The lysine specific demethylase 6B (KDM6B) has been implicated as a coregulator in the expression of proinflammatory mediators, and in the pathogenesis of inflammatory and arthritic pain. However, the role of KDM6B in neuropathic pain has yet to be studied. In the current study, the neuropathic pain was determined by assessing the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) following lumbar 5 spinal nerve ligation (SNL) in male rats. Immunohistochemistry, Western blotting, qRT-PCR, and chromatin immunoprecipitation (ChIP)-PCR assays were performed to investigate the underlying mechanisms. Our results showed that SNL led to a significant increase in KDM6B mRNA and protein in the ipsilateral L4/5 dorsal root ganglia (DRG) and spinal dorsal horn; and this increase correlated a markedly reduction in the level of H3K27me3 methylation in the same tissue. Double immunofluorescence staining revealed that the KDM6B expressed in myelinated A- and unmyelinated C-fibers in the DRG; and located in neuronal cells, astrocytes, and microglia in the dorsal horn. Behavioral data showed that SNL-induced mechanical allodynia and thermal hyperalgesia were impaired by the treatment of prior to i.t. injection of GSK-J4, a specific inhibitor of KDM6B, or KDM6B siRNA. Both microinjection of AAV2-EGFP-KDM6B shRNA in the lumbar 5 dorsal horn and sciatic nerve, separately, alleviated the neuropathic pain following SNL. The established neuropathic pain was also partially attenuated by repeat i.t. injections of GSK-J4 or KDM6B siRNA, started on day 7 after SNL. SNL also resulted in a remarkable increased expression of interleukin-6 (IL-6) in the DRG and dorsal horn. But this increase was dramatically inhibited by i.t. injection of GSK-J4 and KDM6B siRNA; and suppressed by prior to microinjection of AAV2-EGFP-KDM6B shRNA in the dorsal horn and sciatic nerve. Results of ChIP-PCR assay showed that SNL-induced enhanced binding of STAT3 with IL-6 promoter was inhibited by prior to i.t. injection of GSK-J4. Meanwhile, the level of H3K27me3 methylation was also decreased by the treatment. Together, our results indicate that SNL-induced upregulation of KDM6B via demethylating H3K27me3 facilitates the binding of STAT3 with IL-6 promoter, and subsequently mediated-increase in the expression of IL-6 in the DRG and dorsal horn contributes to the development and maintenance of neuropathic pain. Targeting KDM6B might a promising therapeutic strategy to treatment of chronic pain.

赖氨酸特异性脱甲基酶 6B (KDM6B) 被认为是促炎介质表达以及炎症和关节炎疼痛发病机制中的共同调节因子。然而，KDM6B 在神经性疼痛中的作用还有待研究。在目前的研究中，通过评估雄性大鼠腰椎第 5 脊神经结扎 (SNL) 后的缩爪阈值 (PWT) 和缩爪潜伏期 (PWL) 来确定神经性疼痛。进行免疫组织化学、蛋白质印迹、qRT-PCR 和染色质免疫沉淀 (ChIP)-PCR 测定以研究潜在机制。我们的研究结果表明，SNL 导致同侧 L4/5 背根神经节 (DRG) 和脊髓背角中 KDM6B mRNA 和蛋白质显着增加；这种增加与同一组织中 H3K27me3 甲基化水平的显着降低相关。双免疫荧光染色显示 KDM6B 在 DRG 的有髓 A 和无髓 C 纤维中表达；位于背角的神经元细胞、星形胶质细胞和小胶质细胞中。行为数据显示，在注射 GSK-J4（KDM6B 的特异性抑制剂或 KDM6B siRNA）之前，SNL 诱导的机械异常性疼痛和热痛觉过敏受到损害。AAV2-EGFP-KDM6B shRNA 分别在腰椎 5 背角和坐骨神经显微注射，减轻了 SNL 后的神经性疼痛。从 SNL 后第 7 天开始，通过重复注射 GSK-J4 或 KDM6B siRNA 也部分减轻了既定的神经性疼痛。SNL 还导致 DRG 和背角中白细胞介素 6 (IL-6) 的表达显着增加。但这种增加被注射 GSK-J4 和 KDM6B siRNA 显着抑制。并通过在背角和坐骨神经中显微注射 AAV2-EGFP-KDM6B shRNA 之前进行抑制。ChIP-PCR 测定结果表明，在注射 GSK-J4 之前，SNL 诱导的 STAT3 与 IL-6 启动子的增强结合受到抑制。同时，H3K27me3甲基化水平也因处理而降低。总之，我们的结果表明，SNL 通过去甲基化 H3K27me3 诱导的 KDM6B 上调促进了 STAT3 与 IL-6 启动子的结合，随后介导的 DRG 和背角中 IL-6 表达的增加有助于发育和维持的神经性疼痛。