基于TLR4/NF-κB/NLRP3通路介导滑膜细胞焦亡探讨宣痹通络膏对急性痛风性关节炎的作用机制

Exploring the Mechanism of Action of Xuanbi Tongluo Ointment(宣痹通络膏)on Acute Gouty Arthritis Based on TLR4/NF-κB/NLRP3 Pathway-Mediated Pyroptosis of Synovial Cells

目的:探究宣痹通络膏对急性痛风性关节炎(AGA)大鼠滑膜细胞焦亡的作用及机制。方法:将40只雄性Wistar大鼠随机分为对照组、模型组、宣痹通络膏组(36 g/kg)、秋水仙碱组(阳性对照药,0.3 mg/kg),每组各10只。除对照组大鼠外,其余各组大鼠均采取踝关节腔内注射尿酸钠构建AGA模型,并于造模成功后当天对大鼠进行分组灌胃给药干预,1次/d,连续3 d。分别于4、24、48 h测定各组大鼠踝关节肿胀指数;苏木素-伊红(HE)染色观察各组大鼠踝关节滑膜组织病理学变化;TUNEL染色检测滑膜细胞焦亡指数;免疫组化及酶联免疫吸附法(ELISA)检测滑膜组织及血清IL-1β和IL-18表达;免疫荧光及Western blotting检测滑膜组织TLR4/NF-κB/NLRP3信号通路相关蛋白表达。结果:与对照组相比,模型组大鼠踝关节肿胀指数明显升高(P<0.05),滑膜组织明显增厚,并伴有炎性细胞大量浸润,滑膜细胞焦亡指数明显增加(P<0.05),且滑膜组织IL-1β、IL-18蛋白及TLR4/NF-κB/NLRP3信号通路蛋白(TLR4、MyD88、NF-κB、NLRP3、ASC、Caspase-1)表达均明显升高(P<0.05),血清IL-1β和IL-18水平明显升高(P<0.05)。与模型组相比,宣痹通络膏组和秋水仙碱组大鼠踝关节肿胀指数明显降低(P<0.05),滑膜组织损伤减轻,滑膜细胞焦亡指数明显降低(P<0.05),且滑膜组织IL-1β、IL-18蛋白及TLR4/NF-κB/NLRP3信号通路蛋白表达均明显下调(P<0.05),血清IL-1β和IL-18水平明显降低(P<0.05)。结论:宣痹通络膏可能通过调节TLR4/NF-κB/NLRP3通路,抑制滑膜细胞焦亡,从而发挥抗AGA的作用。

Objective:To explore the effect and mechanism of Xuanbi Tongluo Ointment on pyroptosis of synovial cells in rats with acute gouty arthritis(AGA).Methods:A total of 40 male Wistar rats were randomly divided into control group,model group,Xuanbi Tongluo Ointment group(36 g/kg)and colchicine group(positive control drug,0.3 mg/kg),with 10 rats in each group.Except for the rats in the control group,the rats in the other groups were injected with sodium urate into the ankle joint cavity to construct the AGA model.On the day after successful modeling,the rats were given intragastric administration once a day for 3 consecutive days.The ankle swelling index of rats in each group was measured at 4,24 and 48 h,respectively.HE staining was used to observe the pathological changes of synovium of ankle joint in each group.The pyroptosis index of synovial cells was detected by TUNEL staining.The expressions of IL-1βand IL-18 in synovial tissue and serum were detected by immunohistochemistry and ELISA.The expression of TLR4/NF-κB/NLRP3 signaling pathway related proteins in synovial tissue was detected by immunofluorescence and Western blotting.Results:Compared with the control group,the ankle swelling index of the model group was increased(P<0.05),the synovial tissue was significantly thickened,accompanied by a large number of inflammatory cells infiltration,and the pyroptosis index of synovial cells was increased(P<0.05).The expression of IL-1β,IL-18 protein and TLR4/NF-κB/NLRP3 signaling pathway proteins(TLR4,MyD88,NF-κB,NLRP3,ASC,Caspase-1)in synovial tissue were significantly increased(P<0.05),and the levels of serum IL-1βand IL-18 were increased(P<0.05).Compared with the model group,the ankle swelling index of rats in Xuanbi Tongluo Ointment group and colchicine group decreased(P<0.05),the damage of synovial tissue was alleviated,and the pyroptosis index of synovial cells decreased(P<0.05).The expression of IL-1β,IL-18 protein and TLR4/NF-κB/NLRP3 signaling pathway protein in synovial tissue were significantly down-regulated(P<0.05),and the levels of serum IL-1βand IL-18 were decreased(P<0.05).Conclusion:Xuanbi Tongluo Ointment may play an anti-acute gouty arthritis role by regulating TLR4/NF-κB/NLRP3 pathway and inhibiting pyroptosis of synovial cells.