenerative diseases such as AD and PD and that NO and superoxide anion products could induce apoptosis, death and dysfunctions of the nerve cells because of the oxidative damages to DNA, protein and lipid in brain tissues.
Our previous study revealed that METH can cause neurotoxicity, dopamine exhaustion, increases of NO and NOS and microglia activation in different brain regions of rat. By using proteomic methods,30differentially expressed proteins and3NO induced nitrated proteins were identified. In addition, PC12cell model of acute METH poisoning displayed that oxidative stress, disturbance of energy metabolism, dysfunction of proteasomes, apoptosis and nitrification of key proteins are involved in the METH induced neurotoxicity.
Protein nitration is a special post-translational modification which belongs to oxidative stress reactions. With the presence of over produced NO, a huge amount of reactive nitrogen species (RNS) are induced in vivo, which could result in deactivation of key proteins by elevated levels of protein nitration. It was reported that, in many typical recessive diseases such as AD and PD, an up-regulated levels of nitrated proteins were found. Thus, we would like to propose that protein nitration which is mediated by DDAH1/ADMA/NOS pathway plays a critical role in the METH-induced neurotoxicity.
In order to further understand the relationship between protein nitration and METH induced neurotoxicity, in vitro PC12cell model of acute METH poisoning is used in this research. When induced by nerve growth factor (NGF), PC12cells can differentiate toward sympathetic neurons both morphologically and functionally. PC12cells are widely used as an in vitro neurons model for researches of neurobiology, central nervous system diseases and neurotoxin. It is also a useful proteomic model for METH induced neurotoxicity. Therefore in this research PC12cells model of METH poisoning together with proteomic and molecular biology methods are used to investigate the relationship between protein nitration and METH induced neurotoxicity. The effect of nitration on target proteins, which is mediated by DDAH1/ADMA/NOS pathway, is analyzed qualitatively and quantitatively. On the other hand, L-257, the novel inhibitor of DDAH1, is used to confirm the causation of the pathway and protein nitration and verify the nitrated proteins identified by mass spectrum (MS) by repressing the pathway directly.
OBJECTIVES
In this research cytobiological methods, proteomic methods and stable isotope labeling with amino acids in cell culture (SILAC) was used to:(1) establish PC12 cells models of acute METH poisoning with or without L-257treatment,(2) investigate the METH induced neurotoxicity and the protective effect of L-257,(3) determine the nitration related factors which could cause neurotoxicity including NOS and NO levels,(4) quantitatively and qualitatively analyze the target proteins with elevated nitration levels,(5) discuss the relationship between protein nitration mediated by DDAH1/ADMA/NOS pathway and METH induced neurotoxicity.
METHODS
1. Protective effect of L-257on PC12cells treated with METH
Differentiated PC12cells are cultured in high glucose DMEM containing10%FBS and double antibiotics in37℃incubator with5%CO2. PC12cells are divided into control group and experimental groups. The experimental groups are divided into subgroups according to the L-257amount added as0μmol/L,10μmol/L,50μmol/L and100μmol/L. The amount of METH added into every group is2.0mmol/L. When the cells are in logarithmic phase and grow to80%density, the regents are added into each group as described above. The same amount of DMEM is added into the control group. All the groups are cultured for24h. The morphological changes of PC12cells are observed by invert microscope. The cell viability is tested by MTT method. The apoptosis rate is determined by Annexin V-FITC/PI stain associated with flow cytometer. NO level in supernatant is determined by nitrate reductase method. NOS activity is determined by NOS kit. The expressions of total nitrated proteins in the downstream of NO are determined by Western blot.
2. Using SILAC to indentify nitrated proteins in PC12cells treated with METH
In order to recognize different cell groups in the mass spectra, the total proteins in each group is labeled by isotopes which ensure4-6Da or6-8Da shift in molecular weight. Differentiated PC12cells are divided into3groups as (1) light chain group (L, control group) cultured with light solution (high glucose DMEM+10%FBS),(2) middle chain group (METH+L-257, M) cultured with middle solution (high glucose DMEM lack of L-arginine and L-lysine+10%FBS with2H4-L-arginine and13C6-L-lysine) to ensure4-6Da shift,(3) heavy chain group (METH, H) cultured with heavy solution (high glucose DMEM lack of L-arginine and L-lysine+10% FBS with13C6-15N2-arginine and13C6-15N4-L-lysine) to ensure8-10Da shift.
All the three group of PC12cells are planted into6-wells palates and after at least6times of transfers are planted into10cm2dishes. When the cells are in exponential growth phase with80%confluence, culture solution is discarded and treatments as follows are performed. The light chain group is added with high glucose DMEM. The middle chain group is added with middle chain solution containing2.0mmol/L METH and50μmol/L L-257. The Heavy chain group is added with heavy chain solution containing2.0mmol/L METH. After24h of couture, total proteins are abstracted form the three groups respectively. Then all the total proteins are mixed together for co-immunoprecipitation (IP) abstraction. By using IP with anti3-NT antibody the total nitrated proteins from the three groups are abstracted into one EP tube. The nitrated proteins are separated by SDS electrophoresis and the protein blots on the gel are cut off and digested by protease into peptides. At last, the collected peptides are analyzed by LC-MS/MS qualitatively and quantitatively.
3. Inhibitory effect of L-257on GSTP1protein nitration induced by METH
PC12cells are^divided into control group and experimental groups. The experimental groups are divided into4subgroups according to the concentration of L-257as0μmol/L group,10μmol/L group,50μmol/L group, and100μmol/L group. All the experimental groups are treated with2.0mmol/L METH. Western blot is used to analyze the expression of GSTP1protein in each group.
RESULTS
1.1Toxic effect of METH on the morphology of PC12cells and protective effect of L-257
Invert microscope revealed that in the METH associated with L-257groups the shape of PC12cells become round and atrophy. The cytoplasm displayed round and transparent regions. The nervous process became shorter, disrupted and disappeared. The net shape of cytoplasm disappeared gradually. The cell border became unclear because of atrophic processes around the surface. Abradant and floating cells are found in the solution. With the increase of L-257the poisonous injury to cell morphology became enhanced. In the50μmol/L and100μmol/L group, PC12cells mainly displayed round shape and transparent cytoplasm. In the25μmol/L group, PC12cells the above phenomenon became obvious and speculated processes were found. In the0μmol/L group and10μmol/L group, the above phenomenon is most obvious and the net-like structures were hardly to seen.
1.2The protective effect of L-257on the cell viability of PC12cells treated with METH
With the increase of L-257, MTT analysis revealed that OD value and cell viability of PC12cells elevated gradually with treatment of2.0mmol/L METH. Compared with the control group, significant difference was found (P<0.001, F=816.687).
1.3The protective effect of PC12cells against apoptosis
With the increase of L-257the apoptosis rate decreased gradually in PC12cells treated with2.0mmol/L METH. Compared with the control group, significant difference was found (P<0.001, F=119.436).
1.4The protective effect of L-257on NO concentration in the culture solution of PC12cells treated with METH
With the increase of L-257, the NO concentration in the culture solution of PC12cells treated with METH decreased gradually. Compared with the control group, significant difference was found in each experiment group expect the100μmol/L group (P<0.001, F=50.030).
1.5The protective effect of L-257on NOS activity of PC12cells treated with METH
With the increase of L-257, the NOS activity of PC12cells treated with METH decreased gradually. Compared with the control group, significant difference was found in each experiment group expect the100μmol/L group (P<0.008, F=29.043).
1.6Inhibitory effect of L-257against the expression of nitrated proteins in PC12cells treated with METH
Western blot revealed that compared with the control group the expression of nitrated proteins increased widely in15-175kDa. L-257has a significant inhibitory effect against protein nitration. With the increase of L-257the expressions of total nitrated proteins decreased gradually.
2.1Identification and comparison of nitrated proteins by SILAC
There were totally42proteins identified by SILAC, among which27nitrated proteins showed increased levels of nitration including5nitrated proteins with0-10%increased,12nitrated proteins with10%-20%increase,6nitrated proteins with20%-30%increase and1nitrated proteins with over30%increase. L-257revealed obvious inhibitory effect to those nitrated proteins at various levels. Among those identified proteins,15are ribosomal proteins with different elevated levels of nitration. The nitration levels of Heat shock cognate71kDa protein, Alpha actinin1and Glutathione S-transferase Pi (GSTP1) increased significantly.
2.2The expression of GSTP1protein in PC12cells treated with METH and L-257
Western blot revealed that compared with the control group the expression of GSTP1protein decreased significantly. L-257has a dramatic protective effect on the reduction of GSTP1protein. Under the inhibitory effect of L-257, the expression of GSTP1increased gradually.
CONCLUSION
1. METH induces neurotoxicity to PC12cells and causes morphological changes. L-257has significant protective effect to METH induced neurotoxicity.
2. METH causes elevated levels of NOS activity and NO concentration in PC12cells through the activation of DDAH1/ADMA/NOS pathway. METH induced oxidative stress leads to increased levels of protein nitration, which is responsible for the induced cell viability and apoptosis.
3. L-257prevents PC12cells from death and apoptosis and displays significant protective effect against METH induced neurotoxicity by inactivating the DDAH1/ADMA/NOS pathway and inhibiting the flowing protein nitration.
4. METH induced protein nitration is involved in oxidative stress, apoptosis, cytoskeleton damage and nerve jury and plays a key role in neurotoxicity.
甲基苯丙胺(Methamphetamine, METH)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,外观状如冰,俗称“冰毒”,属于一种新型毒品,也是目前危害最为严重和广泛的毒品之一。冰毒具有中枢神经兴奋性、致幻、食欲抑制和拟交感能效应等药理学、毒理学特性。过去曾作为抗疲劳剂、减肥药广泛使用,但随后发现其具有严重的精神依赖性,已被列为联合国精神药品公约管制的精神活性药物。由于METH具有见效快、兴奋作用持续时间长,价格低廉,化学合成技术简单,多途径摄取等特点,导致快速地滥用及蔓延。冰毒号称“毒品之王”,其最大的特点是致瘾性强,戒断困难。近年国际和国内大中城市的滥用情况十分严峻,吸毒不仅造成吸毒者机体和精神损害,而且造成艾滋病等多种疾病传播,严重影响人体身心健康。毒品相关的刑事犯罪和社会治安等问题,对社会的稳定造成极大危害。因此,METH的神经毒性损伤、成瘾机制的研究,是全世界面临的重大课题和研究热点。
METH具有多种药理、毒理学特性,尤其是具有很强的中枢兴奋作用和强烈的药物依赖性,长期滥用可以引起中枢神经系统神经化学、神经病理学和行为学的改变。对METH成瘾者而言,毒品影响的不仅是精神,还包括神经结构和功能的损伤性改变。近年来的研究显示,METH有较强的神经毒性,可导致大脑出现与阿尔茨海默病(Alzheimer's disease, AD)和帕金森病(Parkinson's disease, PD)等退行性神经病变类似的病理学改变。现阶段的研究结果显示,其可能的机制主要包括：氧化应激损伤作用、体温平衡失调、离子稳态破坏以及细胞凋亡相关通路的激活等。但是,现有的研究结果仍不足以充分阐释METH的神经毒性机制,对其毒性作用尚缺乏有效的防治手段。
在我们前一阶段的研究中,对METH处理大鼠的额叶皮质、纹状体、海马等部位的差异表达蛋白质进行鉴定和分析,发现氧化应激、能量代谢障碍、蛋白酶体功能失调以及凋亡等可能是参与METH神经毒性的主要机制。其中,一种新型的一氧化氮(nitric oxide, NO)生成调节酶二甲基精氨酸二甲基氨基水解酶1(Nω, Nω-dimethyl-L-arginine dimethylaminohydrolase, DDAH1)在纹状体和额叶皮质的高表达尤其令人瞩目。DDAH是近年发现的一种蛋白酶,在体内存在生物学活性相似的两种亚型DDAH1和DDAH2,其中DDAH1主要分布于中枢神经系统。DDAH主要参与不对称二甲基精氨酸(asymmetric dimethylated L-arginine, ADMA)的分解代谢,体内95%以上的ADMA均由DDAH分解代谢,调控正常生理情况下不同的组织器官内的ADMA浓度维持在相应的水平。研究发现,UDMA是含有甲基化精氨酸蛋白质的分解产物,是内源性的一氧化氮合酶(nitric oxide synthase, NOS)的抑制剂,对三种亚型的NOS均有抑制作用。蛋白质甲基化是生物体内普遍存在的一种蛋白质翻译后修饰现象,蛋白质的甲基化和甲基化蛋白质的降解,是机体内存在的正常生理过程。某些病理情况下,体内ADMA浓度下降可导致其对NOS的抑制作用减弱,引起组织内NO合成增加。可见,DDAH可以通过调节组织内ADMA的降解率,从而调节组织中NOS的活性和NO的合成。正常生理条下,中枢神经系统中的NO主要通过神经型NOS (nitric oxide synthase, nNOS)产生,作为神经递质发挥生理作用并可调节神经轴突的可塑性。而在氧化应激等病理条件下,产生的过量NO,则有可能对组织造成损害。研究证明,氧化应激可在损伤过程中产生大量的NO,NO和超氧阴离子的产物可造成脑组织DNA、蛋白质和脂质氧化损伤而诱导神经细胞损伤、功能丧失和死亡,在衰老和AD等多种神经退行性疾病的发病机制中起重要作用。
我们前一阶段的体内实验研究结果显示METH可引起大鼠脑组织多部位神经毒性损伤,多巴胺及代谢产物的耗竭,纹状体NOS活性升高,NO含量提高,小胶质细胞激活等。运用蛋白质组学的方法,对METH注射后大鼠纹状体、皮质、海马等部位部位的差异表达蛋白质进行鉴定和分析,寻找和鉴定出30个差异表达蛋白点,3个NO相关蛋白即硝化蛋白。同时,我们采用PC12细胞的体外急性METH中毒模型的研究表明,氧化应激、能量代谢障碍、蛋白酶体功能失调、凋亡及重要结构蛋白的硝化等可能是METH神经毒性的主要机制。
蛋白质硝基化作用是一种特殊的蛋白质翻译后修饰作用,属于氧化应激反应的通路之一。在大量NO介导下,组织内产生大量的活性氮类物质(reactive nitrogen species, RNS)可对相关蛋白质的3位酪氨酸产生硝基化作用,生成3-硝基酪氨酸(3-NT),从而导致蛋白功能的失活。研究表明,许多典型的神经退行性疾病中可发生大量的蛋白质硝基化水平升高现象。因此我们首次提出,DDAH1/ADMA/NOS通路介导的蛋白质硝基化作用可能是METH神经毒性机制的重要组成部分。
为了更深入的研究蛋白质硝基化作用与METH的神经毒性机制之间的关系,本次研究采用多巴胺能神经元体外模型PC12细胞系进行体外实验。PC12细胞在神经生长因子等诱导后,形态学及功能方面向交感神经元分化。由于PC12细胞的神经元分化特性,目前已做为一个广泛使用的神经元体外模型,用于神经生物、中枢神经系统疾病、神经毒素等方面的研究,也是一个可行的METH神经蛋白组学研究模型。因此,我们运用PC12细胞系进行METH体外损伤实验,探讨METH对PC12细胞的毒性损伤作用,并在此基础上运用分子生物学、蛋白质组学及质谱分析技术,对METH作用PC12后的硝基化蛋白质进行定性、定量分析,进一步探讨DDAH1/ADMA/NOS通路介导的蛋白质硝基化作用对靶蛋白的功能影响。同时,我们采用DDAH1的经典抑制剂L-257对该通路进行有效抑制,通过与阴性对照组进行比较,分析DDAH1/ADMA/NOS通路与蛋白质硝基化作用间的因果关系,并对硝基化蛋白进行验证。
目的：
建立单纯METH急性中毒和经L-257预处理的METH急性中毒PC12细胞模型,运用细胞生物学、蛋白质组学、稳定同位素标记氨基酸细胞培养技术(stable isotope labeling with amino acids in cell culture, SILAC)等方法,了解METH的神经毒性作用及L-257预处理对METH神经毒性的保护作用,检测PC12细胞内与METH毒性作用密切相关的硝基化因素NOS、NO含量的表达变化,对硝基化水平升高的蛋白进行定性、定量分析,探讨DDAH1/ADMA/NOS通路介导的蛋白质硝基化作用与METH神经毒性之间的关系。
方法：
1.METH对PC12细胞毒性损伤及L-257的保护作用
采用已分化的PC12细胞,用高糖DMEM培养基(含10%胎牛血清,1：100双抗)进行培养,将细胞置于37℃,5%C02的培养箱中。实验分为空白对照组(阴性对照)和实验组,实验组根据L-257浓度的不同而分为4组,L-257含量分别为0μmol/L(阳性对照)、10μmol/L、50μmol/L、100μmol/L,各组METH含量均为2.0mmol/L。细胞生长至对数生长期,达80%汇合后各实验组分别用上述不同浓度METH和L-257处理,对照组加入等体积的DMEM培养液,继续培养24h。倒置显微镜下观察PC12细胞形态改变,MTT法检测细胞存活率,nnexin V-FITC/PI双染法结合流式细胞术检测细胞凋亡率,NO检测试剂盒硝酸还原酶法检测细胞上清液NO含量,NOS检测试剂盒检测NO上游NOS活力,Western blot技术检测NO下游产物总3-硝基酪氨酸表达水平。
2.应用SILAC技术对PC12细胞内METH诱导的硝基化蛋白的鉴定
采用同位素对PC12细胞进行标记,以使各实验组内总蛋白分子量分别增多4-6和8-10,通过不同分子量对各组间的蛋白质进行区分。已分化的PC12细胞分为3组：(1)轻链组(对照组,L),采用轻链培养液(高糖DMEM培养基+10%胎牛血清)进行培养；(2)中链组(METH+L-257, M),采用中链培养液(缺乏L-精氨酸-L赖氨酸的高糖DMEM培养基+10%的富含2H4-L-赖氨酸和13C6-L-精氨酸的胎牛血清)进行培养,分子量偏移增加4至6；(3)重链组(METH, H),采用重链培养液(缺乏L-赖氨酸-L-赖氨酸的高糖DMEM培养基+10%的富含13C6-’N2-L-赖氨酸和13C6-15N4-L-精氨酸的胎牛血清)进行培养,分子量偏移增加8至10。
将3组PC12细胞接种于6孔板中,给予对应的同位素培养液进行培养,稳定传代6代以上,再次接种于10cm2培养皿中,每组2个培养皿。当细胞生长至对数生长期,达80%汇合时弃去上层培养液,并进行以下处理：轻链组加入高糖DMEM培养基；中链组加入含2.0mmol/L METH和50μmol/L抑制剂L-257的中链DMEM;重链组加入含2.0mmol/L METH的重链DMEM。以上三组细胞继续培养24小时,分别提取各组总蛋白。然后将3组蛋白混合,采用免疫共沉淀法提取总硝基化蛋白。SDS电泳分离总硝基化蛋白,对各个条带进行切胶、酶切和肽段抽提。采用LC-MS/MS质谱对肽段进行扫描,对各组内的硝基化蛋白进行定性、定量分析。
3. METH作用下L-257对GSTP1蛋白表达的影响
实验分为空白对照组(阴性对照)和实验组,实验组根据L-257浓度的不同而分为4组,L-257含量分别为Oμmol/L (阳性对照)、10μml/L、50μmol/L、100μmo/L各实验组均采用2.0mmol/LMETH处理。采用Western-blot技术对各组GSTP1蛋白表达进行定量分析。
结果：
1.1METH对PC12细胞形态学影响及L-257的保护作用
METH+L-257处理组PC12细胞胞体逐渐萎缩变圆,变圆细胞的胞质透亮度增加,胞浆内可见环形透亮区,细胞突起变短、断裂、消失,细胞间神经网络结构逐渐消失,因细胞突起萎缩可在细胞表面形成毛刺状,细胞边界不清晰,并可见细胞脱壁漂浮现象。随着L-257浓度降低,细胞形态损伤呈增强趋势,100μmol/L及50μmol/L处理组主要以胞体变圆及胞浆透亮增加为主,而降低至25μmol/L时突起的改变开始变明显,并有毛刺状结构形成,此现象在10μmol/L及Oμmol/L组尤为明显,细胞突起及网络结构基本消失。
1.2METH作用下L-257对PC12细胞存活率的保护作用
结果显示,随着L-257浓度逐渐增加,MTT法测得经METH(2.0mmol/L)处理的PC12细胞OD值逐渐升高,细胞存活率逐渐升高,各实验组与对照组相比有显著性差异(P<0.001,F=816.687)。
1.3METH作用下L-257对PC12细胞凋亡的保护作用
结果显示,随着L-257浓度的不断升高,经METH (2.0mmol/L)处理的PC12细胞凋亡率逐渐下降,各实验组与对照组相比有显著性差异(P<0.001,F=119.436)。
1.4METH作用下L-257对PC12细胞培养液NO浓度的影响
结果显示,随着L-257浓度逐渐增加,经METH (2.0mmol/L)处理的PC12细胞培养上清液中NO含量逐渐减少。各实验组与对照组相比,除100剂量组与对照组比较无显著性差异(P=0.170),其余各组均有显著性差异(P<0.001,F=50.030)。
1.5METH作用下L-257对PC12细胞NOS活性的影响
结果显示随着L-257浓度逐渐增加,经METH(2.0mmol/L)处理的PC12细胞总NOS活力逐渐减少。各实验组与对照组相比,除100剂量组与对照组比较无显著性差异(P=0.246),其余实验组与对照组相比均有显著性差异(P<0.008,F=29.043)。
1.6METH作用下L-257对PC12细胞总硝基化蛋白表达的影响
结果显示,阴性对照组与阳性对照组相比总硝基化蛋白表达在15-170KD范围内普遍升高。L-257对硝基化作用可产生明显的抑制效果：随着L-257浓度的升高,硝基化蛋白的总体表达水平逐渐降低。
2.1SILAC方法对硝基化蛋白的鉴定和比较
SILAC方法共鉴定出蛋白42个,其中硝基化水平升高的共27个：升高0-10%的为6个,升高10%-20%的为12个,升高20%-30%的为6个,30%以上的为1个,L-257对蛋白质的硝基化作用具有不同程度的抑制效果。其中共有15种核糖体蛋白硝基化水平不同程度地升高,71kDa热休克同源蛋白、辅肌动蛋白α1和谷胱甘肽S转移酶P1的硝基化水平明显升高。
2.2METH作用下GSTP1蛋白表达变化
结果显示,阴性对照组与阳性对照组(单纯METH)相比,GSPT1蛋白表达显著降低。L-257对GSPT1蛋白的下降趋势可起到明显的抑制作用：随着L-257浓度的升高,GSTP1蛋白的表达逐渐上升。
结论：
1. METH对PC12细胞具有神经毒性作用,能导致细胞形态学改变,L-257对METH的神经毒性具有明显的保护作用。
2. METH可通过DDAH1/ADMA/NOS通路使PC12细胞NOS活力和NO含量增加,通过氧化应激引起靶蛋白硝基化水平升高,造成PC12细胞活力下降,诱导细胞凋亡。
3.L-257可通过抑制DDAH1/ADMA/NOS通路而有效抑制靶蛋白的硝基化作用,从而降低PC12细胞的凋亡率和死亡率,发挥显著的神经毒性保护作用。
4. METH诱导的蛋白质硝基化作用参与了氧化应激、凋亡和细胞骨架损伤等神经损伤过程,可能是METH神经毒性的重要机制。
BACKGROUND
Methamphetamine (METH), also known as "ice" for its ice-like and crystalline appearance, belongs to amphetamine-type stimulants (ATS). METH is one of the most popular drugs which have brought about great hazard to the society. METH is characterized by the toxicological and pharmacological features of central excitability, hallucinations, inhibited appetite and sympatheticomimetic effects. It was first used as antifatigue and anti-obesity agents. But soon it was put onto the list of controlled psychoactive drugs by the United Nations Convention on Psychotropic Drugs because of its psychological dependence. In recent years, the use of METH spreads quickly and wildly because it is easy to synthesis with little cost. METH is called "the king of drugs" because it is impossible to get rid of METH addiction once someone has used it. Nowadays the problem of METH addiction has been getting serious in big cities all around the world. METH not only results in damages to the body and the spirit, but also gives rise to many diseases such as AIDS. The METH related crimes also impair the order and the stability of the entire society. Thus, the mechanism study of METH induced neurotoxicity and addiction has become one of the most important topics for researchers.
METH has many pharmacological and toxicological features among which the significant central excitability is the most important one. Long-term abuse of METH can lead to pathological, praxiological and neurochemistry changes to the central nerve system. For those who abuse METH, it is not only the spirit but also the structures and the functions of the CNS that have been affected. Previous studies show that METH induced neurotoxicity can cause disease recessive neuropathies such as Alzheimer's (AD) and Parkinson's disease (PD) in the brain. In recent years it has been revealed that the possible mechanism of METH induced neurotoxicity could be consist of oxidative stress, disturbance of energy metabolism, disorder of proteasomes and apoptosis. It has been reported that the up-regulation of Nω, Nω-dimethyl-L-arginine dimethylaminohydrolase (DDAH1), a newfound regulatory enzyme of NO, in corpus striatum and cortex of frontal lobe has caused the attentions from researchers all over the world. DDAH is a protease which has two isoforms with similar biologic activity in vivo, DDAH1and DDAH2. DDAH1is mainly seen in the CNS. DDAH is chiefly in charge of the metabolism of asymmetric dimethylated L-arginine (ADMA). About95%ADMA are catabolized by DDAH in order to maintain its balance in vivo. ADMA is the decomposed products of methylated Arginine which is common Protein Post-translational Modification. Protein methylation and decomposition of methylated proteins are normal physiological processes in human and animals. ADMA is the inhibitor of endogenous nitric oxide synthase (NOS) and has dramatic inhibitory effect to all the three isoforms. In certain pathological conditions, with the decrease of ADMA and its inhibitory effect the production of NO increases significantly. While in normal conditions, the production of NO, which serves as neurotransmitters performing physiological effects and modulating the plasticity of neuritis, is mainly induced by the neurologic nitric oxide synthase (nNOS) in the CNS. It has been indicated that the production of NO induced by oxidative stress plays a key role in process of aging and some degenerative diseases such as AD and PD and that NO and superoxide anion products could induce apoptosis, death and dysfunctions of the nerve cells because of the oxidative damages to DNA, protein and lipid in brain tissues.
Our previous study revealed that METH can cause neurotoxicity, dopamine exhaustion, increases of NO and NOS and microglia activation in different brain regions of rat. By using proteomic methods,30differentially expressed proteins and3NO induced nitrated proteins were identified. In addition, PC12cell model of acute METH poisoning displayed that oxidative stress, disturbance of energy metabolism, dysfunction of proteasomes, apoptosis and nitrification of key proteins are involved in the METH induced neurotoxicity.
Protein nitration is a special post-translational modification which belongs to oxidative stress reactions. With the presence of over produced NO, a huge amount of reactive nitrogen species (RNS) are induced in vivo, which could result in deactivation of key proteins by elevated levels of protein nitration. It was reported that, in many typical recessive diseases such as AD and PD, an up-regulated levels of nitrated proteins were found. Thus, we would like to propose that protein nitration which is mediated by DDAH1/ADMA/NOS pathway plays a critical role in the METH-induced neurotoxicity.
In order to further understand the relationship between protein nitration and METH induced neurotoxicity, in vitro PC12cell model of acute METH poisoning is used in this research. When induced by nerve growth factor (NGF), PC12cells can differentiate toward sympathetic neurons both morphologically and functionally. PC12cells are widely used as an in vitro neurons model for researches of neurobiology, central nervous system diseases and neurotoxin. It is also a useful proteomic model for METH induced neurotoxicity. Therefore in this research PC12cells model of METH poisoning together with proteomic and molecular biology methods are used to investigate the relationship between protein nitration and METH induced neurotoxicity. The effect of nitration on target proteins, which is mediated by DDAH1/ADMA/NOS pathway, is analyzed qualitatively and quantitatively. On the other hand, L-257, the novel inhibitor of DDAH1, is used to confirm the causation of the pathway and protein nitration and verify the nitrated proteins identified by mass spectrum (MS) by repressing the pathway directly.
OBJECTIVES
In this research cytobiological methods, proteomic methods and stable isotope labeling with amino acids in cell culture (SILAC) was used to:(1) establish PC12 cells models of acute METH poisoning with or without L-257treatment,(2) investigate the METH induced neurotoxicity and the protective effect of L-257,(3) determine the nitration related factors which could cause neurotoxicity including NOS and NO levels,(4) quantitatively and qualitatively analyze the target proteins with elevated nitration levels,(5) discuss the relationship between protein nitration mediated by DDAH1/ADMA/NOS pathway and METH induced neurotoxicity.
METHODS
1. Protective effect of L-257on PC12cells treated with METH
Differentiated PC12cells are cultured in high glucose DMEM containing10%FBS and double antibiotics in37℃incubator with5%CO2. PC12cells are divided into control group and experimental groups. The experimental groups are divided into subgroups according to the L-257amount added as0μmol/L,10μmol/L,50μmol/L and100μmol/L. The amount of METH added into every group is2.0mmol/L. When the cells are in logarithmic phase and grow to80%density, the regents are added into each group as described above. The same amount of DMEM is added into the control group. All the groups are cultured for24h. The morphological changes of PC12cells are observed by invert microscope. The cell viability is tested by MTT method. The apoptosis rate is determined by Annexin V-FITC/PI stain associated with flow cytometer. NO level in supernatant is determined by nitrate reductase method. NOS activity is determined by NOS kit. The expressions of total nitrated proteins in the downstream of NO are determined by Western blot.
2. Using SILAC to indentify nitrated proteins in PC12cells treated with METH
In order to recognize different cell groups in the mass spectra, the total proteins in each group is labeled by isotopes which ensure4-6Da or6-8Da shift in molecular weight. Differentiated PC12cells are divided into3groups as (1) light chain group (L, control group) cultured with light solution (high glucose DMEM+10%FBS),(2) middle chain group (METH+L-257, M) cultured with middle solution (high glucose DMEM lack of L-arginine and L-lysine+10%FBS with2H4-L-arginine and13C6-L-lysine) to ensure4-6Da shift,(3) heavy chain group (METH, H) cultured with heavy solution (high glucose DMEM lack of L-arginine and L-lysine+10% FBS with13C6-15N2-arginine and13C6-15N4-L-lysine) to ensure8-10Da shift.
All the three group of PC12cells are planted into6-wells palates and after at least6times of transfers are planted into10cm2dishes. When the cells are in exponential growth phase with80%confluence, culture solution is discarded and treatments as follows are performed. The light chain group is added with high glucose DMEM. The middle chain group is added with middle chain solution containing2.0mmol/L METH and50μmol/L L-257. The Heavy chain group is added with heavy chain solution containing2.0mmol/L METH. After24h of couture, total proteins are abstracted form the three groups respectively. Then all the total proteins are mixed together for co-immunoprecipitation (IP) abstraction. By using IP with anti3-NT antibody the total nitrated proteins from the three groups are abstracted into one EP tube. The nitrated proteins are separated by SDS electrophoresis and the protein blots on the gel are cut off and digested by protease into peptides. At last, the collected peptides are analyzed by LC-MS/MS qualitatively and quantitatively.
3. Inhibitory effect of L-257on GSTP1protein nitration induced by METH
PC12cells are^divided into control group and experimental groups. The experimental groups are divided into4subgroups according to the concentration of L-257as0μmol/L group,10μmol/L group,50μmol/L group, and100μmol/L group. All the experimental groups are treated with2.0mmol/L METH. Western blot is used to analyze the expression of GSTP1protein in each group.
RESULTS
1.1Toxic effect of METH on the morphology of PC12cells and protective effect of L-257
Invert microscope revealed that in the METH associated with L-257groups the shape of PC12cells become round and atrophy. The cytoplasm displayed round and transparent regions. The nervous process became shorter, disrupted and disappeared. The net shape of cytoplasm disappeared gradually. The cell border became unclear because of atrophic processes around the surface. Abradant and floating cells are found in the solution. With the increase of L-257the poisonous injury to cell morphology became enhanced. In the50μmol/L and100μmol/L group, PC12cells mainly displayed round shape and transparent cytoplasm. In the25μmol/L group, PC12cells the above phenomenon became obvious and speculated processes were found. In the0μmol/L group and10μmol/L group, the above phenomenon is most obvious and the net-like structures were hardly to seen.
1.2The protective effect of L-257on the cell viability of PC12cells treated with METH
With the increase of L-257, MTT analysis revealed that OD value and cell viability of PC12cells elevated gradually with treatment of2.0mmol/L METH. Compared with the control group, significant difference was found (P<0.001, F=816.687).
1.3The protective effect of PC12cells against apoptosis
With the increase of L-257the apoptosis rate decreased gradually in PC12cells treated with2.0mmol/L METH. Compared with the control group, significant difference was found (P<0.001, F=119.436).
1.4The protective effect of L-257on NO concentration in the culture solution of PC12cells treated with METH
With the increase of L-257, the NO concentration in the culture solution of PC12cells treated with METH decreased gradually. Compared with the control group, significant difference was found in each experiment group expect the100μmol/L group (P<0.001, F=50.030).
1.5The protective effect of L-257on NOS activity of PC12cells treated with METH
With the increase of L-257, the NOS activity of PC12cells treated with METH decreased gradually. Compared with the control group, significant difference was found in each experiment group expect the100μmol/L group (P<0.008, F=29.043).
1.6Inhibitory effect of L-257against the expression of nitrated proteins in PC12cells treated with METH
Western blot revealed that compared with the control group the expression of nitrated proteins increased widely in15-175kDa. L-257has a significant inhibitory effect against protein nitration. With the increase of L-257the expressions of total nitrated proteins decreased gradually.
2.1Identification and comparison of nitrated proteins by SILAC
There were totally42proteins identified by SILAC, among which27nitrated proteins showed increased levels of nitration including5nitrated proteins with0-10%increased,12nitrated proteins with10%-20%increase,6nitrated proteins with20%-30%increase and1nitrated proteins with over30%increase. L-257revealed obvious inhibitory effect to those nitrated proteins at various levels. Among those identified proteins,15are ribosomal proteins with different elevated levels of nitration. The nitration levels of Heat shock cognate71kDa protein, Alpha actinin1and Glutathione S-transferase Pi (GSTP1) increased significantly.
2.2The expression of GSTP1protein in PC12cells treated with METH and L-257
Western blot revealed that compared with the control group the expression of GSTP1protein decreased significantly. L-257has a dramatic protective effect on the reduction of GSTP1protein. Under the inhibitory effect of L-257, the expression of GSTP1increased gradually.
CONCLUSION
1. METH induces neurotoxicity to PC12cells and causes morphological changes. L-257has significant protective effect to METH induced neurotoxicity.
2. METH causes elevated levels of NOS activity and NO concentration in PC12cells through the activation of DDAH1/ADMA/NOS pathway. METH induced oxidative stress leads to increased levels of protein nitration, which is responsible for the induced cell viability and apoptosis.
3. L-257prevents PC12cells from death and apoptosis and displays significant protective effect against METH induced neurotoxicity by inactivating the DDAH1/ADMA/NOS pathway and inhibiting the flowing protein nitration.
4. METH induced protein nitration is involved in oxidative stress, apoptosis, cytoskeleton damage and nerve jury and plays a key role in neurotoxicity.
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