基于MAPK通路调控脊髓胶质细胞活化研究益气活血通络方防治糖尿病神经病理性疼痛的作用机制

Diabetic Neuropathic Pain (DNP ) is one of the most common and difficult complications in diabetes mellitus. In recent years, blocking the MAPK pathway and regulating the activity of spinal glial cells have become a new target for the prevention and treatment of DNP. Yiqi Huoxue Tongluo Formula is an effective prescription for the clinical treatment of diabetic peripheral neuropathy. Earlier studies showed that Yiqi Huoxue Tongluo Formula can reduce inflammatory response, improve diabetic peripheral nerve injury and relieve pain symptoms by regulating JNK pathway (one of MAPK classic pathways). At the same time, it was found that this recipe could inhibit the activation of astrocytes in the spinal cord of DNP rats. Therefore, we put forward the hypothesis that the Yiqi Huoxue Tongluo Formula can regulate the activity of spinal glial cells through MAPK signaling pathway, thereby inhibiting the release of inflammatory factors, reducing the central sensitization caused by neuroinflammatory reaction and improving the role of DNP. This project intends to use immunofluorescence confocal microscopy, modern molecular biology experimental techniques combining in vitro and in vivo cell culture with spinal glial cells as the start point. To observe the effect of Yiqi Huoxue Tongluo Formula on glial cells in DNP rats and its relationship with MAPK signaling pathway, and further explore the molecular mechanism of this formula in preventing and treating DNP, so as to provide experimental evidence for improving the efficacy of TCM in preventing and treating DNP.

糖尿病神经病理性疼痛(DNP)是糖尿病最常见最难治的并发症之一。近年来，阻断MAPK通路，调节脊髓胶质细胞活性成为防治DNP新的研究靶点。益气活血通络方为临床治疗糖尿病周围神经病变的有效方药。前期我们的研究显示益气活血通络方可通过调控JNK通路（MAPK经典通路之一），减轻炎症反应，改善糖尿病周围神经损伤和缓解疼痛症状。同时发现该方可抑制DNP大鼠脊髓星形胶质细胞活化。鉴此，我们提出假说：益气活血通络方可通过MAPK信号通路调节脊髓胶质细胞活性，进而抑制炎症因子释放，从而减轻神经炎症性反应导致的中枢敏化而发挥改善DNP的作用。本项目拟以胶质细胞为切入点，运用免疫荧光双标、激光共聚焦等方法和现代分子生物学技术，体内和体内研究结合，观察益气活血通络方对DNP大鼠脊髓胶质细胞的影响及与MAPK信号通路的关系，深入探讨该方防治DNP的分子机制，为提高中医药防治DNP疗效提供实验依据。

糖尿病神经病理性疼痛(DNP)是糖尿病最常见最难治的并发症之一，目前对 DNP发生发展的机制尚未完全阐明，无确切有效的治疗方法。益气活血通络方为临床治疗糖尿病周围神经病变的有效方药，可有效缓解糖尿病患者周围神经系统异常引起的疼痛。本研究探讨了益气活血通络方对DNP的作用及其机制。主要研究内容与结果如下：1）高脂高糖饲养联合低剂量（35mg/kg）STZ腹腔注射诱导建立糖尿病大鼠模型，2周后Von Frey电子测痛仪评估大鼠机械痛阈(MWT)，全自动热痛测试仪评估大鼠热缩足反应潜伏期(TWL)，二者均下降至基础值85%以下则判定为DNP模型大鼠。此动物造模方法更能符合人类2型糖尿病特点。2）利用免疫荧光双标染色、Western blotting、RT-PCR等技术，检测DNP模型大鼠L4-6脊髓组织中星形胶质细胞、小胶质细胞特异性标志物GFAP、OX42及MAPK通路相关磷酸蛋白p-JNK、p- p38MAPK、p-ERK的表达，酶联免疫吸附法检测促炎因子IL-1β、IL-6、TNF-α和趋化因子 CCL2、CXC3L1及其受体 CCR2、CXC3R1的表达，发现JNK、ERK与星形胶质细胞共表达，p38MAPK与小胶质细胞共表达，证实了益气活血通络方能抑制脊髓星型胶质细胞和小胶质细胞的活性，进而抑制MAPK信号通路的表达，从而缓解神经炎症，明确了益气活血通络方防治DNP的作用靶点。3）体外实验采用益气活血通络方含药血清作用于高糖诱导的CTX-TNA2星形胶质细胞和BV2小胶质细胞，证实益气活血通络方可抑制高糖诱导的CTX-TNA2星形胶质细胞和BV2小胶质细胞激活，促进胶质细胞促炎表型向抗炎表型极化，抑制MAPK 信号通路激活，同时降低炎性因子的表达。项目以实验动物为研究对象，以细胞实验验证其机制，环环相扣，验证了预定设想，完成了预期计划。