NOS抑制剂对METH神经毒性的保护作用及GSTP1过表达载体的构建
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摘要
研究背景：
近年来,新型毒品种类不断增多,为毒品的防治提出新的课题和挑战。甲基苯丙胺(Methamphetamine, METH or MA)俗称“冰毒”,属于苯丙胺类兴奋剂,是最具代表性的和最常见新型毒品。因其具有见效快、兴奋作用维持时间长,价格低廉,化学合成技术简单,多途径摄取等特点,使它滥用及蔓延速度极快,成为当今我国危害最为严重和滥用的两大毒品之一。
法医在接触因吸食冰毒死亡或与吸毒有关的其它暴力死亡的尸检和在戒毒所治疗的病人过程中,发现其死亡与心脑重要器官及其血管的代谢损伤相关。有大量的资料表明,冰毒对大脑神经细胞产生直接的损害作用,引起中枢神经系统神经化学、神经病理改变,导致神经细胞变性、坏死和异常的膜性结构改变,出现急性和慢性精神障碍和行为改变；可以导致心肌细胞肥大、萎缩、变性、收缩带坏死、小血管内皮细胞损伤和小血管痉挛,从而导致急性心肌缺血、心肌病和心律失常,成为吸毒者突然死亡的原因。因此,对冰毒吸食者毒性损伤机制乃至成瘾机制及猝死机制的研究和探索成为国内外法医关注的问题,也是本实验室近十年来的重点研究方向。
目前,关于METH的神经毒性机制国内外研究结果尚未完全明确,现阶段的研究结果显示多种机制参与了METH的神经毒性,主要包括氧化应激、神经元凋亡、兴奋性毒性、线粒体功能障碍等,其中氧化应激是METH所致神经毒性损伤的重要机制作用之一。
本实验室运用蛋白质组学的方法,对METH注射后大鼠纹状体、额叶皮质、海马等部位的差异表达蛋白质进行鉴定和分析,发现了一种新型的NO合成调节酶二甲基精氨酸二甲基氨基水解酶1(DDAH1)表达上调,提出了DDAH/ADMA/NOS系统可能是NO过表达引起的中枢神经系统损伤的重要途径。本课题组前期研究应用稳定同位素标记氨基酸细胞培养技术(SILAC),对METH作用后PC12细胞进行分析,发现谷胱甘肽S-转移酶蛋白(GSTP1)硝基化增强,提出了GSTP1-P35/P25-CdK5可能是氧化应激损伤的重要调节途径。
本研究拟建立METH中毒细胞模型和动物模型,通过形态学、NOS含量、DA含量及凋亡等指标的检测,进一步验证METH引起的中枢性毒害作用,并通过给予NOS抑制剂(L-NAME和7-NI)反证氧化应激过程中NOS在METH中毒模型中的作用及其与METH神经毒性的关系,进一步完善DDAH1/ADMA/NOS通路的神经毒性作用机制,并为研究GSTP1-P35/P25-CDK5这一可能的NO氧化应激下游通路的提供依据。
本研究拟利用体外研究手段,通过腺病毒载体在PC12细胞上建立GSTP1过表达模型,通过调控GSTP1表达,研究其基因水平的改变对METH氧化应激过程中上述通路的调控变化及作用,探索其是否是介导氧化应激致细胞骨架结构损伤和细胞凋亡死亡通路的中间环节。本研究预期结果不仅为阐明METH的毒性损伤作用机制提供基础,也为METH中毒的防治、METH戒毒药物靶点的选择乃至为毒品戒断治疗奠定基础。
本研究还将应用mRNA表达谱芯片技术,检测METH作用PC12细胞引起的全基因mRNA的变化,以期筛选出差异表达mRNA,以明确差异表达mRNA和METH神经毒性之间的关系,为今后的METH神经毒性机制研究提供新的研究靶点。
目的：
建立METH中毒细胞模型和动物模型,并给予NOS抑制剂,通过形态学、NOS含量、DA含量及凋亡等指标的检测,验证氧化应激过程中NOS的作用及其与METH神经毒性的关系；通过腺病毒载体在PC12细胞上建立GSTP1过表达模型,进而研究GSTP1在METH氧化应激过程中的作用；应用mRNA表达谱芯片技术,明确差异表达mRNA和METH神经毒性之间的关系,为METH神经毒性机制研究提供新的研究靶点。
方法：
1. METH中毒模型的建立及NO抑制剂的保护作用
1.1METH中毒动物模型的建立及7-NI的保护作用
SD大鼠雄性24只,随机分为四组,每组6只：生理盐水组、METH组、METH+7-NI组、7-NI组。动物单笼饲养,自由饮水、取食。实验前先在饲养室适应3天。第一组(生理盐水组)：腹腔注射生理盐水,早8：30、晚5：00各一次,连续注射3天；第二组(METH组)：腹腔注射METH,早8：30、晚5：00各一次,连续注射3天；第三组(METH+7-NI组)：注射方法同第二组,提前30min在腹腔注射7-NI；第四组(7-NI组)：注射方法同第一组,提前30min腹腔注射7-NI,腹腔注射生理盐水。制成动物模型后,观察行为学变化情况,对大鼠的刻板行为进行评分。应用western blot检测大鼠纹状体nNOS表达、ELISA法检测纹状体多巴胺含量、应用Tunel荧光法检测细胞凋亡。
1.2METH中毒细胞模型的建立及L-NAME的保护作用
已分化的PC12细胞培养于含10%FBS、双抗(1：100)的高糖DMEM培养基中,37℃、5%CO2培养箱中培养。PC12细胞培养至对数生长期,达约80%汇合时进行实验。实验分为空白对照组(阴性对照)和实验组,实验组根据L-NAME(N-硝基-L-精氨酸甲酯)浓度的不同而分为5组,各组METH含量均为2.0mmol/L, L-NAME含量分别为0μmol/L(阳性对照)、1μmol/L、10μmol/L、50μmol/L、100μmol/L。当细胞生长至80%汇合时,倒去原培养液,实验组加入含上述浓度METH和L-NAME的无血清培养基,对照组换成等体积不含血清和METH的培养基,细胞继续培养24h后。倒置显微镜下观察PC12细胞形态改变,PE Annexin试剂盒、流式细胞术检测细胞凋亡率,NOS检测试剂盒检测细胞总NOS活力,Western blot技术检测GSTP1表达变化。
2.GSTP1过表达载体的构建
首先通过PCR扩增大鼠全长GSTP1cDNA序列,然后将PCR产物与pshuttle-IRES-hrGFP-1体外重组并转化大肠杆菌,测序验证。然后又将穿梭质粒pshuttle-GSTP1与腺病毒骨架质粒pAdEasy-1重组。通过Lipo2000介导将重组体腺病毒在AD293细胞中的包装扩增。最后通过qPCR和western blotting验证转染效率。
3.PC12细胞METH毒性损伤mRNA表达谱的差异分析及验证
按照前述方法进行培养PC12细胞并进行药物处理,实验分为空白对照组(阴性对照)和METH组,METH+L-NAME组。METH浓度为2.0mmol/L,L-NAME浓度100μmol/L当细胞生长至80%汇合时,倒去原培养液,实验组加入含上述浓度METH和L-NAME的无血清培养基,对照组换成等体积不含血清和METH的培养基,细胞继续培养24h后。将待检样品抽提RNA后进行mRNA表达谱芯片检测。
结果：
1. METH中毒模型的建立及NO抑制剂的保护作用
1.1动物模型的建立及7-NI的保护作用
本实验METH组和7Ni+METH组的动物刻板行为评分均显著高于7Ni和Control组(P均<0.001),METH、7Ni+METH组间无显著性差别(P=0.769),7Ni、Control组间无显著性差别(P=0.190)。Western blot结果显示METH组大鼠纹状体中nNOS蛋白表达水平明显升高,而METH+7-NI组nNOS表达水平较METH组明显降低。ELISA结果显示METH组DA显著降低(P<0.001),而METH+L-NAME组、L-NAME组与对照组比较,DA无显著差异(P>0.01)。采用Tunel法检测各组大鼠脑组织纹状体内神经细胞凋亡情况,METH组与对照组相比凋亡细胞数显著升高(P<0.001),而7-NI对凋亡的增高表现出明显的保护作用(与METH组相比P<0.001)。
1.2细胞模型的建立及L-NAME的保护作用
结果发现与正常对照组细胞相比,METH处理的细胞萎缩变圆,变圆细胞的胞质透亮度增加,可见环形透亮区,突起变短、断裂、消失,神经网络结构消失,因细胞突起萎缩可在细胞表面形成毛刺状,边界不清晰。加入L-NAME处理后,细胞形态学改变随浓度升高损伤呈减弱趋势,中、高浓度处理主要主要以胞体变圆及胞浆透亮增加为主,突起无明显改变。METH处理后细胞凋亡率显著增加,加入L-NAME后细胞总凋亡率随着浓度增加逐渐减少,各实验组与对照组相比具有显著性差异(P<0.001),尤其是中、高浓度L-NAME具有显著的抑制细胞凋亡作用。METH作用后PC12细胞内总NOS活性增加,加入L-NAME后NOS活力逐渐下降,各实验组与对照组相比有显著性差异(P<0.02)。同时采用western-blot技术对细胞内GSTP1表达变化进行检测,发现单独METH处理PC12细胞GSTP1水平显著下降,加入L-NAME后对GSPT1蛋白的下降趋势可起到明显的抑制作用：随L-NAME浓度的升高,GSTP1蛋白的表达逐渐上升,并出现接近正常水平的趋势。
2.GSTP1过表达载体的构建
本实验通过脂糖凝胶电泳跑胶,证明了携带GSTP1序列穿梭载体构建成功,在Ad-293细胞中观察到绿色荧光的表达证明病毒在细胞内进行包装,绿色荧光在PC12细胞中大面积的出现,显示pAdEasy-1重组腺病毒在PC12细胞中成功表达。qPCR以及western blotting的结果显示腺病毒骨架质粒pAdEasy-1重组的转染效率高。
3. METH作用下大鼠脑组织mRNA表达谱的差异分析
本实验通过9张芯片比较正常对照组与METH组、METH+L-NAME组三组间的差异表达mRNA,结果表明METH可导致184个基因表达上调,518个基因表达下调；而NOS抑制剂L-NAME可对其中的470下调基因和2个上调基因发生有效的保护作用,约占全部差异基因的67.24%。METH组与正常对照组之间差异表达的基因中,我们发现与凋亡相关的基因有16个。
结论：
1.在大鼠动物模型上,METH明显增加大鼠不自主的刻板运动,而应用7-NI预处理后,对刻板行为也无明显改善。METH可导致nNOS蛋白表达水平增高、DA含量的降低及细胞凋亡增多等多种神经毒性表现,nNOS特异抑制剂7-NI能部分减轻其神经毒性作用。这些结果为下一阶段应用此动物模型进一步研究NOS下游通路的作用机制奠定了良好的基础。
2.L-NAME对METH导致的神经毒性损伤作用具有保护作用,能减弱细胞形态学损伤、抑制凋亡、增加GSTP1表达水平,通过阻断氧化应激的上游信号NOS活性发挥抗氧化、抗凋亡作用。中、高浓度L-NAME可用于METH神经毒性损伤保护机制的研究。
3.证明了携带GSTP1序列穿梭载体构建成功,在Ad-293细胞中观察到绿色荧光的表达,证明病毒在细胞进行包装,转染PC12细胞中发现绿色荧光,证明转染成功。经过qPCR以及western blotting的结果显示腺病毒骨架质粒pAdEasy-1重组的转染效率高。以上证明了GSTP1过表达载体的构建成功。为下一步研究GSTP1下游调控基因提供可靠的细胞模型。
4. mRNA表达谱经过GO分析,差异表达的基因主要参与的生物过程有核糖体合成、炎症反应、抗凋亡的正向调节、过氧化氢代谢、线粒体电子传递泛醌-细胞色素C、氧化应激反应、线粒体钙离子转运、多巴胺能突触传递负向调节、过氧化氢反应、凋亡的正向调节、线粒体电子传递细胞色素C氧化、通过细胞色素C的caspase的活化、高热反应等。Pathway分析显示,METH组与正常对照组相比,差异基因主要参与了Alzheimer's disease、Parkinson's disease、糖酵解／糖异生、核糖体、氧化磷酸化、P53信号通路、凋亡、自然杀手细胞介导的细胞毒性、肌动蛋白细胞骨架的调节、钙信号通路等信号通路。
Background
In recent years, new types of drugs is increasing, new issues and challenges for the prevention and treatment of drug. Methamphetamine (Methamphetamine, METH or MA) are amphetamine-type stimulants, commonly known as "ice", is the most representative and the most common new drugs. Because of its quick, excitement role in METHintaining a long time, inexpensive, simple chemical synthesis techniques, multi-channel intake characteristics, METHking it the abuse and the spread of fast become one of the China today against the most serious and the abuse of the two drugs.
Forensic contact related death due to methamphetamine or other drug-related violent death autopsy and patient drug rehabilitation treatment process, metabolic injury death and the vital organs of the heart and brain and its blood vessels. There are a large number of data indicate that methamphetamine produce direct damage to brain cells, causing the central nervous system neurochemical and neuropathological changes, leading to nerve cell degeneration, necrosis and abnormal membranous structure change, acute and chronic mental disorders and behavior change; myocardial cell hypertrophy, atrophy, degeneration, contraction band necrosis, small vascular endothelial cell damage and small blood vessels spasm, leading to acute myocardial ischemia, cardiomyopathy and arrhythmia, become addicts sudden death. Therefore, research and exploration of methamphetamine abusers toxicity mechanisms of injury and even addiction mechanisms and sudden death mechanisms become the forensic attention at home and abroad, is also a key research direction over the past decade in this laboratory.
About METH neurotoxicity research results at home and abroad is not yet fully clear, at this stage of the research results show that a variety of mechanisms involved in METH neurotoxicity, including oxidative stress, neuronal apoptosis, excitotoxicity, mitochondrial dysfunctionetc, wherein the oxidative stress is one of the important mechanism for the role of METH-induced neurotoxic injury.
Use of proteomics methods in the laboratory, after injection of METH rat striatum, frontal cortex, hippocampus and other parts of the differences expressed proteins were identified and analysis, and found a new type of NO synthesis regulating enzyme dimethyl fineacid dimethyl-amino hydrolase1(DDAH1) of upregulation of dimethylarginine dimethylaminohydrolase in/ADMA/NOS system METHy be an important way of central nervous system damage caused by NO overexpression. Previous study by our group application of stable isotope labeled amino acids cell culture (SILAC), PC12cells after METH role analysis found that the enzyme protein of glutathione S-transferase (GSTP1) nitro enhancement of GSTP1-P35/the P25-CdK5important regulatory pathway for oxidative stress injury.
This study to establish METH poisoning cells and animal models, central toxic effects of METH caused by the detection of the morphology, NOS content, DA content and apoptosis index, further validation, and by giving the NOS inhibitor (L-NAME/7-NI) NOS METH poisoning model disprove oxidative stress and its relationship with METH neurotoxicity, further perfect DDAH1/ADMA/NOS path the neurotoxicity mechanism of action, and to study GSTP1-P35/P25-NO oxidation of CDK5this may provide the basis for stress downstream pathway.
This study means of in vitro studies, GSTP1overexpression METH intoxication cell lines, through the regulation of GSTP1expression of altered levels of gene regulation change and the role of the passage METH oxidative stress process, to explore whether it is mediatedoxidative stress caused the cytoskeletal structural damage and apoptosis death pathway intermediate links. The expected results of this study provide a basis not only to elucidate the mechanism of action of METH toxic injury METH poisoning prevention, METH drug selection of drug targets and even to lay the foundation for the treatment of drug withdrawal.
Objective
METH poisoning cells and animal models, and give the NOS inhibitor Verify the NOS's role in the process of oxidative stress and its relationship with METH neurotoxicity by morphology, NOS is DA content, and apoptosis index detection;establishment of GSTP1overexpression METH poisoning cell lines to study the role of GSTP1METH oxidative stress process; application mRNA expression microarray technology, a clear difference in the expression of the relationship between the mRNA and METH neurotoxicity, METH neurotoxicity mechanism of the newResearch targets.
Methods
1.METH poisoning model and the protective effect of NO inhibitor
1.1METH poisoning animal model and the protective effect of7-NI
Male SD rats were24(n=6), were randomly divided into four groups:normal saline, METH, METH+7-NI group,7-NI group. The animal single cages, free access to water, feeding. Experimental adapt before feeding room for three days. First group (saline group):intraperitoneal injection of saline,8:30, the second group (METH group):intraperitoneal injection of METH and00pm each injection of3consecutive days;8:30,5:00each time, three days of continuous injection; third group (METH+7-NI group):injection method with the first group, ahead of30min in the abdominal cavity injection of7-NI; fourth group (7-NI group):injection method with the first group,30min prior to intraperitoneal injection of7-NI, intraperitoneal injection of saline. Made of animal models, the application of nNOS expression in rat striatum detected by western blot, ELISA method detected striatal dopamine content, applications the Tunel assay in cell apoptosis.
1.2protective effect of L-NAME on PC12cells treated with methamphetamine
Differentiated PC12cells were cultured in high glucose DMEM containing10%FBS and double antibiotics in37℃incubator with5%CO2. PC12cells was divided into control group and experimental groups. The experimental groups were divided into subgroups according to the L-NAME amount added as0μmol/L,1μmol/L,10μmol/L,50μmol/L and100μmol/L. The amount of METH added into every group was2.0mmol/L. When the cells were in logarithmic phase and grow to80%density, the regents were added into each group as described above. The same amount of DMEM was added into the control group. All the groups were cultured for24h. The morphological changes of PC12cells were observed by invert microscope. The apoptosis rate was determined by Annexin V-PE stain associated with flow cytometer. NOS activity was determined by NOS kit. The expressions of GSTP1was determined by Western blot.
2.GSTP1over-expression vector construction
Rat full-length sequence of GSTP1cDNA is first amplified by PCR, the PCR product was then pshuttle-IRES-hrGFP-1in vitro recombinant and transformed into E. coli and sequenced. Then turn the shuttle plasmid pshuttle-GSTP1adenovirus backbone plasmid pAdEasy-1recombinant. The recombinant through Lipo2000mediated packaging of the adenovirus in the AD293cells amplified. Last verify the efficiency of transfection through Qpcr and western blotting.
3.Analysis and validation of PC12cells METH toxic injury mRNA expression profile differences
Cultured PC12cells and drug treatment in accordance with the aforementioned method, the experiment was divided into blank control group (negative control) and METH METH and+L-NAME group. The METH concentration of2.0mmol/L, L-NAME concentration100μmol/L, when the cells were grown to80%confluence, culture liquid decanted original experimental group joined serum-free medium containing the above concentrations of METH and L-NAME, the control group converter into an equal volume of medium without serum and METH cells were cultured for24h. The sample to be assayed RNA was extracted and mRNA expression microarray detection.
Results
1.METH poisoning model and the protective effect of NO inhibitor
1.1Establishment of animal models and the protective effect of7-NI
METH can cause the striatum of rat brain tissue nNOS expression and DA concentrations were significantly increased at the same time lead to neuronal cell extensive apoptosis; NOS inhibitor7-Ni effective inhibition of nNOS and DA increased, while the effective protection of the withereddeath of occurrence.
1.2protective effect of L-NAME on PC12cells treated with methamphetamine
Contrast to the control group,we found that the morphological changes of PC12. cells treated by METH2.0mmol/L were featured by shrinkage of the cell bodies, brighter cell cytoplasm, disruption of the dendrite and disappearance of cell reticular formation,as well as cell edges bristle-like formation,when treated with METH+L-NAME of1-100μmol/L, morphological injury change of PC12was attenuated following the increase of concentration The cell apoptosis rates of PC12cells treated by METH2.0mmol/L were increased,when added by L-NAME with the concen ra tion1-100μmol/L, the cell apoptosis rate began to decrease gradually, there were significant differences between the METH+L-NAMEgroup and the control group (P<0.001). The TNOS activation was significantly increased after treatment of2.0mmol/L METH, then was decreased in dose dependent manner after the cells treated with1-100μmol/L L-NAME,and was significantly different between the experiment group and the control group (P<0.02). At the same time,we applied west-blot technology to detect GSTP1expression level, and found that compared with control group,, the GSTP1level of PC12cells exposed to2.0mmol/L METH was decreased, but treated by METH+L-NAME GSTP1level was gradually increased, and get close to common level.
2.GSTP1over-expression vector construction
Run on a gel by agarose gel electrophoresis, the experimental proof of the shuttle vector was successfully constructed with GSTP1sequence, expression of green fluorescence observed in the Ad-293cells that virus packaging cells, a large area of green fluorescence in PC12cellsappear pAdEasy-1recombinant adenovirus successfully expressed in PC12cells. Of Qpcr and western blotting results show high transfection efficiency of adenovirus backbone plasmid pAdEasy-1recombinant.
3.The METH effect in rat brain tissue mRNA expression profile differences analysis
9chip compared to normal control group METH group, METH and L-NAME group differences among the three groups, the experimental expression mRNA, results showed that METH can lead to184genes were up-regulated and518genes were down-regulated; NOS inhibitor L-NAME470down the genes and two up-regulated genes effective protection, accounting for67.24%of all the differences in gene. Genes differentially expressed between the METH group and normal control group, we found that16of the apoptosis-related genes.
Conclusion
1. In the rat model, METH can lead to increased nNOS activity reduced DA content and an increase in apoptosis and other neurotoxic manifestations, nNOS intentionally inhibitor7-NI can partially mitigate its neurotoxic effects. These results for the next phase of the application of this animal model mechanism has laid a good foundation for further study of the role of NOS downstream pathways.
2. L-NAME can protect against METH-induced neurotoxicity including attenuating damage to cell morphology、depressing apoptosis、increasing level of GSTP1by obstructing NOS activation which is the upper stream passway of oxidative stress。Medium dose or high concentration L-NAME was suitable for further investigation of protection mechanism on METH-induced neurotoxicity.
3.The shuttle vector was constructed successfully proved that with a GSTP1sequence was observed in the Ad-293cells, the expression of green fluorescent prove the virus in the cells can be packaged in PC12cells transfected with green fluorescent prove successful transfection after Qpcr and western blotting resultsadenoviral backbone plasmid pAdEasy-1recombinant high transfection efficiency. Above proved GSTP1overexpression vector was constructed successfully. GSTP1downstream regulatory genes for further research can study cell model.
4.Ribosome biogenesis after GO analysis of differentially expressed genes involved in biological processes, inflammatory response, positive regulation of anti-apoptotic, hydrogen peroxide metabolism, mitochondrial electron transport ubiquinone-cytochrome C, oxidative stress, mitochondrialcalcium ion transport dopaminergic synaptic transmission in the negative regulation, the reaction of hydrogen peroxide, apoptosis positive regulation of cytochrome C oxidase, mitochondrial electron transport, cytochrome C, caspase activation, fever reaction. Pathway analysis showed that the the METH and group compared with the control group, the differences in gene involved in Alzheimer's disease, of Parkinson's disease, glycolysis/gluconeogenesis ribosome, oxidative phosphorylation, p53signaling pathway, apoptosis, natural killer cell mediated cytotoxicity, actin cytoskeleton regulation, calcium signaling pathway and other signal paths.
近年来,新型毒品种类不断增多,为毒品的防治提出新的课题和挑战。甲基苯丙胺(Methamphetamine, METH or MA)俗称“冰毒”,属于苯丙胺类兴奋剂,是最具代表性的和最常见新型毒品。因其具有见效快、兴奋作用维持时间长,价格低廉,化学合成技术简单,多途径摄取等特点,使它滥用及蔓延速度极快,成为当今我国危害最为严重和滥用的两大毒品之一。
法医在接触因吸食冰毒死亡或与吸毒有关的其它暴力死亡的尸检和在戒毒所治疗的病人过程中,发现其死亡与心脑重要器官及其血管的代谢损伤相关。有大量的资料表明,冰毒对大脑神经细胞产生直接的损害作用,引起中枢神经系统神经化学、神经病理改变,导致神经细胞变性、坏死和异常的膜性结构改变,出现急性和慢性精神障碍和行为改变；可以导致心肌细胞肥大、萎缩、变性、收缩带坏死、小血管内皮细胞损伤和小血管痉挛,从而导致急性心肌缺血、心肌病和心律失常,成为吸毒者突然死亡的原因。因此,对冰毒吸食者毒性损伤机制乃至成瘾机制及猝死机制的研究和探索成为国内外法医关注的问题,也是本实验室近十年来的重点研究方向。
目前,关于METH的神经毒性机制国内外研究结果尚未完全明确,现阶段的研究结果显示多种机制参与了METH的神经毒性,主要包括氧化应激、神经元凋亡、兴奋性毒性、线粒体功能障碍等,其中氧化应激是METH所致神经毒性损伤的重要机制作用之一。
本实验室运用蛋白质组学的方法,对METH注射后大鼠纹状体、额叶皮质、海马等部位的差异表达蛋白质进行鉴定和分析,发现了一种新型的NO合成调节酶二甲基精氨酸二甲基氨基水解酶1(DDAH1)表达上调,提出了DDAH/ADMA/NOS系统可能是NO过表达引起的中枢神经系统损伤的重要途径。本课题组前期研究应用稳定同位素标记氨基酸细胞培养技术(SILAC),对METH作用后PC12细胞进行分析,发现谷胱甘肽S-转移酶蛋白(GSTP1)硝基化增强,提出了GSTP1-P35/P25-CdK5可能是氧化应激损伤的重要调节途径。
本研究拟建立METH中毒细胞模型和动物模型,通过形态学、NOS含量、DA含量及凋亡等指标的检测,进一步验证METH引起的中枢性毒害作用,并通过给予NOS抑制剂(L-NAME和7-NI)反证氧化应激过程中NOS在METH中毒模型中的作用及其与METH神经毒性的关系,进一步完善DDAH1/ADMA/NOS通路的神经毒性作用机制,并为研究GSTP1-P35/P25-CDK5这一可能的NO氧化应激下游通路的提供依据。
本研究拟利用体外研究手段,通过腺病毒载体在PC12细胞上建立GSTP1过表达模型,通过调控GSTP1表达,研究其基因水平的改变对METH氧化应激过程中上述通路的调控变化及作用,探索其是否是介导氧化应激致细胞骨架结构损伤和细胞凋亡死亡通路的中间环节。本研究预期结果不仅为阐明METH的毒性损伤作用机制提供基础,也为METH中毒的防治、METH戒毒药物靶点的选择乃至为毒品戒断治疗奠定基础。
本研究还将应用mRNA表达谱芯片技术,检测METH作用PC12细胞引起的全基因mRNA的变化,以期筛选出差异表达mRNA,以明确差异表达mRNA和METH神经毒性之间的关系,为今后的METH神经毒性机制研究提供新的研究靶点。
目的：
建立METH中毒细胞模型和动物模型,并给予NOS抑制剂,通过形态学、NOS含量、DA含量及凋亡等指标的检测,验证氧化应激过程中NOS的作用及其与METH神经毒性的关系；通过腺病毒载体在PC12细胞上建立GSTP1过表达模型,进而研究GSTP1在METH氧化应激过程中的作用；应用mRNA表达谱芯片技术,明确差异表达mRNA和METH神经毒性之间的关系,为METH神经毒性机制研究提供新的研究靶点。
方法：
1. METH中毒模型的建立及NO抑制剂的保护作用
1.1METH中毒动物模型的建立及7-NI的保护作用
SD大鼠雄性24只,随机分为四组,每组6只：生理盐水组、METH组、METH+7-NI组、7-NI组。动物单笼饲养,自由饮水、取食。实验前先在饲养室适应3天。第一组(生理盐水组)：腹腔注射生理盐水,早8：30、晚5：00各一次,连续注射3天；第二组(METH组)：腹腔注射METH,早8：30、晚5：00各一次,连续注射3天；第三组(METH+7-NI组)：注射方法同第二组,提前30min在腹腔注射7-NI；第四组(7-NI组)：注射方法同第一组,提前30min腹腔注射7-NI,腹腔注射生理盐水。制成动物模型后,观察行为学变化情况,对大鼠的刻板行为进行评分。应用western blot检测大鼠纹状体nNOS表达、ELISA法检测纹状体多巴胺含量、应用Tunel荧光法检测细胞凋亡。
1.2METH中毒细胞模型的建立及L-NAME的保护作用
已分化的PC12细胞培养于含10%FBS、双抗(1：100)的高糖DMEM培养基中,37℃、5%CO2培养箱中培养。PC12细胞培养至对数生长期,达约80%汇合时进行实验。实验分为空白对照组(阴性对照)和实验组,实验组根据L-NAME(N-硝基-L-精氨酸甲酯)浓度的不同而分为5组,各组METH含量均为2.0mmol/L, L-NAME含量分别为0μmol/L(阳性对照)、1μmol/L、10μmol/L、50μmol/L、100μmol/L。当细胞生长至80%汇合时,倒去原培养液,实验组加入含上述浓度METH和L-NAME的无血清培养基,对照组换成等体积不含血清和METH的培养基,细胞继续培养24h后。倒置显微镜下观察PC12细胞形态改变,PE Annexin试剂盒、流式细胞术检测细胞凋亡率,NOS检测试剂盒检测细胞总NOS活力,Western blot技术检测GSTP1表达变化。
2.GSTP1过表达载体的构建
首先通过PCR扩增大鼠全长GSTP1cDNA序列,然后将PCR产物与pshuttle-IRES-hrGFP-1体外重组并转化大肠杆菌,测序验证。然后又将穿梭质粒pshuttle-GSTP1与腺病毒骨架质粒pAdEasy-1重组。通过Lipo2000介导将重组体腺病毒在AD293细胞中的包装扩增。最后通过qPCR和western blotting验证转染效率。
3.PC12细胞METH毒性损伤mRNA表达谱的差异分析及验证
按照前述方法进行培养PC12细胞并进行药物处理,实验分为空白对照组(阴性对照)和METH组,METH+L-NAME组。METH浓度为2.0mmol/L,L-NAME浓度100μmol/L当细胞生长至80%汇合时,倒去原培养液,实验组加入含上述浓度METH和L-NAME的无血清培养基,对照组换成等体积不含血清和METH的培养基,细胞继续培养24h后。将待检样品抽提RNA后进行mRNA表达谱芯片检测。
结果：
1. METH中毒模型的建立及NO抑制剂的保护作用
1.1动物模型的建立及7-NI的保护作用
本实验METH组和7Ni+METH组的动物刻板行为评分均显著高于7Ni和Control组(P均<0.001),METH、7Ni+METH组间无显著性差别(P=0.769),7Ni、Control组间无显著性差别(P=0.190)。Western blot结果显示METH组大鼠纹状体中nNOS蛋白表达水平明显升高,而METH+7-NI组nNOS表达水平较METH组明显降低。ELISA结果显示METH组DA显著降低(P<0.001),而METH+L-NAME组、L-NAME组与对照组比较,DA无显著差异(P>0.01)。采用Tunel法检测各组大鼠脑组织纹状体内神经细胞凋亡情况,METH组与对照组相比凋亡细胞数显著升高(P<0.001),而7-NI对凋亡的增高表现出明显的保护作用(与METH组相比P<0.001)。
1.2细胞模型的建立及L-NAME的保护作用
结果发现与正常对照组细胞相比,METH处理的细胞萎缩变圆,变圆细胞的胞质透亮度增加,可见环形透亮区,突起变短、断裂、消失,神经网络结构消失,因细胞突起萎缩可在细胞表面形成毛刺状,边界不清晰。加入L-NAME处理后,细胞形态学改变随浓度升高损伤呈减弱趋势,中、高浓度处理主要主要以胞体变圆及胞浆透亮增加为主,突起无明显改变。METH处理后细胞凋亡率显著增加,加入L-NAME后细胞总凋亡率随着浓度增加逐渐减少,各实验组与对照组相比具有显著性差异(P<0.001),尤其是中、高浓度L-NAME具有显著的抑制细胞凋亡作用。METH作用后PC12细胞内总NOS活性增加,加入L-NAME后NOS活力逐渐下降,各实验组与对照组相比有显著性差异(P<0.02)。同时采用western-blot技术对细胞内GSTP1表达变化进行检测,发现单独METH处理PC12细胞GSTP1水平显著下降,加入L-NAME后对GSPT1蛋白的下降趋势可起到明显的抑制作用：随L-NAME浓度的升高,GSTP1蛋白的表达逐渐上升,并出现接近正常水平的趋势。
2.GSTP1过表达载体的构建
本实验通过脂糖凝胶电泳跑胶,证明了携带GSTP1序列穿梭载体构建成功,在Ad-293细胞中观察到绿色荧光的表达证明病毒在细胞内进行包装,绿色荧光在PC12细胞中大面积的出现,显示pAdEasy-1重组腺病毒在PC12细胞中成功表达。qPCR以及western blotting的结果显示腺病毒骨架质粒pAdEasy-1重组的转染效率高。
3. METH作用下大鼠脑组织mRNA表达谱的差异分析
本实验通过9张芯片比较正常对照组与METH组、METH+L-NAME组三组间的差异表达mRNA,结果表明METH可导致184个基因表达上调,518个基因表达下调；而NOS抑制剂L-NAME可对其中的470下调基因和2个上调基因发生有效的保护作用,约占全部差异基因的67.24%。METH组与正常对照组之间差异表达的基因中,我们发现与凋亡相关的基因有16个。
结论：
1.在大鼠动物模型上,METH明显增加大鼠不自主的刻板运动,而应用7-NI预处理后,对刻板行为也无明显改善。METH可导致nNOS蛋白表达水平增高、DA含量的降低及细胞凋亡增多等多种神经毒性表现,nNOS特异抑制剂7-NI能部分减轻其神经毒性作用。这些结果为下一阶段应用此动物模型进一步研究NOS下游通路的作用机制奠定了良好的基础。
2.L-NAME对METH导致的神经毒性损伤作用具有保护作用,能减弱细胞形态学损伤、抑制凋亡、增加GSTP1表达水平,通过阻断氧化应激的上游信号NOS活性发挥抗氧化、抗凋亡作用。中、高浓度L-NAME可用于METH神经毒性损伤保护机制的研究。
3.证明了携带GSTP1序列穿梭载体构建成功,在Ad-293细胞中观察到绿色荧光的表达,证明病毒在细胞进行包装,转染PC12细胞中发现绿色荧光,证明转染成功。经过qPCR以及western blotting的结果显示腺病毒骨架质粒pAdEasy-1重组的转染效率高。以上证明了GSTP1过表达载体的构建成功。为下一步研究GSTP1下游调控基因提供可靠的细胞模型。
4. mRNA表达谱经过GO分析,差异表达的基因主要参与的生物过程有核糖体合成、炎症反应、抗凋亡的正向调节、过氧化氢代谢、线粒体电子传递泛醌-细胞色素C、氧化应激反应、线粒体钙离子转运、多巴胺能突触传递负向调节、过氧化氢反应、凋亡的正向调节、线粒体电子传递细胞色素C氧化、通过细胞色素C的caspase的活化、高热反应等。Pathway分析显示,METH组与正常对照组相比,差异基因主要参与了Alzheimer's disease、Parkinson's disease、糖酵解／糖异生、核糖体、氧化磷酸化、P53信号通路、凋亡、自然杀手细胞介导的细胞毒性、肌动蛋白细胞骨架的调节、钙信号通路等信号通路。
Background
In recent years, new types of drugs is increasing, new issues and challenges for the prevention and treatment of drug. Methamphetamine (Methamphetamine, METH or MA) are amphetamine-type stimulants, commonly known as "ice", is the most representative and the most common new drugs. Because of its quick, excitement role in METHintaining a long time, inexpensive, simple chemical synthesis techniques, multi-channel intake characteristics, METHking it the abuse and the spread of fast become one of the China today against the most serious and the abuse of the two drugs.
Forensic contact related death due to methamphetamine or other drug-related violent death autopsy and patient drug rehabilitation treatment process, metabolic injury death and the vital organs of the heart and brain and its blood vessels. There are a large number of data indicate that methamphetamine produce direct damage to brain cells, causing the central nervous system neurochemical and neuropathological changes, leading to nerve cell degeneration, necrosis and abnormal membranous structure change, acute and chronic mental disorders and behavior change; myocardial cell hypertrophy, atrophy, degeneration, contraction band necrosis, small vascular endothelial cell damage and small blood vessels spasm, leading to acute myocardial ischemia, cardiomyopathy and arrhythmia, become addicts sudden death. Therefore, research and exploration of methamphetamine abusers toxicity mechanisms of injury and even addiction mechanisms and sudden death mechanisms become the forensic attention at home and abroad, is also a key research direction over the past decade in this laboratory.
About METH neurotoxicity research results at home and abroad is not yet fully clear, at this stage of the research results show that a variety of mechanisms involved in METH neurotoxicity, including oxidative stress, neuronal apoptosis, excitotoxicity, mitochondrial dysfunctionetc, wherein the oxidative stress is one of the important mechanism for the role of METH-induced neurotoxic injury.
Use of proteomics methods in the laboratory, after injection of METH rat striatum, frontal cortex, hippocampus and other parts of the differences expressed proteins were identified and analysis, and found a new type of NO synthesis regulating enzyme dimethyl fineacid dimethyl-amino hydrolase1(DDAH1) of upregulation of dimethylarginine dimethylaminohydrolase in/ADMA/NOS system METHy be an important way of central nervous system damage caused by NO overexpression. Previous study by our group application of stable isotope labeled amino acids cell culture (SILAC), PC12cells after METH role analysis found that the enzyme protein of glutathione S-transferase (GSTP1) nitro enhancement of GSTP1-P35/the P25-CdK5important regulatory pathway for oxidative stress injury.
This study to establish METH poisoning cells and animal models, central toxic effects of METH caused by the detection of the morphology, NOS content, DA content and apoptosis index, further validation, and by giving the NOS inhibitor (L-NAME/7-NI) NOS METH poisoning model disprove oxidative stress and its relationship with METH neurotoxicity, further perfect DDAH1/ADMA/NOS path the neurotoxicity mechanism of action, and to study GSTP1-P35/P25-NO oxidation of CDK5this may provide the basis for stress downstream pathway.
This study means of in vitro studies, GSTP1overexpression METH intoxication cell lines, through the regulation of GSTP1expression of altered levels of gene regulation change and the role of the passage METH oxidative stress process, to explore whether it is mediatedoxidative stress caused the cytoskeletal structural damage and apoptosis death pathway intermediate links. The expected results of this study provide a basis not only to elucidate the mechanism of action of METH toxic injury METH poisoning prevention, METH drug selection of drug targets and even to lay the foundation for the treatment of drug withdrawal.
Objective
METH poisoning cells and animal models, and give the NOS inhibitor Verify the NOS's role in the process of oxidative stress and its relationship with METH neurotoxicity by morphology, NOS is DA content, and apoptosis index detection;establishment of GSTP1overexpression METH poisoning cell lines to study the role of GSTP1METH oxidative stress process; application mRNA expression microarray technology, a clear difference in the expression of the relationship between the mRNA and METH neurotoxicity, METH neurotoxicity mechanism of the newResearch targets.
Methods
1.METH poisoning model and the protective effect of NO inhibitor
1.1METH poisoning animal model and the protective effect of7-NI
Male SD rats were24(n=6), were randomly divided into four groups:normal saline, METH, METH+7-NI group,7-NI group. The animal single cages, free access to water, feeding. Experimental adapt before feeding room for three days. First group (saline group):intraperitoneal injection of saline,8:30, the second group (METH group):intraperitoneal injection of METH and00pm each injection of3consecutive days;8:30,5:00each time, three days of continuous injection; third group (METH+7-NI group):injection method with the first group, ahead of30min in the abdominal cavity injection of7-NI; fourth group (7-NI group):injection method with the first group,30min prior to intraperitoneal injection of7-NI, intraperitoneal injection of saline. Made of animal models, the application of nNOS expression in rat striatum detected by western blot, ELISA method detected striatal dopamine content, applications the Tunel assay in cell apoptosis.
1.2protective effect of L-NAME on PC12cells treated with methamphetamine
Differentiated PC12cells were cultured in high glucose DMEM containing10%FBS and double antibiotics in37℃incubator with5%CO2. PC12cells was divided into control group and experimental groups. The experimental groups were divided into subgroups according to the L-NAME amount added as0μmol/L,1μmol/L,10μmol/L,50μmol/L and100μmol/L. The amount of METH added into every group was2.0mmol/L. When the cells were in logarithmic phase and grow to80%density, the regents were added into each group as described above. The same amount of DMEM was added into the control group. All the groups were cultured for24h. The morphological changes of PC12cells were observed by invert microscope. The apoptosis rate was determined by Annexin V-PE stain associated with flow cytometer. NOS activity was determined by NOS kit. The expressions of GSTP1was determined by Western blot.
2.GSTP1over-expression vector construction
Rat full-length sequence of GSTP1cDNA is first amplified by PCR, the PCR product was then pshuttle-IRES-hrGFP-1in vitro recombinant and transformed into E. coli and sequenced. Then turn the shuttle plasmid pshuttle-GSTP1adenovirus backbone plasmid pAdEasy-1recombinant. The recombinant through Lipo2000mediated packaging of the adenovirus in the AD293cells amplified. Last verify the efficiency of transfection through Qpcr and western blotting.
3.Analysis and validation of PC12cells METH toxic injury mRNA expression profile differences
Cultured PC12cells and drug treatment in accordance with the aforementioned method, the experiment was divided into blank control group (negative control) and METH METH and+L-NAME group. The METH concentration of2.0mmol/L, L-NAME concentration100μmol/L, when the cells were grown to80%confluence, culture liquid decanted original experimental group joined serum-free medium containing the above concentrations of METH and L-NAME, the control group converter into an equal volume of medium without serum and METH cells were cultured for24h. The sample to be assayed RNA was extracted and mRNA expression microarray detection.
Results
1.METH poisoning model and the protective effect of NO inhibitor
1.1Establishment of animal models and the protective effect of7-NI
METH can cause the striatum of rat brain tissue nNOS expression and DA concentrations were significantly increased at the same time lead to neuronal cell extensive apoptosis; NOS inhibitor7-Ni effective inhibition of nNOS and DA increased, while the effective protection of the withereddeath of occurrence.
1.2protective effect of L-NAME on PC12cells treated with methamphetamine
Contrast to the control group,we found that the morphological changes of PC12. cells treated by METH2.0mmol/L were featured by shrinkage of the cell bodies, brighter cell cytoplasm, disruption of the dendrite and disappearance of cell reticular formation,as well as cell edges bristle-like formation,when treated with METH+L-NAME of1-100μmol/L, morphological injury change of PC12was attenuated following the increase of concentration The cell apoptosis rates of PC12cells treated by METH2.0mmol/L were increased,when added by L-NAME with the concen ra tion1-100μmol/L, the cell apoptosis rate began to decrease gradually, there were significant differences between the METH+L-NAMEgroup and the control group (P<0.001). The TNOS activation was significantly increased after treatment of2.0mmol/L METH, then was decreased in dose dependent manner after the cells treated with1-100μmol/L L-NAME,and was significantly different between the experiment group and the control group (P<0.02). At the same time,we applied west-blot technology to detect GSTP1expression level, and found that compared with control group,, the GSTP1level of PC12cells exposed to2.0mmol/L METH was decreased, but treated by METH+L-NAME GSTP1level was gradually increased, and get close to common level.
2.GSTP1over-expression vector construction
Run on a gel by agarose gel electrophoresis, the experimental proof of the shuttle vector was successfully constructed with GSTP1sequence, expression of green fluorescence observed in the Ad-293cells that virus packaging cells, a large area of green fluorescence in PC12cellsappear pAdEasy-1recombinant adenovirus successfully expressed in PC12cells. Of Qpcr and western blotting results show high transfection efficiency of adenovirus backbone plasmid pAdEasy-1recombinant.
3.The METH effect in rat brain tissue mRNA expression profile differences analysis
9chip compared to normal control group METH group, METH and L-NAME group differences among the three groups, the experimental expression mRNA, results showed that METH can lead to184genes were up-regulated and518genes were down-regulated; NOS inhibitor L-NAME470down the genes and two up-regulated genes effective protection, accounting for67.24%of all the differences in gene. Genes differentially expressed between the METH group and normal control group, we found that16of the apoptosis-related genes.
Conclusion
1. In the rat model, METH can lead to increased nNOS activity reduced DA content and an increase in apoptosis and other neurotoxic manifestations, nNOS intentionally inhibitor7-NI can partially mitigate its neurotoxic effects. These results for the next phase of the application of this animal model mechanism has laid a good foundation for further study of the role of NOS downstream pathways.
2. L-NAME can protect against METH-induced neurotoxicity including attenuating damage to cell morphology、depressing apoptosis、increasing level of GSTP1by obstructing NOS activation which is the upper stream passway of oxidative stress。Medium dose or high concentration L-NAME was suitable for further investigation of protection mechanism on METH-induced neurotoxicity.
3.The shuttle vector was constructed successfully proved that with a GSTP1sequence was observed in the Ad-293cells, the expression of green fluorescent prove the virus in the cells can be packaged in PC12cells transfected with green fluorescent prove successful transfection after Qpcr and western blotting resultsadenoviral backbone plasmid pAdEasy-1recombinant high transfection efficiency. Above proved GSTP1overexpression vector was constructed successfully. GSTP1downstream regulatory genes for further research can study cell model.
4.Ribosome biogenesis after GO analysis of differentially expressed genes involved in biological processes, inflammatory response, positive regulation of anti-apoptotic, hydrogen peroxide metabolism, mitochondrial electron transport ubiquinone-cytochrome C, oxidative stress, mitochondrialcalcium ion transport dopaminergic synaptic transmission in the negative regulation, the reaction of hydrogen peroxide, apoptosis positive regulation of cytochrome C oxidase, mitochondrial electron transport, cytochrome C, caspase activation, fever reaction. Pathway analysis showed that the the METH and group compared with the control group, the differences in gene involved in Alzheimer's disease, of Parkinson's disease, glycolysis/gluconeogenesis ribosome, oxidative phosphorylation, p53signaling pathway, apoptosis, natural killer cell mediated cytotoxicity, actin cytoskeleton regulation, calcium signaling pathway and other signal paths.
引文
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