Effects Of CCK-8on Arc/Arg3.1mRNA And Protein Expression In SH-SY5Y Cell With Treatment Of Chronic Morphine And Withdrawl

Drug addiction is a chronic, relapsing brain disease that results from theprolonged effects of drugs on the brain. Drug addiction characterized bycompulsive drug seeking and use can impact society’s overall health andsocial safe. The study of drug addiction is an important item in forensictoxicological. Opioids are a class of important representative drug of addiction,the repeated apioids exposure elicits changes in neuronal and synapticplasticity during the development of addiction, sharing the similarities with thesynaptic changes observe in physiological learning and memory. Thecontinuity and robustness of opioids addiction is due to forming ‘addictionmemory’ result from the changes in synaptic plasticity.Induction of immediate early genes （IEGs） is viewed as an important stepin the formation of long-lasting neuroadaptations underlying learning andmemory as well as the persistence of drugs. Activity-regulated cytoskeleton-associated protein （Arc/Arg3.1） is a unique IEG that may be induced byneuronal activity and specifically trafficked and localized to recentlypotentiated symapses, which is critical to synaptic plasticity. That may play animportant part during the form of ‘addiction memory’.Cholecystokinin octapeptide （CCK-8） is the most potent endogenousanti-opioid peptide, In food memory, passive avoidance response and activeavoidance reaction model experiments, CCK-8shows the romotion of memory,to prevent forgetting effect, and has obvious dose-effect relationship and timedependence. different CCK receptors for the memory effect is not consistent.More CCK1receptors focused forgotten, while CCK2receptor focusedmore on the memory facilitation. Our past research indicate：ExogenousCCK-8given intracerebroventricularly inhibited the acquisition of morphine induced CPP and significantly attenuated the reinstatement of CPP elicited bylow dose morphine, as well as blocked morphine induced locomotorsensitization. Above results reveal significant regulation of exogenous CCK-8on the process of psychological morphine dependence and relapse. Arc/Arg3.1is an important factor to study neural adaptation of learning and memory anddrug addiction mechanism, whether it is involved in the mechanism of CCK-8mediating opioid addiction is not clear.Objective: to further explore the interaction between CCK and opioidsystem from the aspects of learning and memory mechanism though observingthe effects of CCK-8on Arc/Arg3.1mRNA and protein expression inSH-SY5Y cell model of morphine dependence and withdrawal, for theapplication of prevention and control of the CCK-8in morphine dependentproviding experimental evidence.Methods: Make cAMP overshoot level as the evaluation index toestablish morphine dependent and withdrawal cell model. With100μ Mmorphine effecting on all-trans RA differentiating6d SH-SY5Y cells for48h,and then with10μM naloxone effecting15min for acute withdrawal toestablish morphine dependent and withdrawal cell model; With100μ Mmorphine effecting on all-trans RA differentiating6d SH-SY5Y cells for48h,then stopping the effects of morphine for6h to establish spontan-eouswithdrawal cell model. At the same time, observing the effects of differentconcentrations of CCK-8and selective CCK1/2receptor antago-nist onmorphine dependent, and detecting the expression of Arc/Arg3.1mRNA andprotein in different groups of cell models by RT-PCR and We-stern blottingtechnology.Data were presented as mean±standard deviation （x±s）, and analyzedwith one way ANOVA and significant difference test（LSD） by SPSS16.0statistical program. A level of P<0.05was considered stastically significant.Results:1With RA differentiating SH-SY5Y cells to establish morphine dependent andwithdrawal SH-SY5Y cell model. 1.1In the SH-SY5Y cells with the differentiation of RA for6d, dendriticprotrusions increased significantly, plasmogamy similar to ganglion struc-tureand a lot of nerve fiber structure is connected forming a network, consistentwith the reported literature. With the effects of100μM morphine on theSH-SY5Y cells for48h, the level of cAMP in cells increased significantly,then given10μM naloxone withdrawal for15min, further increased as the205.49±6.24times as the control group, forming a clear cAMP overshootstate, which indicated that morphine dependence and acute withdrawalSH-SY5Y cell model was established successfully.1.2In the SH-SY5Y cells with the differentiation of RA for6d, With theeffects of100μM morphine on the SH-SY5Y cells for48h, and then stop theeffects of morphine to observed the cAMP for48h. We found the cAMP levelincreased significantly at0h compared with0h, forming a distinct cAMPovershoot state, and then gradually decreased, but still remain at a high level,which indicated that morphine spontaneous withdrawal SH-SY5Y cell modelwas established successfully.2Effects of CCK-8on the expression of Arc/Arg3.1aloneThe10^-8and10^-10mol/L CCK-8can upregulated the Arc/Arg3.1mRNAexpression level and the10^-610^-12mol/L CCK-8upregulated the Arc/Arg3.1protein level, compared with the control group （con）（P<0.05）.3Effects of CCK-8and CCK receptor antagonist on the Arc/Arg3.1mRNAand protein expression in morphine dependence SH-SY5Y cell model3.1During morphine dependence process, Arc/Arg3.1mRNA and proteinlevels were increased significantly at22.5min point, compared with thecontrol group（con）（P<0.05）, then down, reaching the lowest point at90minpoint （P<0.05）, then began to recover to basal level at3h point, and then keptrising trend, reaching the peak at48h point （P<0.01）.3.2①10^-8and10^-10mol/L CCK-8can increase the Arc/Arg3.1mRNAexpression （P<0.001）;10^-610^-12mol/L CCK-8up-regulated Arc/Arg3.1prote-in expression （P<0.001）.②10^-6and10^-8mol/L CCK1receptor antagonist（L-364718） up-regulated Arc/Arg3.1mRNA and protein expression （P<0.001）. ③10^-6and10^-8mol/L CCK2receptor antagonist （LY-288513） down-regulatedArc/Arg3.1mRNA and protein expression （P<0.001）.4Effects of CCK-8and CCK receptor antagonist on the Arc/Arg3.1mRNAand protein expression in morphine acute withdrawal SH-SY5Y cell model4.1Naloxone withdrawal group （mor+nal） up-regulated Arc/Arg3.1mRNAand protein expression, compared with morphine dependent group （mor）（P<0.05）; naloxone up-regulated Arc/Arg3.1mRNA and protein expression,compared with the control group （con）（P <0.01）.4.210^-610^-12mol/L CCK-8down-regulated Arc/Arg3.1mRNA and proteinexpression （P<0.001）;②10^-810^-12mol/L increased Arc/Arg3.1mRNAexpression （P<0.001）,10^-610^-12mol/L CCK1receptor antagonist （L-364718）increased Arc/Arg3.1protein expression （P<0.001）;③10^-6and10^-8mol/LCCK2receptor antagonist （LY-288513） down-regulated Arc/Arg3.1mRNAand protein expression （P<0.001）5Effects of CCK-8and CCK receptor antagonist on the Arc/Arg3.1mRNAand protein expression in morphine spontaneous withdrawal SH-SY5Y cellmodel5.1Stop the effects of morphine to observed the Arc/Arg3.1expression for48h. we found the mRNA and protein levels declined sharply to reach thelowest point at3h, compared with the morphine dependent group （0h）（P<0.001）, then increased and reached the highest point at6h, compared withthe morphine dependent group （0h）（P<0.001）, and then almost down to thebasal level, then kept rising trend to48h （P<0.001）.5.2①10^-6and10^-8mol/L CCK-8down-regulated Arc/Arg3.1mRNA express-ion, while10^-10and10^-12mol/L upregulated the expression （P<0.001）;10^-610^-12mol/L CCK-8down-regulated Arc/Arg3.1protein expression（P<0.001）;②10^-6and10^-8mol/L CCK1receptor antagonist （L-364718）significantly inhibited Arc/Arg3.1mRNA expression （P<0.001）;10^-610^-12mol/L inhibited the expression of Arc/Arg3.1protein （P<0.001）;③10^-610^-12mol/L CCK2receptor antagonist （LY-288513） inhibited the expressi-on of Arc/Arg3.1mRNA and protein （P<0.001）. Conclusions：1CCK-8alone can enhance the expression of Arc/Arg3.1mRNA andprotein.2Chronic morphine dependence can up-regulated Arc/Arg3.1expression,while acute morphine effect can down-regulate the expression; During themorphine dependence, the different effects of CCK receptor antagonists on theexpression of Arc/Arg3.1may be associated with the different regulattedfunction in memory and morphine dependence; CCK-8may play a synergisticeffect with morphine, further increase the expression of Arc/Arg3.1.3During the withdrawal process, naloxone precipitated withdrawalupregulation the expression of Arc/Arg3.1, a synergistic effect of naloxoneand morphine may exist; CCK-8and CCK1receptor antagonistdown-regulated the expression of Arc/Arg3.1, which may affect thewithdrawal symptoms.4During the process of spontaneous withdrawal,CCK-8and CCKreceptor antagonists down-regulated Arc/Arg3.1expression. So CCK systemmay play an important role in the consolidation and retrieval of morphineaddiction memory. Arc/Arg3.1may be involved in morphine addictionmemory later consolidation process.