Protective Effects Of NOS Inhibitors On METH Induced Neurotoxicity And Construction Of GSTP1Over-expression Vectors

BackgroundIn 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. ObjectiveMETH 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.Methods1.METH poisoning model and the protective effect of NO inhibitor1.1METH poisoning animal model and the protective effect of7-NIMale 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 methamphetamineDifferentiated 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 constructionRat 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 differencesCultured 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.Results1.METH poisoning model and the protective effect of NO inhibitor1.1Establishment of animal models and the protective effect of7-NIMETH 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 methamphetamineContrast 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 constructionRun 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.Conclusion1. 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.