The Roles Of GSTP1and ROCK2in METH-induced Neurotoxicity And Its Relative Mechanism

BACKGROUNDAmphetamines (Amphetamine, AMPH) is a class of addictive drugs, the use of such drugs can make people a new thrill,anorexia and a variety of fantasy. Core structure of AMPH drugs are (β-phenylethylamine, such drugs have easily through the blood brain barrier,resist biotrans formation and stimulate the nerve endings release monoamine neurotransmitters characteristics. AMPH drugs can also inhibit monoamine oxidase (monoamine oxidase, MAO) of the amine oxide material Methamphetamine (Mettamine, METH), commonly known as "ice", is a new synthetic drugs, belonging to amphetamine-type stimulant (Amphetamine-Typed Stimulant, ATS) and having a quick, excitatory effects of long duration,low prices, simple chemical synthesis techniques, multi-channel intake and other characteristics, as the world’s second most widely used illicit drug categories (Vearrier et al.,2012). There likely to cause a range of adverse symptoms after inhaling methamphetamine, such as cardiovascular disease, mental illness, neuropsychiatric symptoms, dehydration, rhabdomyolysis, and liver and kidney failure (Zawilska and Wojcieszak,2013). These harmness not only give painful experience to the individuals, but also heavy burdens to families and society.The biggest feature of METH is the use of it will once addicted. Another feature is that the addiction will cause structural damage to the vital organs of the huMETHn body, especially the toxic effects on the central nervous cell damage which compared with other drugs abnormal outstanding(Carvalho et al.,2012). METH can cause injury in human brain substantia nigra, striatum, hippocampus, cortex and other parts, as well as stellate cell disease, brain swelling and degeneration, globus pallidus, and spinal cord gray matter necrosis (Grant et al,2012.; Berman et al,2008;. Chang et al,2007;. Kiyatkin and Sharma,2009; Krasnova and Cadet,2009; Quan et al,2005). Research reports METH abusers have abnormal brain structure, such as the increased volume of striatum, the reduction volume of hippocampal volume, the increases in white METHtter, the decreases volume of cerebral cortex, the selective daMETHge of medial temporal lobe and the edge of the cingulate area (London et al.,2005) and so on. These parts of the brain are related to metabolic disorders, memory disorders, cognitive disorders, alertness and other related dysfunction (Carvalho et al.,2012). Clinically METH abusers manifest memory loss, hallucinations, paranoia, mental irritability, violence, sexual violence, suicide, and homicide etc.(Xu Jianxiong et al,2012;. Grant et al,2012.). It has been confirmed that criminal METH abuseres caused more prominent, which is different from heroin addicts for more drug money of crime, while METH abuseers was mainly due to the criminal acts of violence under the direct toxic effects. These suggest that the abusing of METH-induced toxicity are more stronger and the greater the danger of social security (Liu Zhimin and Lvxian Xiang,2004).The mechanism of how METH cause these injuries remains unclear. Stage study results show a variety of mechanisms involved in METH neurotoxicity, including oxidative stress, neuronal apoptosis, excitotoxicity, mitochondrial dysfunction. Among which, oxidative stress is an important mechanism of METH induced neurotoxicity. These mechanisms are not independent. METH caused nerve damage can be divided into two categories:one is led to scavenging oxygen free radicals, nitrogen radicals and other reactive substances weakened nerve damage which means inadequate protection; another is injury-related pathways activation of damaged organelles and promote further aggravate nerve injury, including some positive apoptotic pathway activation.Our previous study found that METH activated nNOS in multi-brain regions, promoting the formation of a large amount of protein nitration. Glutathione S-transferase (Glutathione S-transferase Pi, GSTP1), which has a certain toxicologically importance can catalyze nucleophilic reactions of glutathione binding affinity of various electronic exogenous chemicals, many exogenous chemicals in the biotrans formation can easily form a first reaction intermediate product of some biological activity. They can be covalently bound to the cells of biological micro molecules essential component causing damage to the body. Combination with glutathione can prevent the occurrence of such covalent bonding and show detoxification. GSTP1nitration may lead to the catalytic ability of exogenous GSH electron affinity binding of the chemical resulting in decreased resistance to oxidative stress. Recent discovery shows that GSTP1, which is a new gene regulating the CDK5can be directly discharged to the inhibition of binding P25/P35CDK5activity as well as eliminate the indirect damage caused by oxidative stress and activation of CDK5(Sun et al.,2011).In addition, METH neurotoxicity is also reflected in the damage of the cytoskeleton, which became the structure basis of addiction and movement dysfunction. Our previous study showed that ROCK2expression increased after METH treatment in vitro experiments. Past studies considered ROCK2participated growth cone collapse and retraction of neurons. ROCK inhibition can protect the brain damage caused by ischemia and prevent necrosis and apoptosis of neurons. Therefore, the second part of the experiment presents ROCK2inhibition may have a protective effect on METH damage to the nervous system. Whether inhibition of ROCK2has a protective effect on neuronal cytoskeleton or injure and the role of ROCK2on METH induced neurotoxicitiy as well as the mechanisms are focused on the study.The purpose of the two parts are to explore the mechanism of the METH induced neurotoxicity and find a reasonable intervention.METHOD The first section of chapter1:Establishment of METH induced in vivo model and inhibition of nNOS by7-NI, using western blot and immunohistochemistry detect GSTP1expression and3-NT/GSTP1ratio after METH or nNOS inhibition group in STR amd SN zone.The second section of chapter1:Successfully constructed the GSTP1overexpression lentivirus, Selecting the appropriate concentration of virus titer, by immunofluorescence and MTT method. To verify the transfection efficiency of LV-3flag-eGFP-GSTP1in PC12and primary neurons by western blot. Compare the apoptosis by METH treatment between overexpressing GSTP1and no-overexpressing in PC12cells and primary neurons. Verify the relationship CDK5and GSTP1by Western blot.The third section fo chapter1:Stereotactic injection of recombinant GSTP1in SN for8weeks. The GSTP1observed in SN regions as well as the projected area of SN region under a fluorescence microscope and by Western blot.The first section fo chapter2:Observed mRNA and Protein level of ROCK2by QPCR and Western after the METH treatment. We observed that ROCK2increasing in METH treated PC12cells in dose-dependent manner. Blocking ROCK2expression by constructing siROCK2. siROCK2transfection efficiency and interference ratio in PC12cells were estimated via western blot, Qpcr and flow cytometry The anti-apoptosis effect of ROCK2inhibiton were test via flow cytometry techniques Annexin V-FITC/PI double staining and TUNEL staining on METH-treated PC12cells. Meantime, ROS level was detected by chemical detection assay in METH treated PC12cells with or without siROCK2. As RhoA is considered to be upregulator of ROCK2, Qpcr was applied to test RhoA transcript levels in METH-treated PC12cells. While ROCK2inhition has no detectable effect to RhoA transcriptional level.in METH treated PC12cells. Again ROCK2interference can increase ratio of p-Akt/Akt but no significant change in active-capspase3and cleave PARP level compared with non ROCK2interference in METH treated groups via western blot. Staining.of F-actin and Tubulin were perform to show the dendritic spines and dendritic length and branching of dendrites change revered by ROCK2 interference in METH treated primary nigra neurons and its associate mechanism, ratio of p-MLC/MLC, was detected by western blot.The second section fo chapter2:Estimating the change of ROCK2expression level in PFc, Str, Hip and SN by western blot and immunofluorescence between compared with con group and METH group. Open field was applied to test locomotor activity affected by METH and METH+fasudil compared with control group. Western blot analysis of ROCK2expression was perform to test inhition effect of ROCK2by fasudil token. TUNEL staining Nissl staining, and TH staining were perform to estimate neurotoxic effect of METH in this vivo model. The apoptosis relative protein such like DAT, Bax, Grp78and CDK5were test via immunohistochemistry to estimate the protective mechanism performed by Fasudil.RESULTSThe first section of chapter1:Figure.1-1-1METH subacute model which nNOS/NO was inhibited was successfully constructed. Figure.1-1-2A GSTP1was decreasd in METH group compared with Con group in Str and SN via IHC. Figure.l-1-2B3-NT/GSTP1decresed by58.24%(*p<0.01, n=3) and GSTP1increased by158.0%(#p<0.001, n=3) in METH+7-NI group compared with METH group in Str region. Figure.1-1-2C3-NT/GSTP1decresed by44.3%(*p<0.05, n=3) and GSTP1increased by161.0%(#p<0.001, n=3) in METH+7-NI group compared with METH group in SN region.The second section of chapter1:Figure.1-2-1to Figure.1-2-2GSTP1overexpression lentivirus were successfully constructed. Figure.1-2-7The percentage of TH-ir neuron in primary substantia nigra neurons is31±3%. Figure.1-2-85×106TU/ml is optimal titer of LV-3flag-eGFP-GSTP1to transfect primary substantia nigra neurons. Figure.1-2-91×107TU/ml is optimal titer of LV-3flag-eGFP-GSTP1to transfect PC12cells. Figure.1-2-10Apoptosis rate decreased by66.5%(*p<0.001, n=3) in2.0mM METH treated ExpGSTP1PC12cells compared with METH treated Wt PC12cells for24h via flow cytometry Annexin V/PE. Figure.1-2-11Hoechst possitive staining decreased by50.03%(p<0.001,n=3) in2.3mM METH treated ExpGSTP1primary substantia nigra neurons compared with METH treated primary substantia nigra neurons for48h via Hoechst staining which indicated overexpression of GSTP1protect primary substantia nigra neurons from METH induced apoptosis. Figure.1-2-12A mRNA level of CDK5increased by157.0%(*p<0.001,n=3) in2.0mM METH treated PC12cells compared with Con group via Qpcr. Figure.1-2-12B Overexpression of GSTP1partially attenuated quantity of CDK5increased by METH in PC12cells. Figure.2-13Overexpression of GSTP1attenuated the increaseing level of ROS by5.73%(F=40.83,*p<0.05) in METH treated PC12cells and by6.9%(F=97.88,**p<0.01) in METH treated primary substantia nigra neurons compared with Con group which indicated overexpression may attenuated oxidative stress.The third section of chapter1:Figure.1-3-18weeks after5uL LV-3flag-eGFP-GSTP1stereotactic injection in SN region. SN region and most of the SN projection regions were with green fluorescent protein. Figure.1-3-2Multiple brain regions appear overexpression of GSTP1including PFc,Str,Hip and SN regions via western blot.The first section of chapter2:Figure.2-1-1mRNA level and protein level of ROCK2increased according to METH concentration for24h METH treatment. Figure.2-1-1A mRNA level of ROCK2increased by4.78times and4.99times (F=5.05,*P<0.05, n=3) separately in2.0mM and2.5mM METH treated PC12cells compared with Con group. Figure.2-1-1B Dendriet shorten and green fluorecent(represent ROCK2) strenthen in METH treated PC12cells compared with Con group. Figure.2-1-1C Intensity of ROCK2increased in160kD according to METH concentration. Figure.2-1-2A,B1,B2transfection efficiency of Lipo/siROCK2was68.86%while siROCK2group and Con group was0.66%and0.59%. Figure.2-1-2C,D Optimal concentration and time for siROCK2was100nM and48h. Figure.2-1-2E siNC has no effect to ROCK2inhibiton compared with siROCK2in 100nM. Figure.2-1-3A,A’ Apoptosis rate decreased by52.38%(p<0.001, n=3) in METH+Lipo/siROCK2group compared wtih METH group in METH treated PC12cells. Figure.2-1-3B,B’ TUNEL staining positive PC12cells increased by8.9times in METH group compared with Con group(*P<0.01) which decreased by60.2%, P<0.01in Lipo/siROCK2group. ROCK2inhibiton decrease apoptosis rate via TUNEL staining. Figure.2-1-4ROS level decreased by5.3%and5.1%(*p<0.05, n=3) in Lipo/siROCK2group separately compared with METH group and METH+siNC group which indicated ROCK2inhibition attenuated ROS increased by METH treatment. Figure.2-1-5A mRNA level of RhoA increased according to METH concentration. mRNA of RhoA increased by71.84%(**p<0.05, n=3) in METH group compared with Con groups. Figure.2-1-5B ROCK2inhibiton has no significant effect to RhoA mRNA level*p>0.05. Figure.2-1-5C1,C2,C3,C4The mechanism of ROCK2inhibition to reduced apoptosis rate was mediated by p-Akt/Akt (*p<0.01) but active caspase3and cleave PARP. Figure.2-1-6ROCK2inhibition maintain the branch and the length of dendrite in PC12cells. Figure.2-1-7ROCK2inhibition maintain the branch and the length of dendrite in primary substantia nigra neurons via Tubulin staining. Figure.2-1-8ROCK2inhibition attenuated the decrease of spine density induced by METH via F-actin staining. Figure.2-1-9B1,B2ROCK2inhibition maintain stablization the structure in METH treated primary substantia neurons via F-actin staining which partially mediated by decreasing p-MLC/MLC.The second section of chapter2:Figure.2-2-1Measurement of the expression of ROCK2in different brain regions of rats after METH treatment for4D15mg/kg/D.Figure.2-2-148h after METH withdrawal locomotion capabilities detected by Open field method. The results showed that the impact of locomotor activity of rats in METH group perfermed no differenece compared with the control group(p>0.05). Figure.2-2-3In the SN and Str districts, Fasudil of30mg/kg can effectively reduce the ROCK2which increased by METH via western blot. Figure.2-2-4TUNEL staining showed compareing with Con/saline group, METH group showed no differences in the number of TUNEL-positive cells in PFc, Str, Hip and SN area. Figure.2-2-5Nissl staining showed that compareing with Con/Saline group, METH group showed that in PFc, Str, Hip and SN area, the number of positive neurons of Nissl staining has significantly declined. These results indicated that METH rat model can damage neuron of above mentioned brain region. Figure.2-2-6Furthermore, our results also showed that compared with the Con group, the METH group showed TH staining deletions with a large area in Str, and the combination of Fasudil can significantly reduce the TH staining deletions. Compared with the Control group, METH group results in a large number of missing neurons of TH-ir in SN district, but can be partial restoration by Fadudil. Figure.2-2-7Immunohistochemistry showed METH group can be reduced DAT expression and increased the expression of Grp78, Bax and CDK5, whereas no significant effect on the activation of caspase3found. Fasudil joint METH group showed that Fasudil can partially recover METH caused alteration of DAT, Bax, Grp78and CDK5.CONCLUTIONChapter I1.METH treatment decreased expression of GSTP1in invivo model of rat brain.2.METH can restore GSTP1protein levels and the proportion of GSTP1of nitration partly by inhibiting nNOS/NO.3. GSTP1reduced METH caused neuronal apoptosis and attenuated ROS level in vitro model.4.CDK5may be the downstream regulatory factors of GSTPlin the mechanisms of METH induced neurotoxicity.5.Overexpression of GSTP1in SN and SN projection regions-model was successfully constructed.Chapter II1.The increase of ROCK2has METH concentration-dependent manner.2.High-performance siROCK2successfully synthetized which can knock down the expression of ROCK2in PC12cells and primary neurons. 3.Inhibition of ROCK2can protect PC12cells from METH induced apoptosis.4.Inhibition of ROCK2can partially reduce METH induced oxidative stress in PC12cells.5.RhoA represents upstream of ROCK2in METH induced neurotoxicity. Inhibition of ROCK2protect PC12cells from METH induced neurotoxicity possibly via enhancing p-Akt/Akt, but not via activation of caspase3and the suppression of cleave PARP.6.Inhibition of ROCK2can reverse METH induced cell morphology in PC12and cytoskeleton instability in primary substantia nigra neuronal, by increasing the density of dendritic branching and dendritic spines to maintain the cell contours. The mechanism may mediated by reducing p-MLC/MLC.7.In vivo study confirmed that ROCK2significantly increased in PFc, SN, and Str after METH treatment, while no significant increased in the Hip.8.ROCK2inhibition protect TH-ir nerve terminal and soma in METH invivo study.9. Nissl staining and TH staining may be more sensitivity in the early neuronal damage in vivo model of METH treamtment compared with TUNEL staining.10.In METH invivo model, protective effect of ROCK2inhibition may mediated by maintaining DAT level and attenuating mitochondrial stress, ER stress and oxidative stress.