| BACKGROUNDIn recent years,there is a rapidly increasing trend of new-type synthetic drug abuse in China.The number of new-type synthetic drug abusers have already outgrown traditional drug abusers and shown bigger groups of abusers and younger-age trend.The 2014 China Drug Report shown that nearly 80%of the newly identified 480 thousands addicts were the abusers of new-type synthetic drug.Teenagers under the age of 35 accounted for 79.2%of all registered addicts and the average age of addicts was 28 year-old.The groups of drugs have spread from unemployees,farmers,individual householders and migrant workers to employees of enterprises,freelances,entertainers and even public officials.The addicts of new-type synthetic drug are susceptible to excitement,mania,depression and hallucination that easily lead to out of behaviors,traffic accidents,suicide,self-injury,hurting others and violence which damage seriously public safety and affect social stability.The new-type synthetic drug abuse is becoming a rapidly growing and more and more serious social problem.In terms of traditional drugs,the new-type drugs,also known as"laboratorial drugs" or "synthetic drugs",are became popular in the recent decades which are mainly psychiatric drugs of chemical synthesis.Amphetamines is a group of central nervous stimulants with similar chemical structure which includes amphetamine,methamphetamine(commonly known as "crystal meth"),3,4-methylene dioxymetham-phetamine(commonly known as "head wriggling pills")and other amphetamine-type stimulants(ATS).ATS are the most popular new-type synthetic drugs and abused more and more severely in recent years.ATS have strong central stimulation and easily lead to mental dependence with a long-time use which can damage multiple organs of the body,particularly the central nervous system of the brain.It would appear various mental problems such as depression,anxiety and lack of pleasure after withdrawal of ATS,ATS have strong psychological dependence,high relapse rate and neurotoxicity that cause easily damage of body and psychology,human immunodeficiency virus(HIV)infection and violence which produce serious negative impact on social security.Therefore,studying on ATS addiction is not only an important subject of prevention and treatment of drug dependence but also an impending medical and social mission.The neurobiological mechanisms of ATS addiction are complex and involve the interactions of multiple brain regions and neurotransmitter systems,there is substantial evidence showing that the mesolimbic dopamine system,endogeneous opioid system and y-aminobutyric acid(GABA)/glutamate neuronal system play key roles.Animal and human studies suggest that activation of the mesolimbic dopamine system is the neurological base of ATS addiction.The nature of drug addiction is a kind of pathological memory which is based on the changes of gene expression and neural synaptic plasticity induced by drugs.To date,the researchers have found a large number of differentially expressed genes and proteins and validated various candidate genes and functional proteins which to a certain extent indicated the biological mechanisms of ATS addiction.The complex regulatory mechanisms and mutual relations of multiple genes and various factors on ATS addiction are still unclear.In the human genome,there are a lot of non-coding DNAs,they used to be regarded as "junk DNA".With the deepening of the research,people gradually changed the opinion of "junk DNA",they found that these useless DNAs actually encoded the RNA molecules which played important regulatory roles,and microRNA(miRNA)was one of them.MiRNAs are an important class of endogenous non-coding small RNAs with about 18?25 nucleotides that mediate posttranscriptional negatively regulation of gene expression by targeting specific mRNA sequences to inhibit the translation of mRNAs or degrade the expression of mRNAs.MiRNAs have been found in mammalian nervous system and some of them are rich in the brain which play an important regulatory role on the development of central nervous system,differentiation of neurons and synaptic plasticity.MiRNA is the regulatory factor of synaptic plasticity,synaptic function and shape modification and is related to higher cognitive functions,such as learning and memory.Since Pietrzykowski etc.reported firstly the regulation of miRNAs in drug addiction in 2008,there is an increased rapidly studies of miRNAs in drug addiction.To date,there are many studies of miRNAs on cocaine,alcohol,nicotine and opioid addiction,but fewer studies on ATS addiction.Since Lipp etc.reported that miRNAs in several brain areas changed when exposed to amphetamine in 2011.Saba etc.reported that amphetamine up-regulated miR-181a expression in the hippocampus,nucleus accumbens and ventral midbrain of mice,particularly in the hippocampus and stimulation of dopamine signaling induced miR-181a expression in hippocampus neurons.ATS can induce the change in the expression of miRNAs in addiction-related brain regions which directly involve in the regulation of ATS-induced addictive behaviors.Therefore,to study the regulatory role of miRNAs in ATS-induced addiction has important implications for dependent mechanisms of new-type synthetic drugs and the discovery of the new targets of drug actions.Nowadays,there are mainly opioid agonists(methadone,buprenorphine,dihydroetorphine,etc.),non-opioid agonists(clonidine,lofexidine,etc.)and naltrexone for the treatment of drug addiction.Although opioid agonists can eliminate rapidly early-stage addiction,they are easy to produce dependence by themselves and become drug substitutes.Non-opioid agonists are mainly to relieve symptoms of addicts and have obvious adverse reactions and are easy to cause serious withdrawal symptoms after discontinuation.Naltrexone is mainly used to the prevention of relapse,but can not relieve withdrawal symptoms.Importantly,all of these drugs are mainly used to the treatment of traditional drugs such as opioid and heroin.To date,there has not yet a particularly effective drugs or replacement therapy drugs like methadone for the treatment of new-type synthetic drugs such methamphetamine.Traditional Chinese medicine(TCM)has accumulated rich theoretical knowledge and valuable clinical experience in drug addiction and has a variety of treatments and rich resources of herbal medicines.TCM can strengthen the body resistance and drive out evil spirits;regulate the whole body,resolve the current problems and eliminate the root causes and has low toxicity and dose not produce dependence.The effects of multiple targets,parts and systems in TCM may be more earlier to solve the complex neural mechanisms of drug addiction in some ways than the action of single target and system in western medicine.Therefore,TCM for the treatment of drug addiction has been paid more and more attention by the world.Uncaria rhynchophylla(Miq.)Jacks(Gouteng in Chinese)is a commonly used agent in clinical TCM compounds for the treatment of drug addiction.Rhynchophylline is the major component of Gouteng which accounts for 28%?50%of its total alkaloids.Substantial experimental evidence indicates that rhynchophylline possesses many beneficial effects such as anti-hypertensive,anti-addictive,anti-anxiety,anti-arrhythmic,anticonvulsant,sedative and neuroprotective effects in various models.Rhynchophylline has attracted considerable interest due to its potent effects on the central nervous system such as acting as a noncompetitive antagonist of the N-methyl-D-aspartic acid(NMDA)receptor and a calcium channel blocker.Our previous studies shown that rhynchophylline could reverse amphetamine/methamphetamine-induced conditioned place preference(CPP)effect in rats,mice and zebrafish and did not produce a CPP effect in itself.In addition,rhynchophylline can slow the heart rate,lower the blood pressure,lower the temperature,cross the blood-brain barrier easily and rapidly and has little side effects.Based on the theories of genomics and proteomics and the miRNA chip technology,we measured the miRNAs expression profile in the rat hippocampus of methamphetamine dependence and screened,identified and validated the key miRNA and its target gene which would provide new available biological information for new-type synthetic drugs addiction.Additionally,we investigated the effects of rhynchophylline on the expression of miRNAs in the rat hippocampus of methamphetamine dependence and explored the new action target and molecular mechanism of rhynchophylline.OBJECTIVES1.To establish methamphetamine-induced CPP model and investigate the effects of rhynchophylline.To screen out differential miRNAs in the hippocampus of rat by the miRNA chip technology.2.To validate the differential miRNA by Real-time PCR,predict the target gene by bioinformatics methods and validate the expression of target gene by western blot and immunohistochemistry(We found that miR-181a-5p and its target gene GABAA receptor ?1-subunit(GABRA1)involved in methamphetamine-induced CPP effect in rats and the effects of rhynchophylline).3.To inhibit/over-express miR-181 a-5p by stereotaxic injection of antagomir/agomir in the rat hippocampus.To investigate the effects of inhibition/over-expression miR-181a-5p on methamphetamine-induced CPP and the effects of rhynchophylline.4.To investigate the effects of methamphetamine on the expression of miR-181a-5p and GABRA1 in PC 12 cells and the effects of rhynchophylline.5.To inhibit/over-express miR-181a-5p by transfection of antagomir/agomir in PC 12 cells.To investigate the effects of inhibition or over-expression of miR-181a-5p in PC 12 cells exposed to methamphetamine and the effects of rhynchophylline.METHODS1.To establish methamphetamine-induced CPP model in ratsAfter measurement of baseline,at 8 a.m.,the rats received methamphetamine(2 mg·kg-1,s.c.)in methamphetamine-paired group or the same volume of sterile physiological saline in saline-paired group,and were immediately confined to the white compartment for 1 h.At 4 p.m.,the rats received the same volume of sterile physiological saline in all groups and were immediately confined to the black compartment for 1 h.The rats received methamphetamine and were trained for 4 days.At 8 p.m.on the next day after administration of methamphetamine,the rats received respectively rhynchophylline(60 mg·kg-1)and MK-801(dizocilpine,0.1 mg·kg-1)in the treatment groups for 3 days.The place preference of rats were measured at 24 h after the last administration of methamphetamine.2.To measure the miRNAs expression profile by the miRNA chip technology.To screen out differential miRNAs in the hippocampus of rat by the miRNA chip technology.This part was finished by Guangzhou RiboBio Company.3.To predict the target gene of miR-181a-5pTo predict and analyze the target gene of miR-181a-5p by TargetScan,miRDB and miRanda softwares.4.Stereotaxic injection of miR-181a-5p antagomir/agomir in the rat hippocampusAfter installing stereotaxic instrument and automatic injector,rats were euthanized by anesthesia,shaved off the hair in the brains and exposed skins.Rats were bilaterally infused with 5 ?L antagomir Negative Control/miR-181 a-5p antagomir/agomir Negative Control/miR-181 a-5p agomir using the following coordinates:4 mm posterior to Bregma,2 mm lateral the medial suture,3 mm ventral to the skull surface(hippocampus injection).All injections were performed according to the rat stereotaxic coordinates(Paxinos and Watson,1998;Shu and Bao,1991).5.PC 12 cells culturePC 12 cells were cultivated in RPMI 1640 medium supplemented with 10%heat-inactivated horse serum and 5%fetal bovine serum in a humidified atmosphere of 95%air and 5%CO2 at 37?.Renewing the medium every 2 days and subculturing in the cell density of 70%.6.PC 12 cells exposured to methamphetamine and rhynchophyllineSeeding 1×105 cells into 12-well plates and incubation of cells with methamphetamine and rhynchophylline in the cell density of 50%.After 48 h,the total RNA and protein were extracted in PC 12 cells.7.Transfection of miR-181a-5p antagomir/agomir in PC12 cellsSeeding 1×105 cells into 12-well plates and incubation of cells with miR-181a-5p antagomir/antagomir Negative Control/miR-181a-5p agomir/agomir Negative Control in the cell density of 50%.The cells were continued to cultivate for 24 h.8.Exposure to methamphetamine and rhynchophylline after transfection24 h after transfection,the cells were incubated with methamphetamine and rhynchophylline for 48 h.The total RNA and protein were extracted in PC 12 cells.9.To measure the expression of miR-181a-5pThe expression of miR-181a-5p was measured by Real-time PCR in the rat hippocampus and PC 12 cells.10.To determine GABRA1 protein expressionThe expression of GABRA1 was determined by western blot in the rat hippocampus and PC 12 cells.The expression of GABRA1 was also determined by immunohistochemistry in the rat hippocampus.11.Statistical analysisAll statistical analysis was carried out using the SPSS 13.0 Statistical software package,and all data were presented as means±S.D.If the variances between groups were homogenous,groups were subjected to the multiple comparisons least significant differences(LSD)test.In case of no homogeneity variances,differences were evaluated by Welch and the groups were subjected to the multiple comparisons Dunnett's T3 test.Statistical significance was achieved when P<0.05.RESULTS1.The time spent and total movement distances of rats in methamphetamine-paired compartmentThere were no significant differences in the time spent and total movement distances of rats in methamphetamine-paired compartment among all groups before training(P>0.05).Methamphetamine significantly increased the time spent and total movement distances of rats in the methamphetamine-paired compartment compared with that of the control rats(P<0.01).Therefore,the rat model of methamphetamine-induced CPP was successfully constructed.Compared with those in the model group,the time spent and total movement distances of rats in the methamphetamine-paired compartment were significant decreased in the rhynchophylline and MK-801 groups(P<0.01).2.MiRNAs expression profile in the rat hippocampusCompared to the control group,57 miRNAs changed significantly in the rat hippocampus of the model group.Compared to the model group,68 miRNAs changed significantly in the rat hippocampus of the rhynchophylline group and 86 miRNAs changed significantly in the rat hippocampus of MK-801 group.11 miRNAs changed significantly in the rat hippocampus among the 4 groups.Through the literature,we chose miR-181a-5p that was closely related ATS addiction for further study.3.The expression of miR-181a-5p in the rat hippocampusCompared with those in the control group,the expression of miR-181a-5p in the rat hippocampus was significantly increased in the model group(P<0.01).Compared with those in the model group,the expression of miR-181a-5p in the rat hippocampus was significantly decreased in the rhynchophylline and MK-801 groups(P<0.01).4.The target gene of miR-181a-5p1581 candidate genes were predicted by TargetScan,miRDB and miRanda.61 candidate genes were screened by Wayne chart.Through the literature,we chose GABRA1 that was closely related ATS addiction for further study.There were several conserved binding sites of miR-181a-5p in the 3'UTR region of GABRA1.5.The expression of GABRA1 in the rat hippocampusCompared with those in the control group,the expression of GABRA1 protein in the rat hippocampus was significantly decreased in the model group(P<0.01).Compared with those in the model group,the expression of GABRA1 protein in the rat hippocampus was significantly increased in the rhynchophylline group(P<0.01);there was no significant difference in the expression of GABRA1 protein in the rat hippocampus in the MK-801 group(P>0.05).6.The expression of miR-181a-5p in the rat hippocampus after stereotaxic injection of antagomir/agomirCompared with those in the antagomir Negative Control group,the expression of miR-181a-5p in the rat hippocampus was significantly decreased in the miR-181a-5p antagomir group(P<0.01).Compared with those in the agomir Negative Control group,the expression of miR-181a-5p in the rat hippocampus was significantly increased in the miR-181a-5p agomir group(P<0.01).Therefore,the expression of miR-181a-5p was inhibited/over-expressed successfully in the rat hippocampus.7.The effects of miR-181a-5p inhibition/over-expression on methamphetamine-induced CPP and the effects of rhynchophylline in rats(1)The time spent and total movement distances of rats in methamphetamine-paired compartmentThere were no significant differences in the time spent and total movement distances of rats in methamphetamine-paired compartment among all groups before training(P>0.05).Methamphetamine significantly increased the time spent and total movement distances of rats in the methamphetamine-paired compartment compared with that of the antagomir Negative Control rats(P<0.01).Therefore,the model of methamphetamine-induced CPP in rats after stereotaxic injection of antagomir was successfully constructed.Compared with those in the antagomir Negative Control+methamphetamine group,the time spent and total movement distances of rats in the methamphetamine-paired compartment were significant decreased in the miR-181a-5p antagomir+methamphetamine and miR-181a-5p antagomir+methamphetamine+rhynchophylline groups(P<0.01).Compared with those in the miR-181a-5p antagomir+methamphetamine group,there were no obvious differences in the time spent and total movement distances of rats in the methamphetamine-paired compartment in the miR-181 a-5p antagomir+methamphetamine+rhynchophylline group(P>0.05).Methamphetamine significantly increased the time spent and total movement distances of rats in the methamphetamine-paired compartment compared with that of the agomir Negative Control rats(P<0.01).Therefore,the model of methamphetamine-induced CPP in rats after stereotaxic injection of agomir was successfully constructed.Compared with those in the agomir Negative Control+methamphetamine group,there were no significant differences in the time spent and total movement distances of rats in the miR-181a-5p agomir+methamphetamine group(P>0.05).Compared with those in the agomir Negative Control+methamphetamine and miR-181a-5p agomir+methamphetamine groups,the time spent and total movement distances of rats in the methamphetamine-paired compartment were significant decreased in the miR-181a-5p antagomir+methamphetamine+rhynchophylline group(P<0.01).(2)The expression of miR-181 a-5p in the rat hippocampusCompared with those in the antagomir Negative Control group,the expression of miR-181a-5p in the rat hippocampus was significantly increased in the antagomir Negative Control+methamphetamine group(P<0.01).Compared with those in the antagomir Negative Control+methamphetamine group,the expression of miR-181a-5p in the rat hippocampus was significantly decreased in the miR-181a-5p antagomir+methamphetamine and miR-181a-5p antagomir+methamphetamine+rhynchophylline groups(P<0.01).Compared with those in the miR-181a-5p antagomir+methamphetamine group,there was no significant difference in the expression of miR-181a-5p in the rat hippocampus in the miR-181a-5p antagomir+methamphetamine+rhynchophylline group(P>0.05).Compared with those in the agomir Negative Control group,the expression of miR-181a-5p in the rat hippocampus was significantly increased in the agomir Negative Control+methamphetamine group(P<0.01).Compared with those in the agomir Negative Control+methamphetamine group,the expression of miR-181a-5p in the rat hippocampus was significantly increased in the miR-181a-5p agomir+methamphetamine group(P<0.01).Compared with those in the agomir Negative Control+methamphetamine and miR-181a-5p agomir+methamphetamine groups,the expression of miR-181a-5p in the rat hippocampus was significantly decreased in the miR-181a-5p agomir+methamphetamine+rhynchophylline group(P<0.01).(3)The expression of GABRA1 in the rat hippocampusCompared with those in the antagomir Negative Control group,the expression of GABRA1 in the rat hippocampus was significantly decreased in the antagomir Negative Control+methamphetamine group(P<0.01).Compared with those in the antagomir Negative Control+methamphetamine group,the expression of GABRA1 in the rat hippocampus was significantly increased in the miR-181a-5p antagomir+methamphetamine and miR-181a-5p antagomir+methamphetamine+rhynchophylline groups(P<0.01).Compared with those in the miR-181a-5p antagomir+methamphetamine group,there was no significant difference in the expression of GABRA1 in the rat hippocampus in the miR-181a-5p antagomir+methamphetamine+rhynchophylline group(P>0.05).Compared with those in the agomir Negative Control group,the expression of GABRA1 in the rat hippocampus was significantly decreased in the agomir Negative Control+methamphetamine group(P<0.01).Compared with those in the agomir Negative Control+methamphetamine group,the expression of GABRA1 in the rat hippocampus was significantly decreased in the miR-181a-5p agomir+methamphetamine group(P<0.01).Compared with those in the agomir Negative Control+methamphetamine and miR-181a-5p agomir+methamphetamine groups,the expression of GABRA1 in the rat hippocampus was significantly increased in the miR-181a-5p agomir+methamphetamine+rhynchophylline group(P<0.01).8.Effects of methamphetamine on the expression of miR-181a-5p and GABRA1 in PC12 cellsCompared with those in the control group,the expression of miR-181a-5p was significantly increased and the expression of GABRA1 was significantly decreased in the methamphetamine group in PC 12 cells(P<0.01);there was no significant difference in the expression of miR-181a-5p and GABRA1 in the rhynchophylline group in PC 12 cells(P>0.05).Compared with those in the methamphetamine group,the expression of miR-181a-5p was significantly decreased and the expression of GABRA1 was significantly increased in the rhynchophylline+methamphetamine group in PC12 cells(P<0.01);the expression of miR-181a-5p was significantly decreased(P<0.01)but there was no obvious difference in the expression of GABRA1(P>0.05)in the MK-801+methamphetamine group in PC12 cells.9.The expression of miR-181a-5p after transfection of antagomir/agomirCompared with those in the antagomir Negative Control group,the expression of miR-181a-5p in PC 12 cells was significantly decreased in the miR-181a-5p antagomir group(P<0.01).Compared with those in the agomir Negative Control group,the expression of miR-181a-5p in PC12 cells was significantly increased in the miR-181 a-5p agomir groups(P<0.01).Therefore,the expression of miR-181a-5p was inhibited/over-expressed successfully in PC12 cells.10.Effects of miR-181a-5p inhibition/over-expression on PC 12 cells exposed to methamphetamine and the effects of rhynchophylline(1)The expression of miR-181a-5p in PC 12 cellsCompared with those in the antagomir Negative Control group,the expression of miR-181a-5p in PC 12 cells was significantly increased in the antagomir Negative Control+methamphetamine group(P<0.01).Compared with those in the antagomir Negative Control+methamphetamine group,the expression of miR-181a-5p in PC 12 cells was significantly decreased in the miR-181 a-5p antagomir+methamphetamine and miR-181a-5p antagomir+rhynchophylline+methamphetamine groups(P<0.05).Compared with those in the miR-181a-5p antagomir+methamphetamine group,there was no significant difference in the expression of miR-181a-5p in PC 12 cells in the miR-181a-5p antagomir+rhynchophylline+methamphetamine group(P>0.05).Compared with those in the agomir Negative Control group,the expression of miR-181a-5p in PC 12 cells was significantly increased in the agomir Negative Control+methamphetamine group(P<0.01).Compared with those in the agomir Negative Control+methamphetamine group,the expression of miR-181a-5p in PC 12 cells was significantly increased in the miR-181a-5p agomir+methamphetamine group(P<0.01).Compared with those in the agomir Negative Control+methamphetamine and miR-181a-5p agomir+methamphetamine groups,the expression of miR-181a-5p in PC 12 cells was significantly decreased in the miR-181a-5p agomir+rhynchophylline+methamphetamine group(P<0.01).(2)The expression of GABRA1 in PC 12 cellsCompared with those in the antagomir Negative Control group,the expression of GABRA1 in PC12 cells was significantly decreased in the antagomir Negative Control+methamphetamine group(P<0.01).Compared with those in the antagomir Negative Control+methamphetamine group,the expression of GABRA1 in PC 12 cells was significantly increased in the miR-181a-5p antagomir+methamphetamine and miR-181a-5p antagomir+rhynchophylline+methamphetamine groups(P<0.05).Compared with those in the miR-181a-5p antagomir+methamphetamine group,there was no significant difference in the expression of GABRA1 in PC12 cells in the miR-181a-5p antagomir+rhynchophylline+methamphetamine group(P>0.05).Compared with those in the agomir Negative Control group,the expression of GABRA1 in PC12 cells was significantly decreased in the agomir Negative Control+methamphetamine group(P<0.01).Compared with those in the agomir Negative Control+methamphetamine group,the expression of GABRA1 in PC 12 cells was significantly decreased in the miR-181a-5p agomir+methamphetamine group(P<0.01).Compared with those in the agomir Negative Control+methamphetamine and miR-181a-5p agomir+methamphetamine groups,the expression of GABRA1 in PC 12 cells was significantly increased in the miR-181a-5p agomir+rhynchophylline+methamphetamine group(P<0.05).CONCLUSION1.Compared to the control group,57 miRNAs changed significantly in the rat hippocampus of methamphetamine-induced CPP.Compared to the methamphetamine group,68 miRNAs changed significantly in the rat hippocampus of the rhynchophylline treatment group.The expression of miR-181a-5p was remarkably increased in the rat hippocampus of methamphetamine-induced CPP and rhynchophylline treatment reduced significantly the expression of miR-181a-5p.2.MiR-181a-5p in the rat hippocampus regulated methamphetamine-induced CPP by targeting GABRA1 which was also found in PC12 cells exposed to methamphetamine.3.MiR-181a-5p by targeting GABRA1 involved in the inhibitive effects of rhynchophylline on methamphetamine-induced CPP and the protective effects of rhynchophylline on PC 12 cells exposed to methamphetamine. |
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