The Molecular Mechanism Of Oprm1 Promoter Methylation Regulated By IL-6 In DRG After Peripheral Nerve Injury In Mice

Purpose:Neuropathic pain is a chronic pain disease caused by primary damage or dysfunction of the nervous system,which seriously affects the quality of life in patients.A variety of different injuries,such as trauma,nerve damage or infection,can cause neuropathic pain.Studies have shown that epigenetic mechanisms are involved in neuropathic pain,and the interaction between the nervous system and the immune system may be the key to chronic pain.After the nerve injury,the promoter region of Mu opioid receptor gene in DRG is regulated by DNA methylation modification,the expression of MOR is decreased,the target of opioid drug is reduced,and the analgesic effect of opioid on neuropathic pain mice is reduced.Several studies have shown that IL-6 may play a key role in the development of neuropathic pain.IL-6 can enhance the methylation of promoter regions of various tumor suppressor genes by up-regulating the expression of DNMT,and inhibit the expression of various tumor suppressor genes.Therefore,we propose the following hypothesis:peripheral nerve injury causes IL-6 expression up-regulation,and binds to IL-6R,activates IL-6 classical signaling pathway,up-regulates DNMT3a expression,and promote methylation of the promoter region of the Oprm1,reduce the expression of MOR and the analgesic effect of morphine.This study aimed to determine the relationship between endogenous IL-6 and MOR involved in neuroimmune interactions in the neuropathic pain model DRG after sciatic nerve injury.Methods:We used molecular biology assays to perform the following experiments in animals and cells:Part I:To observe whether changes in IL-6 and DNMT3a expression in DRG-induced DRG,changes in MOR expression in DRG neurons,and behavioral responses to pain;The IL-6 pathway was blocked and DNMT3a inhibitor was administered,respectively,and the changes in MOR expression and the analgesic effect of morphine in DRG neurons were observed.Part II:The chromosomal co-immunoprecipitation technique was used to detect the binding of DNMT3a to the MOR gene promoter,and the DNA methylation level of MOR gene promoter was detected by pyrosequencing to verify the hypothesis.Part III:Cellular levels were further validated against the putative IL-6/DNMT3a/MOR pathway described above.Part IV:Luciferase reporter assay In vitro,we verified whether MBD binds to the MOR gene promoter.Results:Our data show that chronic neuronal damage leads to a significant up-regulation of IL-6 and DNMT3a expression,which is associated with increased methylation of the MOR gene promoter and decreased expression of MOR protein in DRG.Inhibition of DNMT3a catalytic activity by 5-aza-dC significantly reduced the methylation level of the MOR promoter,up-regulated MOR expression and enhance the analgesic effect of morphine in mice with neuropathic pain,but there was no significant change in IL-6 expression.After blocking the IL-6 pathway,DNMT3a expression and methylation levels of the MOR promoter were reduced,MOR expression was increased and enhance the analgesic effect of morphine in mice with neuropathic pain.The CHIP and CO-IP technique was used to detect the binding of DNMT3a to the MOR gene promoter,confirming that IL-6-mediated DNMT3a regulates the methylation of the MOR gene promoter region.Cell experiments further validated the above result.Conclusion:Under conditions of continuous inflammatory challenge.IL-6 signaling induces and activates the DNA methylating enzyme DNMT3a,which is mounted to MOR promoter sites in the presence of MBD,leading to MOR methylation and Mu downregulation.These studies will not only promote our understanding of the epigenetic mechanisms of neuropathic pain,but will also open the door to new strategies for the prevention and treatment of this disease.