电针镇痛对P2X4受体介导的小胶质细胞活化及脊髓背角长时程抑制的影响

Neuropathic pain is the major indication of acupuncture treatment. Spinal microglia activation is the key factor in the process of occurrence and persistent of pain, while purinergic subtype P2X4 receptors (P2X4R) are involved in the microglia-mediated nociceptive transmission. However, it remains unknown if the electroacupuncture (EA) analgesia works though regulating the P2X4R-mediated activation of microglia. Thus，studies focus on the effect of EA on lumber-5 Spinal Nerve Ligation rat models (SNL) from the aspects of morphological, molecular biological and electrophysiological will be involved in this project. In the beginning, to confirm that microglia P2X4R are involved in EA analgesia, we observe the activation of spinal microglia and the expression change of protein and gene of P2X4R, using methods of immunofluorescence, Western-Blot and Q-PCR. Meanwhile, to approach whether the evoked mechanism of EA analgesia is the P2X4R-dependent long-term depression (LTD), we observe the LTD effect of EA on synaptic transmission in spinal dorsal horn, through recording C-fiber evoked field potentials in spinal dorsal horn with electrophysiological techniques. Furthermore, to investigate the effect of EA on the ATP-evoked excitatory postsynaptic currents (eEPSCs) in layer II of spinal dorsal horn, we apply the patch clamp technique in both the isolated spinal cord sections and intact spinal cord segments (S2-3). This study aims to clarify part of the neurobiological mechanisms of effect of EA on chronic neuropathology pain.

神经病理性疼痛是针灸临床常见适应症。脊髓小胶质细胞激活是病理神经性疼痛发生和持续的关键因素，嘌呤能亚型P2X4受体参与了小胶质细胞介导的痛觉信息传导。电针是否影响P2X4 R介导的脊髓小胶质细胞活化发挥镇痛作用尚不清楚。本项目拟建立L5脊神经结扎（SNL）大鼠模型①采用免疫荧光双标、Western-Blot、Q-PCR观察脊髓小胶质细胞活化及小胶质细胞上ATP 受体P2X4R蛋白与基因的表达，证实小胶质细胞P2X4R参与电针镇痛；②运用在体电生理学技术记录脊髓背角C-纤维诱发场电位，观察电针对脊髓背角突触传递的长时程抑制（LTD）的诱导作用，证实为P2X4R依赖性的LTD；③应用膜片钳技术，在离体脊髓切片和整体脊髓节段（S2-3）上观察电针对脊髓背角第Ⅱ板层ATP诱发的兴奋性突触后电流（eEPSCs）的影响；以期阐明电针治疗慢性神经病理痛的部分神经生物学机制。

小胶质细胞活化参与了神经病理性疼痛的发生发展。本研究选用大鼠腰5脊神经结扎模型并对其进行电针治疗，运用生物信息学、蛋白质生物化学、电生理学等方法与技术，对P2X4R参与神经病理性疼痛的生物学机制进行了初步研究。首先通过测量行为学、形态学证明电针能够治疗神经病理性疼痛；进而利用分子生物学技术对P2X4R与小胶质细胞进行分析，证明了电针治疗对P2X4R介导的脊髓小胶质细胞活化具有抑制作用。之后利用在体电生理和膜片钳技术对疼痛信号的产生及传导进行研究，结果提示电针能够抑制中枢敏化，调节脊髓背角突触功能。上述结果表明，P2X4R介导的脊髓小胶质细胞活化参与了神经病理性疼痛的发生发展，同时为探讨电针治疗慢性神经病理性疼痛的生物学机制奠定了坚实的工作基础。