基于脊髓CXCL1-CXCR2脱敏的手针镇痛机制研究

Pain is an enormous problem globally. Until now, analgesic drugs do not lead to an adequate response but associated with various side-effects. Acupuncture, an effective analgesic therapy with fewer side-effects, has been widely used in the different parts of the world. However, its analgesic mechanism is still not clear enough. Previous studies showed that the analgesic and anti-inflammatory effects of acupuncture on rheumatoid arthritis rats were achieved via the adjustment of NEI network, and in which the increase of chemokine CXCL1 played a critical role. Therefore, it is proposed that CXCL1-CXCR2 desensitization may be one of the potential mechanisms in manual acupuncture analgesia. In this study, using Comlete Freund’s adjuvant (CFA) mouse models, treated with manual acupuncture at bilateral ST36 (Zusanli), we will access to the analgesic effect of acupuncture and the content of several algogenic substance in the spinal cord. And then the source of CXCL1 in spinal cord will be clarified by using qPCR, ELISA, immunofluorescence staining and imaging techniques in vivo. Using missing/capture methods, namely exogenous CXCL1 and its blockage, the analgesic effect of acupuncture via CXCL1 will be confirmed. To investigate the role of CXCL1-CXCR2 desensitization in acupuncture analgesia, some key proteins in CXCR2 desensitization signaling and the downstream MAPK pathway will be detected by Western Blot, co-immunoprecipitation and phosphorylation of GPCRs protein chip. The desensitization cell type in spinal cord will be observed by multiple-labeled immunofluorescense staining. Based on the above study, we attempt to provide a novel mechanism for acupuncture analgesia.

疼痛已成为全球性巨大问题，但治疗药物常效果不佳，且伴有诸多副作用。针刺作为一种有效且低副作用的镇痛疗法，已被广泛应用，但作用机制仍需进一步明确。申请者前期研究发现针刺对类风湿性关节炎大鼠的镇痛抗炎效应通过NEI网络调节实现，其中趋化因子CXCL1升高起关键作用，但机制不明。因此，本课题提出受体脱敏机制是手针调控CXCL1-CXCR2镇痛关键机制之一的科学假说，以完全弗氏佐剂诱导小鼠炎性痛为模型，观察针刺足三里穴对痛觉行为和脊髓致痛物质含量的影响；采用Q-PCR、ELISA、免疫荧光染色、小鼠活体成像技术明确脊髓CXCL1升高的来源，并通过缺失/捕获技术明确CXCL1在针刺镇痛的作用；采用Western Blot、免疫共沉淀、GPCRs蛋白磷酸化芯片检测针刺对CXCR2脱敏通路关键蛋白和信号转导下游MAPK等通路影响，通过免疫荧光共染明确脱敏机制发生的细胞，为针刺镇痛学说提供新的科学依据。

疼痛，可见于疾病或疾病的伴随症状，严重影响患者的生活质量。针刺镇痛疗效确切，随着研究不断深入，其机制也在不断更新完善中。申请者前期研究发现针刺对CFA大鼠的镇痛抗炎效应中趋化因子CXCL1升高起关键作用，提出受体脱敏机制是针刺调控CXCL1-CXCR2镇痛关键机制之一的科学假说，本项目围绕科学假说建立针刺治疗炎性痛模型的研究平台，探索针刺引起脊髓CXCL1升高调控镇痛效应与CXCL1升高来源，及针刺升高脊髓CXCL1参与镇痛的机制。结果发现针刺可显著提高CFA小鼠热辐射痛、机械痛痛阈值，降低脊髓局部COX1、PGE2蛋白含量，对COX2蛋白含量无影响；针刺升高脊髓部位CXCL1基因表达和蛋白含量，CXCL1升高来源于脊髓神经元、星形胶质细胞和外周血；鞘内注射低剂量CXCL1中和抗体能降低针刺后足底热痛阈值，拮抗针刺镇痛效应，鞘内注射CXCL1高剂量重组蛋白能显著提高造模后足底热痛阈，模拟针刺镇痛效应，从而明确脊髓升高的CXCL1介导针刺镇痛；在此基础上针刺可降低脊髓部位全蛋白和膜蛋白CXCR2，但对CXCR2脱敏的关键蛋白GRK2、GRK6和β-Arrestin1/2蛋白含量无影响，否认脱敏信号通路是针刺镇痛的关键信号通路；磷酸化蛋白组学结果提示内吞信号通路参与针刺镇痛，而针刺可升高脊髓部位内吞通路关键蛋白Rab7a，使CXCR2运送至溶酶体降解，从而通过CXCL1-CXCR2内吞信号通路影响针刺镇痛。本项目从CXCL1-CXCR2内吞角度阐释针刺镇痛机制，为针刺镇痛学说提供新的科学依据。