The Role Of SCF/c-kit Signaling Pathways In Opioid Dependence And Carving

Objective:Drug addiction is a chronic,relapsing brain disorder characterized by compulsive drug seeking and loss of control over drug use.It affects the development of the social and the health of the individual.Opioid is more common,and it produces a serious harm.There is now considerable evidence that drugs of abuse converge on a common circuitry in the mesolimbic dopamine system.Drugs of abuse produce a strong reward effect and long-term alterations in neural function.Drug abusers often display a strong physical dependence and psychological dependence.Relapse can be caused by exposure to induction factors(environmental cues,drugs or stress)associated with drug use even after long periods of withdrawal.Relapse is a major clinical problem.Reactivation of a previously consolidated memory returns the memory to reconsolidatin,an additional labile phase,the result of which is that it can be modified,disrupted or even erased.In fact,recent studies indicate that disrupting the reconsolidation of drug-related memories will diminish drug addiction,the mechanisms are not clear.c-kit receptor has been observed in thecentral nervous system,including cerebral cortex,hippocampus,nucleus accumbens,amygdala,low brain stem,cerebellum and other parts of the brain.c-kit is an important member of type III receptor tyrosine kinase family.SCF is a ligand of the c-kit receptor,it is the earliest hematopoietic stem cell factor.SCF/c-kit signaling pathway plays an important role in the neural plasticity of the maintenance of normal neurological and nerve injury repair.Previous studies have suggested that SCF/c-kit signaling pathway plays an important role in the maintenance of normal neurological function and neural plasticity of nerve injury repair.SCF and c-kit were activated by phosphorylation,and then scf/c-kit activates a variety of downstream signal transduction pathways,including PI3K/Akt,Ras/ERK,PLC-y/PKC signal pathways and so on.PLC-y/PKC pathway activates intracellular protein,PI3K/Akt pathway is closely related to the protein synthesis of cells,Ras/ERK activates the expression and regulation of the promoter gene.In the previous studies,our groups and other laboratory have found that PI3K/Akt/mTOR,Ras/ERK,PLC-gamma/PKC signaling pathways in the nucleus accumbens and amygdale played a different role in drug reward and memory,and can suppress either signal molecular pathways to interfere with the memoryreconsolidation,making stable morphine,cocaine,alcohol’s reward memory disappear,so as to achieve the effect of eliminating psychological desire to prevent relapse.c-kit is numerous common activated receptor of the downstream signal transduction molecules,and has a expressionin the related neural circuits of drug addiction,whereas SCF/c-kit signaling pathways in the role of drug addiction are not reported.Thus,we speculate that SCF/c-kit signaling pathway is likely to play an important role in the process of drug addiction,c-kit could be the important molecular basis to interve with drug reward memory to prevent relapse.Molecules and behavior method are adopted in this paper,to determine the SCF/c-kit signaling pathways in the role of opioid dependence psychological craving,which confirmed the feasibility that c-kit receptor molecule might be the molecular target in opioid drugs addiction,and their inhibitors imatinib compounds can be potential new treatment towards the drug addictionMethods:Our study consists of two parts of the experiment to confirm the mechanism of SCF/c-kit signaling pathway in opioid addiction formation and memory reconsolidation,including:Part 1:The molecular mechanism of SCF/c-kit signaling pathway in opioid addiction1)The activation of c-kit receptors in the central nervous system was examined by immunohistochemistry after acute morphine administration;2)Western blot was used to detect the inhibitory effect of c-kit inhibitor imatinib mesylate on morphine-induced central addiction-related brain regions;3)Immunofluorescence double labeling co-localization detection of acute morphine-activated c-kit distribution in nucleus accumbences;4)Immunohistochemistry was used to detect the change of c-kit receptors and downstream activation of Akt,ERK,PKC in nucleus accumbences;5)Immunofluorescence double labeling was used to detect the co-distribution of c-kit receptor activation and downstream Akt,ERK and PKC in the brain cells of nucleus accumbences after acute morphine-activated c-kit receptor;6)Using conditioned place preference and sensitization model to detect the role of c-kit in addictionformation;7)In order to confirm that the SCF/c-kit signaling pathway is opioid-specific,Western blot is used to detect the activity of the c-kit in natural reward induced by sucrose;Part 2:the mechanism of SCF/c-kit signaling pathway in reconsolidation of opioid addiction1)Western blot combined with behavioral method was used to detect the activation of c-kit receptors in the re-activation of opioid reward memory;2)Systematic injection of c-kit receptor inhibitor imatinib mesylate,to observe its impact on opioid reward memory reconsolidation,and to determine the potential feasibility of imatinib as a detoxification drug;3)In order to verify the systematic administration of imatinib compounds to interfer reward memory was not caused by its movement ability has been suppressed,our study use the same dose of drugs to test its effect on spontaneous activity;4)Finally,cardiovascular side effects were monitored by heart rate and blood pressure monitoring.Results:Through molecular and behavioral experiments,the mechanism of SCF/c-kit signaling pathway in opioid addiction is as follows:Part 1:The molecular mechanism of SCF/c-kit signaling pathway in opioid addiction1)Immunohistochemistry:After acute morphine administration,activation of c-kit receptors in the central nervous system was enhanced in nucleus accumbens,and there was statistically significant difference;There was no statistically significant difference in the change in prefrontal cortex,hippocampus,amygdala,and the ventral tegmental;There was no significant difference in the activity of hypothalamic arcuate nucleus in food related brain regions;2)Before and after morphine administration,c-kit inhibitor imatinib mesylate was injected intraperitoneally.Western blot quantitative detection:after imatinib mesylate administration,the Akt activity induced by morphine in nucleus accumbens was significantly decreased than morphine group,no obvious changes in other brain regions;3)Immunofluorescence double labeling revealed that acute morphine administration activated c-kit in neurons of the two sub regions of the nucleus accumbens,core and shell.4)Immunohistochemical:morphine activated the c-kit receptor,and downstream Akt,ERK,PKC activation in the nucleus accumbens core and shell in different degree.There was statistically significant differences:Akt is mainly activated in the nucleus accumbens core,ERK is mainly activated in the nucleus accumbens core and shell,PKCzeta is mainly activated in the nucleus accumbens core;5)Immunofluorescence:acute morphine activated c-kit receptors and downstream signaling molecules corresponding colocalized in the same nerve cells.The activition of c-kit receptors is consistent with the activation of PI3K/Akt/mTOR,Ras/ERK,PLC-y/PKC signaling pathway;6)Behavioral experiment:after administration of imatinib mesylate in rats,the preference was not formed;Similarly,the sensitization was not formed.7)In order to confirm the activation of SCF/c-kit signaling pathway is opioid-specific,Western blot detection of c-kit activation after administrated sucrose,there was no significant statistical differences in addiction related brain region.Part 2:the mechanism of SCF/c-kit signaling pathway in reconsolidation of opioid addiction1)Western blot and behavior test showed that the activity of c-kit receptor in opioid reward memory reactivation was higher than that in the control group,and the difference was statistically significant;2)Systemic administration of the c-kit receptor inhibitor imatinib mesylate,the formed conditioned preferences disappeared;3)Systemic administration of the same dose of imatinib compounds has no apparent effect on locomotor activity in rats;4)Heart rate and blood pressure monitoring:the side effects of imatinib mesylate on blood pressure was not significant.Conclusion:Through the above experiments,our study draws the following conclusions:1)SCF/c-kit signaling pathway play an important role in the formation of opioid addiction.Acute morphine administration were activated c-kit receptor in the nucleus accumbens,which is related to drug addiction and reward memory,regulating downstream PI3K/Akt/mTOR,Ras/ERK,PLC-y/PKC signaling pathway,through activating effect molecules,increasing protein synthesis,promote gene expression and regulation in the nucleus accumbens core and shell reverse changes of neural plasticity,promoting the formation of drug reward memory.2)Based on reconsolidation,the test of part 2 indicated that the important role of SCF/c-kit signaling pathway in drug reward memory.Using imatinib compounds can eliminate the memory permanently,the effectiveness and the cardiovascular adverse effects were confirmed.Above all,SCF/c-kit receptor may serve as a new molecular for drug reward memory intervention,imatinib compounds has a potential treatment for drug addiction.In short,the results of our study expands the neurobiological mechanism of drug addiction,provide a new molecular basis for opioid addiction treatment.