核酶PARP-1激活通过核蛋白聚ADP-核糖基化修饰参与神经病理性痛的分子机制

Poly (ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme that is activated by DNA strand breaks and functionally linked to DNA repair via catalized neucleoprotein poly ADP-rebosylation. Emerging evidences reveal that PARP-1 activation, which activated by phospho-ERK directly, involves in the long-term memory formation in brain, and contributes to the development of inflammatory diseases via transcriptional regulation. The central sensitization during the generation of neuropathic pain share same mechanisms with the learning and memory in brain. However, the role of PARP-1 activation in the development and maintenance of neuropathic pain, and the associated-mechanism still remain largely unknown. Recently, our preliminary experiments found that PARP-1 was activated in lumbar 5 dorsal root ganglia (DRG) and spinal cord dorsal horn following lumber 5 spinal nerve ligation (L5 SNL) in rats. Intrathecal administration of Tiq-A , a specific PARP-1 inhibitor, alleviated the mechanical allodynia and thermal hyperalgesia induced by L5 SNL. To further verify the role and the related-mechanism of PARP-1 in neuropathic pain, the following experiments were designed in the present application. (1) The activation of PARP-1 in DRG and spinal cord dorsal horn following L5 SNL in rats. (2) The role of PARP-1 activation in the development and maintenance of L5 SNL induced-neuropathic pain. (3) The role of PARP-1 activation in C-fibre evoked long-term potentiation in rats’ spinal cord dorsal horn. (4) The signal pathways of L5 SNL triggered PARP-1 activation. (5) The molecular mechanism of PARP-1 mediated neuropathic pain. The results might provide a new therapeutic target to the treatment of chronic pain in clinical.

磷酸化ERK直接激活的核酶PARP-1，通过催化核蛋白和自身的聚ADP-核糖基化调控基因转录，参与脑长期记忆形成，并通过调节细胞因子表达参与介导炎性疾病。神经病理性痛中枢敏化形成过程与脑学习记忆具有相似或相同的机制，但PARP-1激活在神经损伤后病理性痛中的作用及机制仍不清楚。我们前期工作发现大鼠腰5脊神经结扎(L5 SNL)可引起PARP-1在背根神经节(DRG)和脊髓背角激活，鞘内注射PARP-1抑制剂可减轻L5 SNL大鼠痛觉过敏。为进一步证明PARP-1的作用及机制，本研究拟开展以下工作：①大鼠L5 SNL后PARP-1在DRG和脊髓的激活；②PARP-1在L5 SNL大鼠病理性痛形成和维持过程的作用；③PARP-1激活在脊髓背角C-纤维诱发电位长时程增强（神经病理性痛中枢敏化模型）中的作用； ④L5 SNL引起PARP-1激活的信号途径；⑤PARP-1激活介导病理性痛的分子机制。

有研究报道胶质细胞激活释放的细胞因子、趋化因子等致炎介质在神经病理性痛的发生和发展过程发挥重要作用。聚ADP-核糖基化酶-1(poly(ADP-ribose) polymerase-1, PARP-1)作为一种调控基因转录的核酶，通过调节致炎因子的表达在多种炎性疾病过程发挥重要作用。但迄今PARP-1在神经病理性痛中的作用及机制仍不清楚。本项目采用大鼠腰5脊神经结扎（SNL）制备神经病理性痛模型，经检测大鼠机械刺激撤足阈值（PWT）和热刺激撤足潜伏期（PWL）评价大鼠痛行为；用免疫荧光双染色、免疫印迹、免疫共沉淀(Co-IP)、染色质共沉淀（ChIP）、qPCR等分子生物学技术，结合鞘内注射PAPR-1抑制剂和PARP-1 siRNA系统探讨了PARP-1激活参与大鼠神经病理性痛的机制。结果显示：（1）SNL可引起PARP-1在大鼠背根神经节（DRG）和脊髓背角的上调和激活；在DRG，PARP-1表达于A和C类神经元，在脊髓，主要表达于背角浅层的神经元和星形胶质细胞。（2）鞘内微量注射PAPR 抑制剂PJ-34或PAPR-1特异性抑制剂Tiq-A能剂量依赖性地减轻SNL引起的大鼠触觉诱发痛和热刺激痛觉过敏；选择SNL 7天后给药，能使已经建立起的神经病理性痛症状得到缓解；实验还采用鞘内注射PARP-1 siRNA抑制PARP-1的合成，结果发现PARP-1 siRNA治疗组大鼠的PWT和PWL明显升高。（3）SNL可引起TNF-α在大鼠DRG和脊髓背角表达增加，但鞘内注射Tiq-A和PARP-1 siRNA都可明显抑制SNL引起的TNF-α表达变化。ChIP和Co-IP结果显示SNL通过激活PARP-1引起组蛋白H1的聚ADP-核糖基化，促进了组蛋白H1从TNF-α启动子区的释放，进而促进了NF-κB p65与TNF-α启动子区的结合，从而增强了TNF-α的转录表达。结论：脊神经损伤引起的PARP-1在DRG和脊髓背角的激活，通过促进p65在TNF-α启动子区结合而调控的TNF-α表达增加，参与了大鼠神经病理性痛的发生和维持。研究成果揭示了慢性痛过程TNF-α表达调控的新机制，PARP-1可作为防治慢性痛的一个极具潜力的药物研究新靶点。