Study On The Role Of Thioredoxin And Thioredoxin Inducers In Methamphetamine Addiction

Methamphetamine (METH) is an illicit psychostimulant which is the most common amphetamine type stimulants (ATS) in the world, considering the appearance of pure white crystals, commonly is called as "ice". The inexpensive production of METH, its low cost, and long duration of action have made it is popular and METH abusers become younger, it is the drugs which is be strictly governed by the United Nations Convention on psychotropic substances. METH abuse is becoming a global problem urgently needed to be solved. METH addiction is a chronic and recurrent brain disease which is characterized by the compromised behaviour of seeking drug, craving and withdrawal syndromes after cessation of drug taking. Long-term use may induce abnormal neuronal compensatory adaptation, leading to tolerance, sensitization, dependence and relapse.It has been known that the mesocorticolimbic dopamine systems, including prefrontal cortex, ventral tegmental area and nucleus accumbens, are the anatomical and functional base of drug addiction, related with enhancement effect, memory, conditional response of drug-associated cues and withdrawal syndrome accompanied by cessation of drug taking. The activation of learning and memory circuits, including ventral tegmental area, nucleus accumbens and hippocampus, play an important role in METH addiction and relapse. METH acts on the brain region selectively, promotes the release of dopamine, increases the excitability of the cerebral cortex, finally results in a series of adaptive change. DR1-mediated cAMP pathway and DR2-mediated PI3K/Akt/GSK-3pathway are involved in METH addiction. cAMP response element binding protein(CREB), ΔFosB and Cyclin-depdent kinase5(Cdk5) play the important regulating roles in the neuron adaptive change by METH. However, the cellular and molecular mechanisms of METH addiction remain largely unknown.So far, it has been shown that oxidative stress is related to METH addiction and toxicity induced by METH. METH administration can decrease the lever of glutathione (GSH) and hydrogen peroxidase, increase lipid peroxidation and protein carbonyl and induce the oxidation of neurotransmitters, such as, DA and serotonin. Some addiction-associated molecules (fos/Jun, calmodulin kinase, NF-κB and CREB) are sensitive to the redox state. Long-term potentiation (LTP) related to plasticity changes induced by METH are regulated by the intra-cellular redox state. At the same time, METH enteres DA axons and induces oxidative stress, promotes the damage of DNA and proteins, resultes in neuron apoptosis. Studies have confirmed, antioxidants can attenuate METH addiction. These observations suggest that maintaining the intra-cellular redox balance may be effective for the METH addiction.Trx-1is an important response protein of oxidation reduction, has a highly conserved redox-active site sequence:-Cys-Gly-Pro-Cys-. Trx system is composed of Trx-1, thioredoxin reductase (TR) and NADPH, plays a key role in maintaining the intra-cellular redox balance. Trx-1has various biological activities, including the reduction of some functional protein, such as, activator protein-1(AP-1), NF-κB, CREB, the promotion of synapse growth, the inhibition of cellular apoptosis and the modulation of inflammation. Thus, Trx-1may be a potential therapeutic target of some neurologic disease including METH addiction. Many studies have showed that geranylgeranylacetone (GGA), neurotropin and temocapril could induce Trx-1expression. This paper choosed GGA as the inducer Trx-1, because it has been reported that GGA is lipophilic and can pass the blood-brain barrier easily, is more effective in areas of the brain. Several studies have showed GGA can protect neurons against MPP+induced neurotoxicity and reduced morphine-induced behave respons, suppressed conditioned place preference and attenuated the withdrawal symptom by naloxone.Based on the association between the biological activities of Trx-1and the mechanisms underlying METH dependence, three hypothesis are proposed:Trx-1is involved in the process of METH. Trx-1inducers could resist addiction by METH. Trx-1inducers could protect against METH-induced neurotoxicity and liver and kidney toxicity. The main results were as follows:(1) Trx-1is related to the actions of METH. The dopaminergic neuronal cell model (rat pheochromocytoma tumor cell line, PC12) was chosed as research cells in this section. Firstly, PC12cells were incubated with0.5,1,2, and4mM METH for24h, MTT assay and LDH release assay showed that METH would cause the decreased cell viability and cell damage from2mM. PC12cells were treated with1mM METH at various times (1,2,4,12,24h), western blot assay showed that Trx-1expression was significantly elevated in response to METH after1h. However, Trx-1expression was significantly decreased from12h after METH treatment. The dose of1mM METH for1h at which there was not neurotoxic effect on PC12cells, but the Trx-1expression has changed significantly. Then1mM METH for1h was chosen to study its roles but not neurotoxicity in this study. In order to determine the pathway on Trx-1induced by METH, the following experiments were performed using0.5mM SQ22536(AC inhibitor),20μM LY294002(a PI3K inhibitor) and5mM Licl (GSK-3pinhibitor) for30min, the result showed only LY294002suppressed METH-induced increase of Trx-1expression, suggesting the effect of METH on Trx-1expression is mediated by PI3K pathway. In order to further verify this conclusion, we examined the downstream molecules expressions of PI3K (Akt and GSK-3P), we found that the expressions of p-Akt and p-GSK-3β were increased, so METH induced Trx-1expression via PI3K/Akt signaling pathway. CREB is an important transcription factor in the action of METH. regulating the expreesion of immediate early protein, in this paper PC12cells were treated with1mM METH for1h, western blotting showed that CREB activity was significantly elevated, and the increased expression of p-CREB was suppressed followed the preliminary treatment of LY294002, suggesting that METH can activate CREB through PI3K pathway, too. Finally, we further examined the relationship between Trx-1and CREB in PC12cells after METH treatment. We found Trx-1siRNA significantly decreased CREB activity by METH, the results showed that, as an upstream molecule, Trx-1mediates the activation of CREB. So, Trx-1might play a key role in the actions of METH.(2) Trx-1inducer GGA inhibited METH addiction. By using the mice model of METH addiction as research model in this section, we first established mice model of METH addiction by chronic METH administration (2.5mg/kg, intraperitoneally injection on alternate days,8day) and, we proved it successfully that METH induecd conditioned placed preference by the experiment. We also detected the relative proteins expression in the region of VTA, NAc, PFC and hippocampus, and found that METH increased the expressions of AfosB and Cdk5, METH decreased Trx-1expression, while CREB activity and Hsp70expression were not changed in VTA and NAc. In order to study the resistant effects of GGA on METH addiction, mice were treated with GGA (800mg/kg, orally,8day) before METH (2.5mg/kg, intraperitoneally injection on alternate days,8day) injection, behavioral tests showed that pretreatment with GGA could reduce the motor activity induced by METH acute administration, suppress conditioned place preference (CPP), inhibite the reinstatement of CPP and behavioral sensitization induced by chronic METH administration. Most of METH abusers are very thin, in this paper, we demonstrated GGA could attenuate mice weight loss induced by METH. In addition, GGA suppressed chronic METH administration-induced increase of expressions of AfosB and Cdk5, the decrease in Trx-1expression, and reduced the increased Cdk5expression in Hippocampus in reinstatement of METH-induced CPP. These results suggest that GGA could be a safe and novel therapeutic agent for METH dependence. The protection may depend on that GGA can induce Trx-1expression.(3) Trx-1inducers GGA protected cells against METH-induced neurotoxicity and liver and kidney toxicity. Not only the central nervous system adaptability change, but also the toxicity accumulation is involved in METH action. METH abuser autopsy reports showed that the accumulation of METH mainly distributed in the brain, liver and kidney. So, we choiced the PC12cell and the liver tissue, kidney tissue of the mice treatmented by METH as research materials in this section. Firstly, we demonstrated that the dose of2mM METH treatment for24h induced apoptosis in PC12cells. Pretreatment with GGA (10μM,30min) improved the decreased cellular viability of PC12induced by METH. Tyrosine hydroxylase is a rate-limiting enzyme in dopamine synthesis considered as an evaluation criterion on dopaminergic neuron activity. We found TH expression was decreased24h after METH treatment. Pretreatment with GGA prevented PC12cells against METH-induced down-regulation of TH expression. Then, we examined the expressions of pro-caspase-9, pro-caspase-12and pro-caspase-3after METH treatment with or without GGA pretreatment by western blotting in the PC12cell and found that the pretreatment with GGA suppressed pro-caspase-9and pro-caspase-3activations caused by METH, while the pro-caspase-12expression had not change. These data suggest that METH activates the mitochondria-mediated apoptosis pathway, and GGA can protect PC12cell against METH toxicity by suppressing the process. We also examined the expressions of pro-caspase-9, pro-caspase-12and pro-caspase-3of the mice liver tissue and kidney tissue after chronic METH administration (2.5mg/kg, intraperitoneally injection on alternate days,8day), consistent with the results of PC12cells, the pretreatment with GGA (800mg/kg, orally,8day) suppressed pro-caspase-9and pro-caspase-3activations caused by METH, while the pro-caspase-12expression had not change. Moreover, either PC12cells or the liver and kidney of METH addiction mice model, the pretreatment with GGA could reverse the reduced expression of Trx-1and Hsp70. The results of this study suggest that GGA may be a safe and novel therapeutic agent for METH-induced neurotoxicity and liver and kidney tissues toxicity. The protection may depend on GGA inducing Trx-1and Hsp70expression.In summary, these findings suggest Trx-1is related to METH addiction. Trx-1inducer has the roles in preventing METH addiction and toxicity.