Promoter Demethylation-induced Upregulation Of CXCR4 Expression Contributes To Inflammatory Hyperalgesia In Rats

BackgroundChronic pain associated with inflammation is a common clinicalproblem,and the underlying mechanisms have only begun to beunraveled.DNA methylation has been implicated in the pathogenesis of chronic pain.However,the specific genes regulated by DNA methylation under inflammatory pain condition remain largely unknown.ObjectiveIn this study,we investigated the role that chemokine receptor CXCR4 have played in inflammatory pain model in rats induced by complete Freund's adjuvant(CFA),and also from the aspects of epigenetics to discover the methylation status of CXCR4 gene promoter CpG island area,then to investigate CXCL12/CXCR4 signaling pathways in genes,molecules,and the overall level to explore the effects and mechanism of CXCL12/CXCR4 signaling pathway in chronic inflammatory pain.Method1.Establishment of rat model of inflammatory pain induced by complete Freund's adjuvant(CFA).2.The behavioral test in rat was applied to determine the extents of mechanical allodynia and thermal hyperalgesia.3.The expression of CXCR4 cell type was dectected by immunofluorescence staining.4.The levels of CXCR4 protein in DRGs were detected by Western blot method5.The expression of CXCLl2 and CXCR4 mRNA was detected by Real-time PCR method6.The methods of methylation specific PCR(MSP)and bisulfite sequencing PCR(BSP)were used to detect the promoter methylation of CXCR4 gene7.The method of chromatin immunoprecipitation assay(ChIP)was used to detect the binding of NF-?Bp65 with the promoter region of CXCR4 gene in CpG.ResultsThe results showed that intraplantar injection of CFA could induce significant hyperalgesia in rats,also can significantly increase expression of CXCR4 mRNA and protein inthe dorsal root ganglion(DRG)and the expression of CXCL12 was also significantly up-regulated.Intrathecal injection of CXCR4 antagonist AMD3100 can significantly relieve hyperalgesia in inflammatory rats,and the effect is time and dose dependent.Further analysis by methylation specific PCR and bisulfite sequence PCR showed that CFA injection led to a significant demethylation of CpG island in CXCR4 gene promoter.Meanwhile,the expression of DNMT3 b was significantly down regulated after CFA injection.There were three binding sites of p65 in the CpG island of CXCR4 gene promoterby online software prediction.The chromatin immunoprecipitation assay confirmed that there were three p65 binding sites,a significant increase in binding activity of p65 with CXCR4 genepromoter in CFA-induced inflammatory rats compared to normal rats.ConclusionOur findings suggest that upregulation of CXCR4 expression was mediated by the enhanced binding capability of p65 with demethylated promoter of CXCR4 gene,thus contributing to inflammatory hyperalgesia.These findings provide a theoretical basis for the treatment of chronic pain from an epigenetic perspective.