| Drug addiction is a serious global public health problem,which greatly damages human physical and mental health,increases medical and economic burdens,and affects social stability.Methamphetamine(METH)is an amphetamine-type central nervous system stimulant with a pure white crystal appearance,commonly known as "methamphetamine".In recent years,new synthetic drugs,represented by METH,are increasingly abused due to their low cost,quick onset and long duration of action.METH addiction is a chronic and recurrent brain disease caused by repeated administration of METH.Its main characteristics are compulsive drug seeking,strong craving and withdrawal symptoms after discontinuation of medication.Long-term use can lead to abnormal compensatory adaptation of neurons in the body,leading to symptoms such as tolerance,sensitization,dependence and relapse.In recent years,METH addiction has become a research hot spot for researchers all over the world,but the exact mechanism of METH addiction has not been clarified,and there is no effective treatment for METH addiction and relapse.In this study,METH induced behavioral sensitization was used to simulate the relapse process of METH,to explore its mechanism and therapeutic method.The details are mainly as follows:1.Model establishment of METH induced behavioral sensitization and the mechanism of it(1)Model establishment of METH induced behavioral sensitizationThe dosage of METH-induced behavioral sensitization was screened from 2mg/kg to 8 mg/kg,and the dosage of METH was determined to be 4 mg/kg and 8mg/kg according to the changes of locomotor activity of mice.After the dosage was determined,we established the model of behavioral sensitization and tested the contents of dopamine(DA),5-hydroxytryptamine(5-HT),glutamate(Glu)and Gamma-aminobutyric acid(GABA)in serum of METH-sensitized mice by ELISA.The results showed that the contents of DA,5-HT and GABA in serum of METH-induced behavioral sensitized mice were decreased significantly during the development period,recovered during the withdrawal period,and returned to decrease significantly after ignition during the expression period.The contents of Glu was increased significantly during the development period,recovered during the withdrawal period,and returned to increase significantly after ignition during the expression period.(2)Bioinformatics analysis of DEGs in the PFC of METH sensitized miceThe differential expressed gene(DEG)in prefrontal cortex(PFC)of mice of METH induced behavioral sensitization were detected by RNA-Seq,and were carried out GO and pathway analysis.We found that Fas existed in the GO function and pathway of DEG,so Fas and its upstream and downstream related genes were selected for further study.The candidate molecules at RNA and protein levels were validated by RT-PCR and Western blot respectively.RT-PCR results showed that the expression levels of Fas,MEK1 and CREB were decreased significantly during the development period,and were returned to increased during the withdrawal period,but were not return to normal level.In the expression phase,the expression levels of Fas,MEK1 and CREB were decreased significantly.Moreover,the expression levels of Erk1/2were increased significantly during the development period,decreased during withdrawal period,and returned to increased significantly after once METH injection.Western blot results showed that the expression levels of Fas,GIT1,MEK1,and p-CREB were significantly decreased during the development period,and were returned to increased during the withdrawal period,but were not return to normal level,and were significantly decreased in the expression phase.Additionally,the expression levels of p-Erk1/2 was significantly increased during the development period,was decreased during the withdrawal period,and was significantly increased during the expression period.(3)Mechanism of Fas and GIT1 mediating METH induced behavioral sensitizationIn order to further verify whether Fas and GIT1 regulate METH induced behavioral sensitization through the MEK1-Erk1/2-CREB signaling pathway,we up-regulated the expression of Fas and GIT1 in PFC of mice by lentivirus vector.We built the model of METH induced behavioral sensitization,tested the contents of DA,5-HT,Glu and GABA in serum of METH-sensitized mice by ELISA,measured the protein levels in MEK1-Erk1/2-CREB pathway by western blot,detected the morphological changes of brain tissues by Hematoxylin-eosin(HE)staining.The results showed that the over-expression of Fas and GIT1 reduced the locomotor activity of mice,inhibited the formation of METH induced behavioral sensitization,reversed the content changes of DA,5-HT,Glu and GABA effectively in serum,changed the protein expression of MEK1-Erk1/2-CREB pathway induced by METH,and reduced the morphological and pathological changes of brain tissue.2.Intervention of human umbilical cord mesenchymal stem cells on METH induced behavioral sensitizationTo investigate whether human umbilical cord mesenchymal stem cell(h UC-MSC)can interfere with METH induced behavioral sensitization,we injected h UC-MSCs into PFC of behavioral sensitized mice,tested the contents of neurotransmitters in serum of METH-sensitized mice by ELISA,measured the protein levels by western blot,detected the morphological changes of brain tissues by HE.The results showed that h UC-MSCs can significantly reduce the locomotor activity of mice;increase the content of DA,5-HT and GABA in serum,reduce the content of Glu;increase the protein expression of Fas,GIT1,BDNF and Bcl-2,reduce the expression of p-Erk1/2;reduce the morphological and pathological changes of brain tissue induced by METH effectively.These results indicate that it has a certain interventive effect of h UC-MSCs on METH induced behavioral sensitization.This study laid an experimental foundation for elucidating the mechanism of METH addiction,and also provided new targets for the treatment of METH addiction and relapse. |
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