CCK-8 Inhibits The Foot Shock-primed Reinstatement Of Morphine-induced CPP Through The Upregulation Of Endogenous Opioid Peptides By CCK1 Receptor

Poppy flowers or fruit in medicine have analgesic effect, and the active ingredient is the opioids, which are widely used for clinic, such as in pethidine, morphine, fentanyl, and so on. However, the opioids have particularly significant side effects e.g. tolerance and addiction. The users can produce euphoric effects after the use of the drugs, and the euphoric effects gives rise to a strong craving that can’t be controlled and forces the users to use the drugs to alleviate the psychological needs periodically or continuously. Psychological dependence and relapse induced by opioids is the emphasis and difficulty of the opiate addiction research at present.Studies have found that the central nervous system may have a variety of "anti-endogenous opioid peptides" on the regulation of homeostasis after long-term use of opioids. And they believe non-opioid receptor system may be an important target for prevention and treatment of opiate addiction today. Cholecystokinin(CCK) is a brain-gut peptide that plays an important role in the regulation of varieties of functions. And it is in the form of a neurotransmitter or neuro-modulator and widely spread in the nervous system. Cholecystokinin octopeptide(CCK-8), is the main form of the anti-opioid peptides in the central nervous system. Our research group has found that chronic intervention which is intracerebroventricular administration of exogenous CCK-8 can significantly reduce the withdrawal symptoms of morphine-dependent rats. And it can significantly inhibit the conformation of chronic morphine-induced conditioned place preference and spiritual morphine-dependent processes. However, whether or not CCK-8 can prevent relapse, which can help achieve the effect to promote the "detoxification", is unclear.CPP model, an experimental model, has been widely used for rewarding effects of drugs and drug craving, drug addiction of memory research on rodent at present. In this study, through the establishment of morphine CPP model, to investigate the effects of different doses of exogenous CCK-8 on the foot shock-primed reinstatement of morphine-induced CPP and to explore its mechanism.Methods:1 Model of the foot shock-primed reinstatement of conditioned place preference(CPP) was established: The rats was divided into morphine group and saline group. The rats were trained 7 days by drug program partner/non-associated drug after subcutaneous injection of morphine(10 mg/kg)/NS(0.2 ml), twice a day, to establish an CPP experimental model of rats, and then naturally extinguished for 10 days. In order to observe whether conditioned place preference is disappear, the rats were tested every five days.During the reinstatement, each group were given 15 mins of foot shock(intensity:0.5m A, ecah time for 0.5s,at the interval of 40s), then test was conducted to observe whether the CPP effect reconstruction again.2 Effect of exogenous CCK-8 on the foot shock-primed reinstatement of morphine-induced CPP: All of the rats were trained after subcutaneous injection of morphine(10 mg/kg)during the conditioning period.During the expression, they were not injected any drugs. According to the CPP scores, the rats were randomly divided into three groups:Mor+saline group,Mor+0.1mg CCK-8 group, Mor+1mg CCK-8 gruop.The rats were injected CCK-8(0.1,1mg) in intracerebroventricular before reinstated CPP, in order to observe the effects of exogenous CCK-8 on the foot shock-primed reinstatement of morphine- induced CPP.3 Mechanism of CCK-8 inhibited the foot shock-primed reinstatement of morphine-induced CPP: The rats were divided into the Mor + CCK-8(1mg) + DMSO group,Mor+ CCK-8+ CCK1 receptor antagonist(L-364,718,10μg),Mor+ CCK-8+ CCK2 receptor antagonist(L-365,260,10μg), Mor+ CCK1 receptor antagonist L-364, 718 group and Mor+CCK2 receptor antagonist L-365, 260 group. 15 minutes before the test of reinstatement, the rats were co-administeredly injected with CCK-8 and CCK receptor antagonist(CCK1 receptor antagonist L-364, 718 and CCK2 receptor antagonist L-365, 260). Or L-364, 718 and L-365, 260 were given singly. Then the CPP test were complete after giving foot stock for 15 minutes.4 Effect of CCK-8 on threshold sensitivity to foot shock: The rats were divided into Mor+CCK-8(1μg)+saline group, Mor+CCK-8+naloxone(5μg) group,Mor+CCK-8+CTAP(10μg) group, Mor+naloxone group and Mor+CTAP group. 15 minutes before the reinstatement, the rats were co-administeredly injected with CCK-8 and the opioid receptor antagonist(non-selective opioid receptor antagonist naloxone, highly selective opioid receptor antagonist CTAP) in intracerebroventricula.Or the naloxone and CTAP were given singly. Then the CPP test were completed after15mins’ foot stock.5 Effect of CCK-8 on threshold sensitivity to foot shock:The rats were injected with NS or different doses of CCK-8. After 15 minutes, they were given foot shock. The initial current is 0.1 m A, and then increases by 0.05 m A every time, and the current value when the rat hind paw off the ground is its sensitive threshold.CPP score were represented as mean ± standard deviation(mean ± Sd), and the data were statistically analyzed by SPSS 16.0 statistical program. Each group mean were subjected to one-way analysis of variance( one-way ANOVA), followed by Bonferroni post hoc test for pairwise comparison. The statistics of establishing CPP reinstatement model were repeated measures design. When measuring between different phases(precondition, expression, extinction, reinstatement), the data were pairwise compared by Bonferroni post hoc test. Values of P<0.05 were considered to be statistically significant.Results:1 After the subcutaneous injection of morphine(10 mg/kg),the CPP test was conducted. CPP scores were significantly increased comparing with pre-conditioning period(P<0.01).Then naturally extinguished for 10 days, conditioned place preference disappeared, CPP scores significantly decreased(P<0.01),recovering back to pre-conditioning level. After 15 mins’ foot shock, conditioned place preference was reinstated.2 Comparing with the saline group, the CPP scores of differente does CCK-8 groups were significantly decreased(P<0.01).3 Comparing with Mor+CCK-8+DMSO group, the CPP scores of Mor+CCK-8+L-364,718 group were significantly decreased(P<0.01),while Mor+CCK-8+L-365,260 group showed no significant difference(P>0.05). However, when the CCK1 receptor antagonist was given singly,it has no significant effect on the foot shock-primed reinstatement(P>0.05).While when the CCK2 receptor antagonist was given singly,it can inhibited the foot shock-primed reinstatement of morphine-induced CPP(P<0.05).4 The CPP scores of the Mor+CCK-8+naloxone group and the Mor+CCK-8+CTAP group were significantly decreased compared with the Mor+CCK-8+Saline group(P<0.01).There were no significant difference among the Mor+naloxone group,the Mor+CTAP group and reinstatement level(P>0.05).5 There was no signicicant difference between each group of rats’ threshold sensitivity to foot shock(P>0.05).Conclusions:1 The model of CPP were successful established.2 CPP was successfully reinstated by foot shock, and 0.1 μg and 1 μg CCK-8 effectively inhibited the process.3 Exogenous CCK-8 inhibited the foot shock-primed reinstatement of morphine-induced CPP by CCK1 receptor and the endogenous CCK-8 promoted the foot shock-primed reinstatement of morphine-induced CPP by CCK2 receptor.4 The inhibiting effect of CCK-8 on the foot shock-primed reinstatement of morphine-induced CPP can be blocked by naloxone and CTAP. But there was no significant effect on the foot shock-primed reinstatement of morphine-induced CPP when naloxone and CTAP administered alone. So, the exogenous CCK-8 inhibited the foot shock-primed reinstatement of morphine-induced CPP related to endogenous opioid system.5 Threshold sensitivity of foot shock was not affected by CCK-8.CCK-8 can inhibit the foot shock-primed reinstatement of morphine-induced CPP through the upregulation of endogenous opioid peptides by CCK1 receptor.