Effects Of Jitai On Opioid-induced Drug-seeking Behavior & Relapse And Research Of DNA Methylation On Heroin Addiction In Dorsal Hippocampus

The opioids are alkaloids extracted from opium and their derivatives, including morphine, codeine, heroin, pethidine, etc. Although the opioids can be used in the therapeutics of analgesia, cardiac asthma, diarrhea and tussis clinically, given the fact that opioids have strong dependence and tolerance, not only are their clinical application limited, but also cause severe commitment to crime and the spread of AIDS, which brings great harm to individuals, families and society. Therefore, to probe the mechanism on addiction of the opiods and to seek the effective drugs have become the urgent priorities.Using the traditional Chinese medicine to cure drug addiction in clinical has had a long history. The clinical study shows that the traditional Chinese medicine has a good therapeutical effect on addiction. But the complexity of their components and the uncertainty of active ingredients, as well as the imperfection of evaluations on their therapeutical effects have limited their applications on the treatment of drug addiction. Jitai have been used on drug addiction for many years, but the preclinical pharmacology study is incomplete. What's more, there hasn't been any report on its therapeutical effect on psychological dependence, which is more severe in drug addiction. Therefore, this study evaluated the therapeutical effect of Jitai on opiods-induced drug seeking and reinstatement by the using rat self-administration and conditioned place preference of mice in the first section.It is possible to provide new target of developing effective therapeutical drugs by studying the addiction mechanism. As recently pointed out, epigenetics not only plays an important role in various physiological and pathological changes, but also closely related with drug addiction process. Hippocampus is broadly viewed as important in contextual conditioning, namely in the processing of contextual cues by which memories can be accessed and retrieved. It is reported that hippocampus induced declarative memory participated in the drug addiction process. However, there is very little report on whether the epigenetics changes in hippocampus participate in the drug addiction process and its mechanism. Therefore, in the second section of this paper, we studied whether the changes of DNA methylation in dorsal hippocampus would affect the drug-seeking behavior of rats by using self-administration of heroin, and took further observation on the changes of the content of the dorsal hippocampus DNA methyltransferase after heroin addiction.PartⅠEffects of Jitai on opioid-induced drug-seeking behavior and relapsePurpose: To study the effects of Jitai, a traditional Chinese medicine, on opioid-induced drug-seeking behavior and relapse by using rat self-administration and conditioned place preference of mice.Methods: 1) Rat self-administration: delivered the rats into three groups randomly, saline group, Jitai of low dosage group and Jitai of high dosage group, and saline, Jitai at the dose of 2.16g/kg or 3.6g/kg was administrated through intragastric way 1 hour before behavior experiment. 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 stop when reached 3h or infusion for 25 times, then put the animals back to their home cages. 2) Conditioned place preference of mice: delivered the mice into four groups randomly, saline group, morphine+saline group, morphine+Jitai of low dosage group and morphine+Jitai of high dosage group. Made the mice to extinct after modeling, then entered the treatment period. The saline group and saline+morphine group were administrated with saline through intragastric way, once a day for 3 days. The morphine+Jitai of low dosage group and morphine+Jitai of high dosage group were administrated with Jitai 0.6g/kg or 4.3g/kg, once a day for 3 days. Test 1h after the last administration. Put the mice into the drug-paired chamber after subcutaneous injections of saline (saline group) or morphine 4mg/kg (morphine group), let them explore the two chambers freely for 15min and record the time each mouse stayed in the black and white chamber.Results: 1) Rat self-administration: when the rats were administrated of Jitai through intragastric way at the dose of 2.16g/kg 1h before the experiment, their self-infusions in the 7-12th days displayed a decreasing tendency compared with the saline group. The heroin consumption also had a decreasing tendency. When the dosage of Jitai increased to 3.6g/kg, the two items have a significant decrease compared with the saline group. 2) Conditioned place preference of mice: when the mice extinct after modeling, administrating of Jitai at the dose of 4.3g/kg through intragastric way for 3d could inhibit morphine-induced reinstatement of CPP.Conclusion: 1) Jitai has a dose-dependent effect on inhibiting the heroin-induced drug-seeking behavior, and Jitai at the dose of 3.6g/kg has a significant inhibition effect. 2) Jitai at the dosage of 4.3g/kg can inhibit morphine-induced reinstatement of CPP in mice.PartⅡResearch of DNA methylation on heroin addiction in dorsal hippocampusPurpose: To study the whether the change of DNA methylation in dorsal hippocampus affect the drug-seeking behavior of rats by using self-administration of heroin, and to further observe the change of the content of the dorsal hippocampus DNA methyltransferase after heroin addiction.Methods: To deliver the rats involved into two groups, vehicle group and 5-Aza-dC group, injected into two sides of dorsal hippocampus with vehicle (0.107‰acetic acid) or 200ng/ul 5-Aza-dC. 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 stop when reached 3h or infusion for 25 times, then put the animals back to their home cages. After the final training, took the brain for nissl staining to exclude the error data produced by the incorrect injection point. The rats using for determine the content of DNMT were only performed the intravenous surgery, but not intracranial surgery or micro-injection. Rats were delivered randomly into saline group and heroin group before the behavior experiment and the training methods are as before. Take the dorsal hippocampus and extract the protein for western blot analysis 24h after the final training.Results: The self-infusions of 5-Aza-dC group were almost as the same as that of vehicle group in the first 1-6 days but increased significantly in the 9-12th days. The average number of self-infusions of 5-Aza-dC group from the 7-12th days were significant increased compared with the vehicle group, which increased from 14±2 to 18±3. The heroin consumption during the 12 days in each rat of 5-Aza-dC group was 3.3±0.5mg, which was much higher than the vehicle group (2.9±0.6mg). The western blot analysis of the protein level of DNMT before and after heroin addiction of rats showed that after 12 days self-administration training, DNMT1 and DNMT3b had no significant change in dorsal hippocampus, but the amount of DNMT3a was significantly decreased compared with saline group.Conclusion: 1) Inhibiting the activity of DNMT in dorsal hippocampus could enhance the heroin-induced drug-seeking behavior in rats. 2) Heroin addiction involved the content change of DNMT, the protein level of DNMT3a of heroin addicted rats was significantly lower than saline group in dorsal hippocampus.