电针对AGA模型小鼠关节部位NLRP3表达的干预及镇痛作用研究

The interventional effect of EA on NLRP3 inflammasome in joints of AGA model mice and EA’s analgesia

摘要:

目的 观察电针对急性痛风性关节炎(AGA)模型小鼠机械痛超敏的干预作用,并研究电针对模型小鼠踝关节组织中炎性小体3(NLRP3)通路的影响,探讨电针缓解急性痛风性关节炎疼痛的相关机制。方法 将SPF级C57/BL6小鼠随机分为对照组(Control)、模型组(MSU)、模型+电针组(MSU+EA)、模型+电针+溶剂组(MSU+EA+Veh)、模型+电针+尼日利亚菌素组(MSU+EA+NG)。除对照组注射对照溶剂外,其余三组于小鼠右后肢踝关节注射MSU混悬液。MSU+EA+Veh、MSU+EA+NG于造模后7.5h、23.5h进行电针治疗,治疗参数：电针频率：2/100Hz,电流强度为0.5mA。MSU+EA+NG组于电针前半小时右踝关节局部注射尼日利亚菌素(100μg/20μl/只)、其余各组注射对照溶剂。采用游标卡尺测量小鼠患侧踝关节肿胀；von Frey法测定小鼠患侧足底50%机械缩足反应阈值(PWTs)；H E染色观察炎性细胞浸润；免疫印迹观察NLRP3信号通路(包括NLRP3、Caspase-1、IL-1β)的蛋白表达。结果 与Control组相比,MSU组小鼠患侧踝关节明显肿胀(P<0.01),机械痛阈显著降低(P<0.01),NLRP3、Caspase-1、IL-1β蛋白表达显著增高(P<0.01),H E染色显示局部有广泛炎性细胞浸润；与MSU组相比,MSU+EA组小鼠患侧踝关节机械痛阈明显升高(P<0.05或P<0.01),NLRP3、Caspase-1、IL-1β蛋白表达显著下降(P<0.05或P<0.01)；与MSU+EA+Veh组相比,MSU+EA+NG组小鼠患侧踝关节50%PWTs明显降低(P<0.01),NLRP3、Caspase-1、IL-1β蛋白表达显著上升(P<0.01)。 结论 电针干预可明显改善AGA模型小鼠痛觉超敏及踝关节肿胀,这一作用可能与抑制踝关节组织中NLRP3通路有关。研究结果提示电针可用做一种缓解AGA的绿色替代疗法。

Abstract:

Objective: To observe the interventional effect of electroacupuncture (EA) on mechanical pain hypersensitivity in the mouse model of acute gouty arthritis (AGA), and to investigate the effect of EA on NLRP3 signaling pathway in ankle joint tissues of model mice, and to explore the mechanism related to the pain relief of AGA by EA. Methods: C57/BL6 (SPF grade) mice were randomly divided into control group (Control), model group (MSU), model+EA group (MSU+EA), model+EA+Veh group (MSU+EA+Veh), and model+EA+ nigericin group (MSU+EA+NG). Except for the control group, which was injected with control solvent, all other 3 groups were injected with monosodium urate crystal (MSU) suspension in the right ankle joints. MSU+EA+Veh and MSU+EA+NG were treated with EA at 7.5 h and 23.5 h after modeling, and the treatment parameters were electroacupuncture frequency of 2/100Hz and current intensity of 0.5mA.The MSU+EA+NG group was locally injected with Nigericin at the right ankle joint (100 μg/20μl/only) 0.5 h before EA, and the other group received corresponding vehicle treatment. The swelling of the affected ankle joint was measured by calipers; the 50% mechanical paw withdrawal (PWTs) were measured by von Frey hairs; the inflammatory cell infiltration was observed by H E staining; the protein expression of NLRP3 signaling pathways (including NLRP3, Caspase-1, IL-1β) was studied by immunoblotting. Results: Compared with the control group, the MSU group showed significantly swollen ankle joint (P<0.01), significantly lower mechanical pain threshold (P<0.01), significantly higher NLRP3, Caspase-1 and IL-1β protein expression (P<0.01), and extensive local inflammatory cell infiltration by H E staining. Compared with MSU, the MSU+EA group showed significantly higher 50% PWTs (P<0.01), and the expression of NLRP3, Caspase-1 and IL-1β was significantly decreased (P<0.05 or P<0.01). Compared with the MSU+EA+Veh group, the 50% PWTs of the affected ankle joint were significantly lower in the MSU+EA+NG group (P<0.05 or P<0.01), and the expression of NLRP3, Caspase-1 and IL-1β was significantly increased (P<0.05 or P<0.01); Conclusion: EA intervention significantly improved mechanical hypersensitivity and ankle swelling in AGA model mice, and this effect may be related to the inhibition of NLRP3 signaling pathway in ankle joint tissues. These findings suggest that EA can be used as a green and alternative therapy to alleviate AGA.