CCK-8与内源性阿片系统在吗啡依赖中的相互作用

Interaction of CCK-8 and endogenous opioid system in opioid dependence

目的通过观察不同剂量的CCK-8对吗啡依赖SH-SY5Y细胞模型内源性阿片系统的影响,初步探讨CCK-8与内源性阿片系统在吗啡依赖过程的相互作用及其机制。方法建立吗啡慢性依赖SH-SY5Y细胞模型,应用放射配基结合技术观察μ阿片受体(MOR)结合特征,应用Real-Time PCR技术检测前脑啡肽原(PENK)、前阿黑皮质素原(POMC)基因的表达,并观察CCK-8对上述指标的影响。结果①10μmol·L-1吗啡作用于全反式维甲酸(RA)分化6d的SH-SY5Y细胞48h,成功建立了慢性吗啡依赖模型;②CCK-8可剂量依赖性地抑制MOR的结合,并且此抑制作用可被CCK1及CCK2受体拮抗剂(L-364,718,LY-288,513)翻转;③10-7、10-6mol·L-1CCK-8使PENK、POMC的表达较正常组分别增加(5.81±0.84)、(7.26±1.55)倍和(4.55±0.73)、(5.16±0.82)倍。吗啡依赖后,PENK、POMC表达较正常组下降(0.43±0.10)倍和(0.37±0.07)倍,10-8、10-7、10-6mol·L-1CCK-8使下调的PENK、POMC表达增加,PENK与正常组的相对表达值为(0.63±0.10)、(0.86±0.21)、(1.17±0.19),POMC与正常组的相对表达值为(0.46±0.10)、(0.60±0.11)、(0.96±0.11)。10-10、10-9mol·L-1CCK-8对PENK、POMC表达无影响。结论低剂量(10-10、10-9mol·L-1)CCK-8可通过激活CCK受体抑制MOR的结合力,对PENK、POMC的表达无影响;高剂量(10-8～10-6mol·L-1)的CCK-8可使PENK、POMC基因表达增加,促进内源性阿片肽的生成,并可改善吗啡依赖后内源性阿片系统的失衡。

Aim To explore the interaction of CCK-8 and endogenous opioid system in the development of opioid dependence and its mechanism by observing the effects of different doses of CCK-8 on endogenous opioid system of morphine dependent SH-SY5Y cell.Methods The SH-SY5Y cell model of chronic mor-phine dependence was established.The binding characteristic of μ opioid receptor （MOR） and expression of PENK and POMC gene were studied by radio-ligand binding assay and Real-Time PCR,respectively.Also,the effects of CCK-8 on above factors were investigated.Results ① The cell model of chronic morphine dependence was successfully established by 10 μmol· L-1 morphine incubation for 48 h on 6 days RA differentiated SH-SY5Y cells.② 10-10～10-6 mol·L-1 CCK-8 suppressed,dose-dependently,the binding activity of MOR,and this effect could be reversed by CCK1 and CCK2 receptor antagonist.③ 10-7 and 10-6 mol· L-1 CCK-8 increased the expressions of PENK and POMC in SH-SY5Y cells to a level of （5.81±0.84）,（7.26±1.55） and （4.55±0.73）,（5.16±0.82） times that of control group.The expressions of PENK and POMC were down-regulated to a level of （0.43 ±0.10） and（0.37 ±0.07） times that of control group by 10 μmol·L-1 morphine incubation for 48 h,and 10-8,10-7 and 10-6 mol·L-1 CCK-8 up-regulated the decreased PENK and POMC expression after morphine dependence,the relative expression of PENK was（0.63±0.10）,（0.86±0.21） and（1.17±0.19） vs control group,and that of POMC was （0.46±0.10） 、（0.60±0.11） 、（0.96±0.11） vs control group.In addition,10-10 and 10-9 mol·L-1 CCK-8 produced no effect on PENK and POMC expression.Conclusion 10-10,10-9 mol·L-1 CCK-8 could suppress μ opioid receptor by activating CCK receptor,but had no effect on PENK,POMC gene expression.10-8～10-6 mol·L-1 CCK-8 could improve endogenous opioid imbalance through increasing endogenous opioid peptide release.