| The opioids are natural alkaloids and derivative extracted from poppy, including morphine, codeine, heroin, pethidine, etc. Although the opioids can be used in the therapeutics of analgesia, cardiac asthma, antidiarrheal and antitussive clinically, overall given the fact that opioids have multiple and severe adverse reactions, especially the tolerance and dependence after repeated applications, not only are their clinical application limited to a great extent, but also cause severe commitment to crime and the widespread of hepatitis, tuberculosis and AIDS, which brings great harm to individuals, families and society. Therefore, to probe the mechanism of the opioids addiction and to seek the effective drugs and target become the urgent priorities.Clinical symptoms of opioid addiction are mainly physical and mental dependency, physical dependence refers to a state that the body has adapted to the long-term use of the drug of dependence, including dependence and the symptoms after withdrawal. Psychological dependence is a sense of satisfaction oreuphoria produced by the drugs and drives the users to continue the drugs mentally, thus leading to a strong craving and compulsive drug use behaviors. Physical dependence may use methadone detoxification treatment, but psychological dependence still has no effective treatment, which caused high relapse rate, thus psychological dependence is a key reason for drug abuse as well as the core issue of the mechanism of opiate addiction research.The physical dependence is mainly related to locus coeruleus, however physical dependence is primarily associated with the limbic system, including prefrontal cortex, midbrain ventral tegmental area, the nucleus accumbens, the arcuate nucleus, amygdala hippocampus and so on. The hippocampus involved in storing and reading process of environmental cues, hippocampal dysfunction is closely related to the process of drug addiction, thus it became our focus.Relapse refers to addicted patients desire to feel the effects of the drug and regain the drugs stimulated by some related factors after withdrawing for a period of time. Relapse is an important feature of drug addiction and the most important issue of treatment of addiction. Generally speaking, relapse after detoxification involved in the following three main factors, contact with addictive drugs again, exposed to the environment or clus accompanied by drugs again and stressful events, in which exposure to the environment is a major factor associated with relapse.Drug addiction is characterized by forced medication of reckless, this anomalous behavior occurs gradually after repeated administration, and continued after withdrawal for a long time, studies have shown that repeated drug use can alter gene or protein expression levels or types in related brain areas, these changes affect not only the function of neurons, but also the relevant neural circuits, eventually lead to drug addiction, so we use proteomics to analyze the changed proteins in dorsal hippocampus after heroin self-administration and then choose some differential proteins to study whether microinjection of associated protein inhibitor in dorsal hippocampus would affect the drug-seeking behavior of rats by self-administration of heroin.Purpose:To analyze the changed proteins-in dorsal hippocampus after heroin addiction by proteomics. To study whether microinjection of Hsp70 or ROCK inhibitor in dorsal hippocampus would affect the drug-seeking behavior of rats by self-administration of heroin.Methods:1)rat self-administration(FR1):delivered the rats into two groups randomly, saline and heroin. The experiment lasted for 12 days,3h/d, FR=1. In the first 6 days, the dosage was 100ug/kg/infusion, whereas 50ug/kg/infusion for the rest 6 days. The experiment was stopped when reached 3h or infusion for 30 times, then put the animals back to their cages. Rats were sacrificed 24h after training for the dorsal hippocampus, which was used for proteomics and western blot analysis.2)rat self-administration(PR):dl-3,an FR1 was applied(one response for one infusion) for training,3h/d;d4-6, an FR2 was used(two response for one infusion) for training,3h/d; from d7, the program was switched to FR5,3h/d, a PR was started until the intake of heroin achieved stability (fluctuations in the frequency of infusion of three days in a row< 10%). When PR was stable(fluctuations in the frequency of infusion of three days in a row<twice) for 6 continuous days,6h/d, all the animals entered the withdrawal. Micro injection of solvent and PES/Y27632 in dorsal hippocampus was before PR. After the final training, took the brain for Nissl staining to exclude the error data produced by the incorrect injection point.Results:There are 421 differential proteins in 3959 proteins identified from proteomics, in which 291 is known. These differential proteins are closely related to energy metabolism, oxidative stress, protein degradation and modification, synaptic function and signal transduction, and other important life processes. The expression of Hsp70 and Rho B within dorsal hippocampus was significantly increased after heroin addiction according to western blot verification. Microinjection of corresponding solvent or PES in dorsal hippocampus 15min before program PR, two-way ANOVA shows F1.85=4.8, P<0.05,indicates that the break point of PES group was significantly lower than the solvent group. Similarly, microinjection of corresponding solvent or PES in dorsal hippocampus 15min before testing environment-induced relapse, the result shows that the solvent group and PES group share no significant difference in the active holes. Microinjection of corresponding solvent or Y-27632 in dorsal hippocampus 30min before program PR, there is no significant difference between the Break Point of the two groups. Similarly, microinjection of corresponding solvent or Y-27632 in dorsal hippocampus 30min before testing environment-induced relapse, the result shows the active holes of Y-27632 were significantly lower than the solvent group.Conclusion:1) Heroin self-administration in rats can cause 421 protein expression changes in dorsal hippocampus, involving 18 signaling pathway, and closely related to energy metabolism, oxidative stress, protein degradation and modification, synaptic function and signal transduction, and other important life processes.2) Heroin addiction induced Hsp70 and Rho B expression in rat dorsal hippocampus.3) Inhibition of Hsp70 in the dorsal hippocampus can significantly reduce drug-seeking motives in rats, but does not affect the environment-induced relapse.4) Inhibition of Rho B pathway in the dorsal hippocampus has no significant effect on drug-seeking motives in rats, but can significantly reduce the environmental-induced relapse. |
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