| Objective: There are three opioid receptor subtypes, which termed as μ,δ and κ opioid receptor. Κ-opioid receptor modulates a variety ofphysiological processes via binding to dynorphins, such as analgesia,neuroendocrine, emotional behavior, cognition, as well as the development ofmorphine dependence and the negative affect after withdrawal. Most offindings verified that dynorphins and κ–opioid receptor play an important rolein morphine dependence and withdrawal by inhibiting the function ofmesocortico limbic dopaminergic system.Cholecystokinin octapeptide, acts as a potent anti-opioid peptide in the CNS,was associated with the processes of pain, cognition, reward, learning andmemory. There is evidence that CCK-8can reverse the analgesia whichinduced by κ–opioid receptors. However, the interaction between CCK-8andκ–opioid receptor system during the morphine dependence and withdrawal isstill unclear.We established a cellular model of morphine dependence and withdrawal byutilizing PC12cell line. The study aimed to investigate the exact effect ofCCK-8on cellular morphine dependence and withdrawal, as well as the roleof PDYN/κ–opioidreceptor system for clear the mechanism of CCK-8modulation on morphine dependence and withdrawal.Methods:1We established the PC12cellular model of morphine dependence with100μM morphine treatment for48h, and10μM naloxone was used forprecipitating morphine withdrawal. We detected the cAMP level within thecells with the time-resolved fluoroisnmunoassay and identified the success ofthe cellular model with the cAMP overshoot as the index. The cells wereco-treated with100μM morphine and CCK-8(10-12-10-6M) for48h, and then naloxone was added for precipitation of morphine withdrawal to observe theinfluence of CCK-8on the morphine withdrawal process. The cells weretreated with CCK-8alone for48h to observe the individual effect of CCK-8on cAMP level.2We applied the Real-Time PCR technique, to observe the change ofmRNA level in κ-opioid receptors, prodynorphin (PDYN) and CCK1andCCK2receptors in PC12cells of the morphine withdrawal model; and also, toobserve the influence of different dosages of CCK-8on the expression ofκ-opioid receptors and prodynorphin mRNA after morphine withdrawal, toinvestigate whether CCK-8participates in the morphine withdrawal processwith the adjustment of dynorphin/κ-receptor system.Results:1.48h after treating PC12cells with morphine (100μM), we treated thecells with the naloxone (10μM) for15min. We observed that the cAMPcontent in the PC12cells was increased to3.98±0.26pM, evidently differentfrom the control group (2.75±0.37pM) and the morphine dependence group(3.16±0.10pM), indicating that and a cAMP overshoot was induced and thewithdrawal cellular model of morphine dependence had been built.248h after treating PC12cells with morphine (100μM) and15min afterthe precipitated withdrawal with naloxone (10μM), we observed that themRNA level of the k-receptor, PDYN and CCK1and CCK2receptors hasbeen distinctly upregulated, showing statistical significance compared with thecontrol group.348hours after treating PC12cells with different concentrations ofCCK-8(10-12-10-6M) and morphine (100μM), we treated the cells withnaloxone (10μM) for15min, inducing a cAMP overshoot. Results show thatCCK-8(10-7and10-6M) can evidently inhibit the cAMP overshoot whichinduced by the precipitated withdrawal of naloxone after the treatment of thechronic morphine, showing statistical significance compared with themorphine withdrawal group; while CCK-8(10-12,10-10and10-8M) has noevident influence on the cAMP content. It has no evident influence on cAMP with the single treatment by different concentrations of CCK-8(10-12-10-6M)for48h and with or without the treatment of naloxone.4It can evidently decrease the expression level of mRNA of κ-receptorsand PDYN with the treatment of PC12cells by CCK-8(10-7M,10-6M) andmorphine, showing statistical significance compared with the morphinedependence group. CCK-8at the concentration of10-12,10-10and10-8M has noevident influence on the expression of mRNA of κ-receptors and PDYN. It candecrease the mRNA level of κ-receptors and PDYN with the single treatmentof PC12cells by CCK-8(10-7,10-6M) for48h, while CCK-8at theconcentration of10-12,10-10and10-8M has no evident influence on themRNA level of κ-receptors and PDYN.548h after treating the PC12cells with the CCK-8(10-7M,10-6M) andmorphine, we treated the cells with naloxone (10μM) for15min. We observedthat CCK-8could evidently inhibit the increase of the mRNA level ofκ-receptors and PDYN after the morphine withdrawal, showing statisticalsignificance compared with the morphine withdrawal group. CCK-8at theconcentration of10-12,10-10and10-8M has no evident influence on themRNA expression of κ-receptors and PDYN.Conclusion:1We have successfully built the cellular models of morphine dependence and withdrawal in the present experiment.2On the cellular models of morphine dependence and withdrawal in thepresent experiment. CCK-8can evidently inhibit the morphine dependenceand withdrawal, with evident dose-effect relationship.3The mRNA expression of k-opiate receptor and prodynorphin incr easesafter the morphine dependence and withdrawal and CCK-8can decrease themRNA expression of κ-opiate receptor and prodynorphin dose-depen dently,indicating that CCK-8may inhibit the morphine withdrawal symptoms viainhibiting the expression of k-opiate receptors and prodynorphin. |
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