硫氧还蛋白及其诱导物在甲基苯丙胺成瘾中的作用研究
发布时间：2018-09-10 12:15
【摘要】：甲基苯丙胺(methamphetamine, METH),属于苯丙胺类中枢神经系统兴奋剂,外观为纯白色结晶体,俗称“冰毒”。因制作成本低廉、起效快、作用时间持久,滥用率逐年增长并趋向低龄化,是联合国精神药品公约明令管制的精神药物。METH成瘾已成为亟待解决的全球性问题。METH成瘾是一种反复服用METH而引起的慢性、复发性脑疾病,主要特点是强迫性觅药、强烈的渴求心理以及中断用药后出现戒断症状。长期使用会引起机体内神经元产生异常的代偿性适应,导致耐受、敏化、依赖及复吸等症状。 中脑边缘多巴胺系统,即腹侧被盖区(Ventral Tegmental Area, VTA)及其投射区伏隔核(nucleus accumbens, NAc)、前额叶皮质(prefrontal cortex, PFC)等其他脑区共同构成的区域,与多数成瘾药物相关,是强迫觅药和复吸行为的重要的神经环路。该回路改变可产生强化效应、记忆、与渴求相关联的条件反应以及戒断症状中的恐惧、焦虑等情绪反应。另外,由VTA/NAc/Hippocampus构成的强化学习记忆回路的激活在METH成瘾及复吸中发挥重要作用。METH选择性地作用于这部分脑区,促进多巴胺(dopamine, DA)j释放,引起机体发生一系列的适应性变化。METH成瘾所涉及的信号通路主要有多巴胺D1受体介导的腺苷酸环化酶(adenylate cyclase, AC)及其下游环磷酸腺苷(Cyclic Adenosine monophosphate, cAMP)通路与多巴胺D2受体介导的phosphatidyl inositol3kinase, PI3K/Akt/Glycogen synthase kinase-3β,GSK-3通路,在METH引起的神经元结构和功能适应性改变的过程中,cAMP反应元件结合蛋白(cAMP response element binding protein, CREB), ΔFosB蛋白和细胞周期依赖蛋白激酶5(Cyclin-depdent kinase5, Cdk5)起到重要的调节作用,但确切的分子机制尚未阐明。 METH成瘾及毒性机制与氧化应激相关。METH会引起脑内谷胱甘肽、过氧化氢酶等水平降低,脂质过氧化物和蛋白羰基的增加；引起DA、5-羟色胺等神经递质的氧化；许多与成瘾药物相关的分子(如：fos/Jun、钙调激酶、NF-κB和CREB等)对神经元内的氧化还原状态敏感。与药物成瘾引起的突触可塑性变化相关的长时程增强(long-term potentiation, LTP)也受机体的氧化还原状态的调节,进入胞内的METH通过破坏神经元氧化还原平衡,导致氧化应激,促使DNA、蛋白质等的损伤,从而加剧多巴胺能神经元凋亡。已有研究证实,抗氧化剂能缓解药物成瘾的发生。这些现象提示我们：维持机体氧化还原平衡可能干预METH成瘾。 硫氧还蛋白(thioredoxin, Trx)是一种重要的氧化还原应答蛋白,具有高度保守的氧化还原活性位点：-Cys-Gly-Pro-Cys-(CGPC)。Trx、NADPH和硫氧还蛋白还原酶(thioredoxinreductase, TrxR)组成的Trx还原系统,在维持细胞内的氧化还原平衡方面起重要调节作用,Trx具有多种生物活性,包括调节多种转录因子的活性,如激活蛋白1(activator protein-1, AP-1)、NF-κB、CREB等,促神经突触生长,调控细胞周期、抗凋亡、抗炎等。因此,我们认为Trx是中枢神经系统疾病预防或治疗的重要靶点。已有研究证明替普瑞酮(Geranylgeranylacetone, GGA)、神经妥乐和萝卜硫素等多种药物都可以诱导Trx-1的表达。本论文选择GGA作为Trx-1的诱导物,因为,GGA具有亲脂性,能够透过血脑屏障,更有效地作用于脑区;并且,已有研究报道：GGA具有保护神经免于帕金森病毒性物的损害作用；可以缓解吗啡引起的条件位置偏爱及戒断症状。 基于METH成瘾及毒性作用机理与Trx-1的生物学功能,本论文提出三个假设：Trx-1参与了METH的作用过程,Trx-1诱导物GGA可以抵抗METH成瘾,Trx-1诱导物GGA可以抵抗METH引起的神经元凋亡以及肝脏肾脏损伤。 本论文的研究结果如下： (1)明确Trx-1参与了METH作用过程。本论文采用多巴胺能神经元模型——大鼠肾上腺嗜铬细胞瘤细胞(1at pheochromocytoma tumor cell line, PC12)作为研究细胞,首先,用浓度分别为0.5、1.0、2.0、4.0mM的METH刺激PC12细胞,通过MTT分析、LDH释放分析检测细胞存活率及损伤程度,结果发现,从2.0mM开始,METH抑制了PC12细胞的存活且引起细胞损伤。用1mM的METH刺激PC12细胞1、2、4、12、24h,通过蛋白免疫印迹(Western Blotting)检测Trx-1表达水平,结果发现,在METH作用1h的情况下,Trx-1表达水平显著升高,而从12h开始,Trx-1表达水平明显降低。由于1mM的METH作用PC12细胞1h未出现凋亡,此时Trx-1分子水平已经发生变化。因此,第二章后续实验均采用的METH的剂量为1mM,作用时间为1h。为了证明METH对Trx-1诱导的分子机制,本论文分别采用0.5mM的SQ22536(AC的抑制剂)、20μM的LY294002(P13K的抑制剂)、5mM Licl (GSK-3β的抑制剂)预刺激,结果发现,只有LY294002降低了METH诱导的Trx-1的表达,说明METH对Trx-1的诱导通过了PI3K通路,为了进一步验证这一结论,本论文检测了PI3K的下游分子Akt和GSK-3β的活性,即p-Akt和p-GSK-3β的表达水平,结果发现,二者均被激活,从而确定了METH是通过PI3K/Akt信号通路诱导Trx-1表达。CREB在METH作用过程中起重要作用,调节即可早期蛋白的表达,本论文发现1mM的METH作用PC12细胞1h引起了CREB活性升高,且升高的活性能够被LY294002预刺激所抑制,说明：METH激活CREB也是通过PI3K/Akt这条信号通路。为了进一步阐明Trx-1与METH作用的关系,本论文采用SiRNA的方法将Trx-1蛋白表达降低,并检测到CREB的活性也明显降低,说明Trx-1作为上游分子介导了CREB的激活。这些数据提示我们,Trx-1确实参与了METH的作用,且发挥了重要的调节作用。 (2)Trx-1诱导物GGA能够抵抗METH成瘾。本论文采用METH成瘾小鼠模型作为研究材料,首先,通过METH慢性给药(2.5mg/kg,隔天腹腔注射,共8天)构建METH成瘾小鼠模型,通过条件性位置偏爱实验验证其构建成功,并检测了小鼠脑内VTA、NAc、PFC及海马区相关分子的变化,结果显示,METH慢性给药引起了VTA和NAc区ΔAFosB和细胞周期依赖蛋白激酶5(Cdk5)表达水平增加,Trx-1表达水平降低,CREB活性和热休克蛋白70(heat shock protein70, Hsp70)表达水平变化不显著；为了研究Trx-1诱导物GGA对METH成瘾的抵抗作用,本论文给予小鼠GGA预处理(800mg/kg/d,灌胃8天),之后METH给药,行为检测结果发现,GGA能降低METH急性作用(2.5mg/kg,一次腹腔注射)所引起的小鼠行为能力的增强,抑制METH慢性给药(2.5mg/kg,隔天腹腔注射,共8天)引起的条件性位置偏爱的形成,条件性位置偏爱消退点燃以及行为敏化。METH成瘾患者伴有明显的消瘦,本论文证明了GGA预处理可以抑制METH慢性给药造成的体重降低；在分子水平上,GGA恢复了METH'慢性给药(2.5mg/kg,隔天腹腔注射,共8天)引起的VTA和NAc区ΔFosB和Cdk5表达的增加及Trx-1表达的降低；降低了METH条件性位置偏爱消退点燃后海马区Cdk5表达的增加。这些数据说明,GGA可以抵抗METH所致的位置偏爱及其消退点燃,行为敏化和运动能力增强,这种抵抗作用主要可能是GGA诱导Trx-1的高表达而实现的。 (3) Trx-1诱导物GGA能够抵抗METH引起的神经元凋亡和肝脏、肾脏损伤。METH成瘾不仅涉及中枢神经系统的适应性改变,同时也是机体对药物毒性积累的过程,对METH成瘾患者的尸检报告显示,METH在大脑、肝脏、肾脏中积累最多,且造成的损伤最大,因此,本论文采用PC12细胞及METH成瘾模型小鼠的肝脏、肾脏为研究材料,首先,通过MTT分析检测到METH(2mM,24h)可引起PC12细胞存活率明显下降,且可以被GGA预刺激(10μM,提前30min)显著缓解。酪氨酸羟化酶是合成多巴胺过程中的限速酶,被认为是多巴胺能神经元活性的标志,为了进一步研究METH对PC12细胞的神经毒性,本论文还检测了TH的表达量,结果发现,METH (2mM,24h)引起了TH表达的降低,同样可以被GGA预刺激(10μM,提前30min)显著缓解。其次,检测了METH处理(2mM,24h)的PC12细胞pro-caspase酶的表达量,结果发现METH能够降低pro-caspase-9、pro-caspase-3,而对pro-caspase-12无明显作用,说明,METH处理激活了线粒体介导的细胞凋亡途径,而pro-caspase-9、pro-caspase-3的降低可以被GGA预刺激(10μM,提前30min)所缓解。同样,本论文还检测了METH慢性给药(2.5mg/kg,隔天腹腔注射,共8天)后小鼠肝、肾脏pro-caspase酶的表达量,与PC12细胞的结果相一致,GGA预处理(800mg/kg/d,灌胃8天)抑制了METH所致pro-caspase-9、pro-caspase-3的降低,而pro-caspase-12无明显变化。更重要的是,无论是PC12细胞还是METH成瘾模型小鼠的肝脏、肾脏,GGA预处理均能够逆转METH引起的Trx-1和Hsp70表达降低。以上数据说明,GGA可以抑制METH对PC12细胞及小鼠肝脏、肾脏的毒性作用,这种保护作用是通过对Trx-1和Hsp70的共诱导而实现的。 综上所述,Trx-1与甲基苯丙胺导致的成瘾密切相关,硫氧还蛋白诱导物具有抵抗甲基苯丙胺成瘾的作用以及抵抗甲基苯丙胺毒性作用。
[Abstract]:Methamphetamine (METH), an amphetamine-type central nervous system stimulant, is a pure white crystal, commonly known as "methamphetamine". METH addiction is a chronic, recurrent brain disease caused by repeated use of METH. It is characterized by compulsive drug seeking, strong craving and withdrawal symptoms after discontinuation of the drug. Long-term use can cause abnormal compensatory adaptation of neurons in the body, resulting in tolerance, sensitization, dependence, and withdrawal. Relapse and other symptoms.
The ventral tegmental area (VTA) and its projection nucleus accumbens (NAc), prefrontal cortex (PFC), together with other brain regions, are associated with most addictive drugs and are important neural circuits for forcing drug seeking and relapse behavior. Additionally, the activation of the reinforcement learning and memory circuit composed of VTA/NAc/Hippocampus plays an important role in the addiction and relapse of MEH. METH selectively acts on this part of the brain to promote dopamine (DA). The major signaling pathways involved in METH addiction include dopamine D1 receptor-mediated adenylate cyclase (AC) and its downstream cyclic adenosine monophosphate (cAMP) pathways and dopamine D2 receptor-mediated phosphatidyl inositol 3 kinase (PI3K). CAMP response element binding protein (CREB), Delta FosB protein and cyclin-depdent kinase 5 (Cdk5) play important roles in the regulation of neuronal structural and functional adaptation induced by METH. The molecular mechanism of cutting has not yet been elucidated.
METH addiction and toxicity are associated with oxidative stress. METH can cause decreased levels of glutathione and catalase, increased lipid peroxides and protein carbonyls in the brain, oxidization of neurotransmitters such as DA, 5-hydroxytryptamine, and many molecules associated with drug addiction (e.g. fos/Jun, calmodulin kinase, NF-kappa B and CREB) in neurons. Long-term potentiation (LTP), which is related to changes in synaptic plasticity induced by drug addiction, is also regulated by the body's redox state. Intracellular METH leads to oxidative stress by disrupting the redox balance of neurons, causing damage to DNA, proteins and so on, thus aggravating dopa. Amine neuron apoptosis. Antioxidants have been shown to alleviate drug addiction. These phenomena suggest that maintaining the body's redox balance may interfere with METH addiction.
Thioredoxin (Trx) is an important redox-responsive protein with highly conserved redox sites: -Cys-Gly-Pro-Cys-(CGPC). Trx, NADPH and thioredoxin reductase (TrxR) constitute a Trx reductive system that plays an important role in maintaining the balance of redox and oxidation in cells. Trx has a variety of biological activities, including regulating the activity of a variety of transcription factors, such as activator protein-1 (AP-1), NF-kappa B, CREB, promoting synaptic growth, regulating cell cycle, anti-apoptosis, anti-inflammation and so on. Therefore, we believe that Trx is an important target for the prevention or treatment of central nervous system diseases. In this study, GGA was selected as the inducer of Trx-1 because of its lipophilicity and its ability to penetrate the blood-brain barrier and act more effectively on the brain region; moreover, it has been reported that GGA can protect nerves from Parkinson's virus. It can relieve the conditioned place preference and withdrawal symptoms induced by morphine.
Based on the mechanism of METH addiction and toxicity and the biological function of Trx-1, three hypotheses are proposed in this paper: Trx-1 participates in the process of METH, Trx-1 inducer GGA can resist METH addiction, Trx-1 inducer GGA can resist METH-induced neuronal apoptosis and liver and kidney injury.
The research results in this paper are as follows:
(1) Trx-1 is involved in the process of METH. In this study, the rat adrenal pheochromocytoma tumor cell line (PC12), a dopaminergic neuron model, was used as the research cell. Firstly, the PC12 cells were stimulated by MTH at concentrations of 0.5, 1.0, 2.0 and 4.0 mM, respectively. The results showed that METH inhibited the survival of PC12 cells from 2.0 mM and caused cell damage. The expression of Trx-1 was detected by Western Blotting at 1,2,4,12,24 h after stimulation with 1 mM METH. The expression of Trx-1 was significantly increased at 1 h after treatment with METH, but from 12 h on. The expression level of Trx-1 was significantly decreased. The molecular level of Trx-1 had changed since 1 mM ETH did not induce apoptosis in PC12 cells for 1 h. Therefore, the dose of METH used in the second chapter was 1 mM and the duration of action was 1 h. In order to prove the molecular mechanism of the induction of Trx-1 by METH, 0.5 mM SQ22536 (AC inhibitor) was used. LY294002 (inhibitor of P13K) and 5mM Licl (inhibitor of GSK-3 beta) pretreatment showed that only LY294002 decreased the expression of Trx-1 induced by METH, indicating that the induction of Trx-1 by METH passed the PI3K pathway. To further verify this conclusion, the activities of Akt and GSK-3 beta downstream molecules of PI3K, i.e. p-Akt and p-GSK-3 beta, were detected. The results showed that both of them were activated, which confirmed that METH could induce Trx-1 expression via PI3K/Akt signaling pathway. CREB played an important role in the process of METH, and regulated the expression of early proteins. In this paper, we found that 1 mM of METH could induce the elevation of CREB activity in PC12 cells for 1 h, and the elevated activity could be preempted by LY294002. In order to further elucidate the relationship between Trx-1 and METH, SiRNA was used to reduce the expression of Trx-1 protein, and the activity of CREB was also significantly decreased, indicating that Trx-1 as an upstream molecule mediated the activation of CREB. Indeed, Trx-1 has played an important role in regulating METH's role.
(2) Trx-1 inducer GGA can resist METH addiction. In this study, METH addiction mice model was used as research materials. First, METH addiction mice model was established by chronic administration of METH (2.5mg/kg, intraperitoneal injection every other day for 8 days). Conditional place preference test was used to verify the success of the model, and VTA, NAc, PFC and hippocampal phase were detected. The results showed that chronic administration of METH increased the expression of AFosB and Cdk5 in VTA and NAc regions, decreased the expression of Trx-1, and had no significant changes in CREB activity and the expression of heat shock protein 70 (Hsp70). In this study, mice were given GGA pretreatment (800mg/kg/d, intragastric administration for 8 days), and then given METH. Behavioral test results showed that GGA could reduce the acute effect of METH (2.5mg/kg, once intraperitoneal injection) induced by the enhancement of behavior in mice, inhibit the chronic administration of METH (2.5mg/kg, intraperitoneal injection every other day, a total of 8 days) induced conditioned place preference. This study demonstrated that GGA pretreatment could inhibit weight loss caused by chronic METH administration; at the molecular level, GGA restored the VTA and NAc FosB and Cdk5 tables induced by chronic METH administration (2.5mg/kg, intraperitoneal injection every other day, for a total of 8 days). These data suggest that GGA can resist METH-induced position preference and its regressive kindling, and enhance behavioral sensitization and motor ability, which may be due to GGA-induced high expression of Trx-1.
(3) Trx-1 inducer GGA can resist neuronal apoptosis and liver and kidney injury induced by METH. METH addiction involves not only the adaptive changes of central nervous system, but also the accumulation of drug toxicity. Autopsy reports of METH addicts showed that METH accumulated most in brain, liver and kidney, and caused the most damage. In this study, we used PC12 cells and the liver and kidney of METH addicted mice as research materials. First, MTT analysis showed that METH (2mM, 24h) could significantly reduce the survival rate of PC12 cells, and could be significantly alleviated by GGA pre-stimulation (10 mu M, 30 min earlier). In order to further study the neurotoxicity of METH on PC12 cells, the expression of TH in PC12 cells was detected. The results showed that the expression of TH was decreased by METH (2mM, 24h) and could also be significantly alleviated by GGA pre-stimulation (10mM, 30min earlier). Secondly, the expression of pro-c in PC12 cells treated with METH (2mM, 24h) was detected. The results showed that METH could decrease the expression of pro-caspase-9, pro-caspase-3, but had no obvious effect on pro-caspase-12, indicating that METH treatment activated the mitochondrial-mediated apoptosis pathway, while the decrease of pro-caspase-9, pro-caspase-3 could be alleviated by GGA pre-stimulation (10 mu M, 30 min earlier). The expression of pro-caspase in liver and kidney of mice after chronic administration (2.5 mg/kg, intraperitoneal injection every other day for 8 days) was consistent with that of PC12 cells. GGA pretreatment (800 mg/kg/d, 8 days after intragastric administration) inhibited the decrease of pro-caspase-9 and pro-caspase-3 induced by METH, while pro-caspase-12 did not change significantly. These data suggest that GGA can inhibit the toxic effects of METH on PC12 cells and mice liver and kidney, and this protective effect is achieved by co-induction of Trx-1 and Hsp70.
In summary, Trx-1 is closely related to methamphetamine-induced addiction, and thioredoxin inducers can resist methamphetamine addiction and methamphetamine toxicity.
【学位授予单位】：昆明理工大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.64
[Abstract]:Methamphetamine (METH), an amphetamine-type central nervous system stimulant, is a pure white crystal, commonly known as "methamphetamine". METH addiction is a chronic, recurrent brain disease caused by repeated use of METH. It is characterized by compulsive drug seeking, strong craving and withdrawal symptoms after discontinuation of the drug. Long-term use can cause abnormal compensatory adaptation of neurons in the body, resulting in tolerance, sensitization, dependence, and withdrawal. Relapse and other symptoms.
The ventral tegmental area (VTA) and its projection nucleus accumbens (NAc), prefrontal cortex (PFC), together with other brain regions, are associated with most addictive drugs and are important neural circuits for forcing drug seeking and relapse behavior. Additionally, the activation of the reinforcement learning and memory circuit composed of VTA/NAc/Hippocampus plays an important role in the addiction and relapse of MEH. METH selectively acts on this part of the brain to promote dopamine (DA). The major signaling pathways involved in METH addiction include dopamine D1 receptor-mediated adenylate cyclase (AC) and its downstream cyclic adenosine monophosphate (cAMP) pathways and dopamine D2 receptor-mediated phosphatidyl inositol 3 kinase (PI3K). CAMP response element binding protein (CREB), Delta FosB protein and cyclin-depdent kinase 5 (Cdk5) play important roles in the regulation of neuronal structural and functional adaptation induced by METH. The molecular mechanism of cutting has not yet been elucidated.
METH addiction and toxicity are associated with oxidative stress. METH can cause decreased levels of glutathione and catalase, increased lipid peroxides and protein carbonyls in the brain, oxidization of neurotransmitters such as DA, 5-hydroxytryptamine, and many molecules associated with drug addiction (e.g. fos/Jun, calmodulin kinase, NF-kappa B and CREB) in neurons. Long-term potentiation (LTP), which is related to changes in synaptic plasticity induced by drug addiction, is also regulated by the body's redox state. Intracellular METH leads to oxidative stress by disrupting the redox balance of neurons, causing damage to DNA, proteins and so on, thus aggravating dopa. Amine neuron apoptosis. Antioxidants have been shown to alleviate drug addiction. These phenomena suggest that maintaining the body's redox balance may interfere with METH addiction.
Thioredoxin (Trx) is an important redox-responsive protein with highly conserved redox sites: -Cys-Gly-Pro-Cys-(CGPC). Trx, NADPH and thioredoxin reductase (TrxR) constitute a Trx reductive system that plays an important role in maintaining the balance of redox and oxidation in cells. Trx has a variety of biological activities, including regulating the activity of a variety of transcription factors, such as activator protein-1 (AP-1), NF-kappa B, CREB, promoting synaptic growth, regulating cell cycle, anti-apoptosis, anti-inflammation and so on. Therefore, we believe that Trx is an important target for the prevention or treatment of central nervous system diseases. In this study, GGA was selected as the inducer of Trx-1 because of its lipophilicity and its ability to penetrate the blood-brain barrier and act more effectively on the brain region; moreover, it has been reported that GGA can protect nerves from Parkinson's virus. It can relieve the conditioned place preference and withdrawal symptoms induced by morphine.
Based on the mechanism of METH addiction and toxicity and the biological function of Trx-1, three hypotheses are proposed in this paper: Trx-1 participates in the process of METH, Trx-1 inducer GGA can resist METH addiction, Trx-1 inducer GGA can resist METH-induced neuronal apoptosis and liver and kidney injury.
The research results in this paper are as follows:
(1) Trx-1 is involved in the process of METH. In this study, the rat adrenal pheochromocytoma tumor cell line (PC12), a dopaminergic neuron model, was used as the research cell. Firstly, the PC12 cells were stimulated by MTH at concentrations of 0.5, 1.0, 2.0 and 4.0 mM, respectively. The results showed that METH inhibited the survival of PC12 cells from 2.0 mM and caused cell damage. The expression of Trx-1 was detected by Western Blotting at 1,2,4,12,24 h after stimulation with 1 mM METH. The expression of Trx-1 was significantly increased at 1 h after treatment with METH, but from 12 h on. The expression level of Trx-1 was significantly decreased. The molecular level of Trx-1 had changed since 1 mM ETH did not induce apoptosis in PC12 cells for 1 h. Therefore, the dose of METH used in the second chapter was 1 mM and the duration of action was 1 h. In order to prove the molecular mechanism of the induction of Trx-1 by METH, 0.5 mM SQ22536 (AC inhibitor) was used. LY294002 (inhibitor of P13K) and 5mM Licl (inhibitor of GSK-3 beta) pretreatment showed that only LY294002 decreased the expression of Trx-1 induced by METH, indicating that the induction of Trx-1 by METH passed the PI3K pathway. To further verify this conclusion, the activities of Akt and GSK-3 beta downstream molecules of PI3K, i.e. p-Akt and p-GSK-3 beta, were detected. The results showed that both of them were activated, which confirmed that METH could induce Trx-1 expression via PI3K/Akt signaling pathway. CREB played an important role in the process of METH, and regulated the expression of early proteins. In this paper, we found that 1 mM of METH could induce the elevation of CREB activity in PC12 cells for 1 h, and the elevated activity could be preempted by LY294002. In order to further elucidate the relationship between Trx-1 and METH, SiRNA was used to reduce the expression of Trx-1 protein, and the activity of CREB was also significantly decreased, indicating that Trx-1 as an upstream molecule mediated the activation of CREB. Indeed, Trx-1 has played an important role in regulating METH's role.
(2) Trx-1 inducer GGA can resist METH addiction. In this study, METH addiction mice model was used as research materials. First, METH addiction mice model was established by chronic administration of METH (2.5mg/kg, intraperitoneal injection every other day for 8 days). Conditional place preference test was used to verify the success of the model, and VTA, NAc, PFC and hippocampal phase were detected. The results showed that chronic administration of METH increased the expression of AFosB and Cdk5 in VTA and NAc regions, decreased the expression of Trx-1, and had no significant changes in CREB activity and the expression of heat shock protein 70 (Hsp70). In this study, mice were given GGA pretreatment (800mg/kg/d, intragastric administration for 8 days), and then given METH. Behavioral test results showed that GGA could reduce the acute effect of METH (2.5mg/kg, once intraperitoneal injection) induced by the enhancement of behavior in mice, inhibit the chronic administration of METH (2.5mg/kg, intraperitoneal injection every other day, a total of 8 days) induced conditioned place preference. This study demonstrated that GGA pretreatment could inhibit weight loss caused by chronic METH administration; at the molecular level, GGA restored the VTA and NAc FosB and Cdk5 tables induced by chronic METH administration (2.5mg/kg, intraperitoneal injection every other day, for a total of 8 days). These data suggest that GGA can resist METH-induced position preference and its regressive kindling, and enhance behavioral sensitization and motor ability, which may be due to GGA-induced high expression of Trx-1.
(3) Trx-1 inducer GGA can resist neuronal apoptosis and liver and kidney injury induced by METH. METH addiction involves not only the adaptive changes of central nervous system, but also the accumulation of drug toxicity. Autopsy reports of METH addicts showed that METH accumulated most in brain, liver and kidney, and caused the most damage. In this study, we used PC12 cells and the liver and kidney of METH addicted mice as research materials. First, MTT analysis showed that METH (2mM, 24h) could significantly reduce the survival rate of PC12 cells, and could be significantly alleviated by GGA pre-stimulation (10 mu M, 30 min earlier). In order to further study the neurotoxicity of METH on PC12 cells, the expression of TH in PC12 cells was detected. The results showed that the expression of TH was decreased by METH (2mM, 24h) and could also be significantly alleviated by GGA pre-stimulation (10mM, 30min earlier). Secondly, the expression of pro-c in PC12 cells treated with METH (2mM, 24h) was detected. The results showed that METH could decrease the expression of pro-caspase-9, pro-caspase-3, but had no obvious effect on pro-caspase-12, indicating that METH treatment activated the mitochondrial-mediated apoptosis pathway, while the decrease of pro-caspase-9, pro-caspase-3 could be alleviated by GGA pre-stimulation (10 mu M, 30 min earlier). The expression of pro-caspase in liver and kidney of mice after chronic administration (2.5 mg/kg, intraperitoneal injection every other day for 8 days) was consistent with that of PC12 cells. GGA pretreatment (800 mg/kg/d, 8 days after intragastric administration) inhibited the decrease of pro-caspase-9 and pro-caspase-3 induced by METH, while pro-caspase-12 did not change significantly. These data suggest that GGA can inhibit the toxic effects of METH on PC12 cells and mice liver and kidney, and this protective effect is achieved by co-induction of Trx-1 and Hsp70.
In summary, Trx-1 is closely related to methamphetamine-induced addiction, and thioredoxin inducers can resist methamphetamine addiction and methamphetamine toxicity.
【学位授予单位】：昆明理工大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.64
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