吴茱萸汤通过热敏通道瞬时电位锚蛋白-1抑制福尔马林内脏痛模型小鼠的疼痛研究

Study of Wuzhuyu decoction, by thermal channel TRPA1, inhibiting formalin-induced visceral pain in mice

目的探查吴茱萸汤减轻福尔马林诱导的内脏痛模型小鼠的疼痛作用机制。方法 40只C57BL/6雄性小鼠,随机分为正常组、模型组、吴茱萸汤低剂量组、吴茱萸汤中剂量组和吴茱萸汤高剂量组。吴茱萸汤各剂量组分别灌以吴茱萸汤水煎液[3.12、6.24、12.48 g/(kg·d)],共4天。第4天,吴茱萸汤各剂量组灌胃后2小时,以瞬时感受器电位锚蛋白-1(transient receptor potential ankyrin 1,TRPA1)激动剂10%福尔马林,10 μL/只灌肠,在福尔马林诱导的急性内脏痛模型小鼠上,观察吴茱萸汤干预后的疼痛行为变化和运用免疫组化染色及免疫印迹法检测吴茱萸汤干预后的小鼠结肠组织中TRPA1的表达变化。结果 HE染色显示吴茱萸汤各剂量组干预均可使小鼠结肠中肌层、黏膜层坏死减轻,炎性浸润减少,受损的隐窝修复;行为学实验观察吴茱萸汤高、中、低剂量预处理4天后与模型组对比,小鼠疼痛行为频次明显降低( P <0.05);免疫组化实验,吴茱萸汤高、中、低剂量预处理后,与模型组对比,肌层的TRPA1阳性表达明显下调( P <0.05);蛋白条带密度分析提示,吴茱萸汤高、中剂量预处理后能使小鼠结肠组织中TRPA1表达下调,与模型组差异明显( P <0.05 )。结论热敏通道TRPA1是吴茱萸汤发挥止痛作用,尤其是缓解内脏痛的重要靶点。

Objective To investigate the analgesic effects of Wuzhuyu decoction(WZYD) on formalin-induced visceral pain in mice. Methods A total of 40 C57BL/6 male mice were randomly divided into normal group,model group, low-dose group[3.12 g/(kg·d)], middle-dose group[(6.24 g/(kg·d)] and high-dose group[12.48 g/(kg·d)]. WZYD was administered respectively to 3 WZYD groups by gavage for 4 days. On the 4 th day, 2 hours after the gavage of WZYD, transient receptor potential ankyrin-1 (TRPA1) agonist 10% formalin, 10 μl/enema, acute viscera modal rats induced by formalin, the changes of pain behavior after WZYD intervention and the expression of TRPA1 in the colon tissue of mice after WZYD intervention were detected by immunohistochemical staining and immunoblotting. Results HE dying showed that different doses of WZYT could alleviate the necrosis of the muscular layer and mucosal layer in the colon of mice, reduce the inflammatory infiltration, and repair the damaged crypt. It was found by the behavioral test that after 4 days of pretreatment with high, middle, and low doses of WZYT, the frequency of pain behavior of mice was significantly reduced, which was significantly different from that of the model group ( P <0.05). In the immunohistochemical experiment, after the pretreatment with high, middle and low doses of WZYT, the positive expression of TRPA1 in the muscular layer was significantly decreased, which was significantly different from that of the model group ( P <0.05). Conclusion Thermal channel TRPA1 plays a key role on the analgesic effect of WZYT.