| Drugs are a major social scourge. The global spread of drug abuse is against social progress and development. The abuse of morphine, heroin and other opioid drugs and other narcotic drugs, psychotropic substances keep continued growth. Especially, teenagers'drug users accounted for the sharp rise in the proportion of the total number. Drug abuse leads to infinite harm.Opiate addiction is a chronic recurrent encephalopathy, because the associated neurons have their adaptability to the body to form a state of addiction after a long-term drug abuse. This process of adaptation and behavior are associated with time and regions in the brain. Therefore, the current researches focus on the role of specific key brain regions in morphine addiction, for example, prefrontal cortex (PFC), hippocampus (Hip), etc. These regions involve in the formation of phychological dependence through the contact of the fibers with the DA system in medium brain. Many substances join in this addiction process. Cholecystokinin (CCK) is a neuropeptide found in many systems. There is a broad distribution the central nervous system, with a variety of physiological functions. CCK-8 is the most potent endo- genous anti-opioid peptides. Cell adaptations induced by opioid abuse include reduced opioid receptor, internalization, uncoupling from G proteins, cyclic adenosine monophosphate signal (cAMP) upregulation and high sensitivity. The rapid regulation of opioid receptors and its down-stream signaling play important roles in opioid dependence. It is well known that CCK functions through its receptors. CCK receptor has two subtypes, CCK1 and CCK2, and in the brain, mainly function through CCK2 receptor. They may participate in pain, anxiety and other emotion regulation and memory processes. Reseach suggests that CCK receptor antagonists can alleviate the naloxone percipitated withdrawl syndrome, also can prevent the relapse of opioid abuse. But its mechanism is not clear. The morphine dependent model was established by subcutaneous injection of morphine in gradually increasing doses. The withdrawal syndrome was precipitated with naloxone. CCK-8, CCK1 and CCK2 receptor antagonists were applied to observe effects of CCK on morphine withdrawal symptoms by i.p. and i.c.v. And then, Bmax and Kd ofμ-opioid receptor were tested in cortex, caudate putamen and hippocampus by radioligand binding assay. The study is to explore the relationship of CCK andμ-opioid receptor and provide new clues for CCK-8 application in drug treatment.Method: Male Wistar rats (200±10g) were used to establish the morphine withdrawal model. The morphine dependent model in rats was establishshed by subcutaneous injection of morphine. The daily dose gradually increased as following: 10, 20, 30, 40, 50mg·kg-1, twice a day, at 8:00 and 20:00, for 5 days. On day 6, 50mg·kg-1 morphine was injected at 8:00. Withdrawal syndromes was precipitated by intraperitoneal injection of naloxone (5mg·kg-1). Bmax and Kd ofμ-opioid receptor were tested in cortex, caudate putamen and hippocampus by radioligand binding assay.To observe effects of CCK on the withdrawl syndrome, CCK-8 (50μg·kg-1, i.p., 0.1μg/rat, i.c.v), L-364,718(1mg·kg-1, i.p., 1μg/rat, i.c.v), LY-288,513(1mg·kg-1, i.p., 1μg/rat, i.c.v) were used 30min before morphine treatment. Moreover, CCK-8 (100μg·kg-1, i.p.; 1μg/rat, i.c.v), L-364,718 (5mg·kg-1, i.p., 10μg/rat, i.c.v), LY-288,513 (5mg·kg-1, i.p., 10μg/rat, i.c.v) were used 30min before naloxone (5mg·kg-1, i.p.) treatment.Data were presented as x±s and analyzed by one way ANOVA and least significant difference test with SPSS 11.5 statistical program. A level of P<0.05 was considered statistically significant.Result:1 Radioligand binding assay optimization for detectingμ-opioid receptor in hippocampus The result was excellent when the protein concentration was 1mg·ml-1, [3H]DAMGO concentrations were among 0.5 nmol·L-18 nmol·L-1 and unlabled DAMGO concentration was 5μmol·L-1, 4℃overnight. The protocal is sparing, reliable and duplicate.2 Effects of CCK-8 and its receptor antagonists on morphine withdrawal symptomsWithdrawal symptoms apppered, such as teeth chattering, wet dog shakes, diarrhea, tears, salivation and jumping, body weight loss and other symptoms, suggesting that the models were successful. We found that chronic CCK-8 and its receptor antagonists treatment could attenuate morphine withdrawal symptoms by i.p. and i.c.v. Acute CCK-8 receptor antagonists treatment also decreased withdrawal symptoms. However, acute treatment of CCK-8 by i.c.v increased withdrawal symptoms, while no effect by i.p.3 Effects of CCK-8 and its receptor antagonists onμ-opioid receptor in cortex, caudate putamen and hippocampus in morphine withdrawal ratsWe detected the change ofμ-opioid receptor in morphine withdrawal rat, and found that chronic morphine treatment decreased Bmax and increased Kd ofμ-opioid receptor in PFC and Hip, but no effect on the Bmax ofμ-opioid receptor in CPu, only increased the Kd. Naloxone could markedly upregulated decreased Bmax, and downregulated increased Kd ofμ-opioid receptor in PFC and CPu, but no effect on Bmax ofμ-opioid receptor in Hip, only decreased Kd.Also, we found that chronic CCK-8, L-364,718 and LY-288,513 treatment could not change Bmax and Kd ofμ-opioid receptor in PFC, and also Bmax of CPu, Kd of Hip in the naloxone-precipitated withdrawal rats. However, they could effectively escalate Kd ofμ-opioid receptor in CPu and Bmax of μ-opioid receptor in Hip of the naloxone-precipitated with- drawal rats.Furthermore, acute CCK-8, L-364,718 and LY-288,513 treatment could not change Bmax and Kd ofμ-opioid receptor in PFC, and Bmax of CPu, Kd of Hip. But L-364,718 and LY-288,513 could effectively escalate Kd ofμ-opioid receptor in CPu and Bmax ofμ-opioid receptor in Hip. Particularly, administration of CCK by i.c.v. could decrease Kd ofμ-opioid receptor in CPu and Bmax ofμ-opioid receptor in Hip, though it had no effect on it by i.p.Conclusion:1 We estalished a safe, sparing and credible experi- mental technique for detectingμ-opioid receptor in brain of rats.2 CCK-8 and CCK receptor antagonists could alleviate morphine withdrawal symptoms by regulatingμ-opioid receptor, with significant specificity in different brain regions. |
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