基于NLRP3炎症小体探讨三石汤治疗痛风性关节炎的机制研究  

Mechanism of Sanshi decoction in the treatment of gouty arthritis by NLRP3 inflammasome

作　　者：朴勇洙[1,2] 齐明明[2] 聂双莲 潘国雄 张皓 王欣波[1,2] PIAO Yong-zhu;QI Ming-ming;NIE Shuang-lian;PAN Guo-xiong;ZHANG Hao;WANG Xin-bo(First Affiliated Hospital,Heilongjiang University of Chinese Medicine,Harbin 150040,China;Heilongjiang University of Chinese Medicine,Harbin 150040,China)

机构地区：[1]黑龙江中医药大学附属第一医院,黑龙江哈尔滨150040 [2]黑龙江中医药大学,黑龙江哈尔滨150040

出　　处：《海南医学院学报》2023年第23期1786-1793,共8页Journal of Hainan Medical University

基　　金：黑龙江省中医药科研项目(ZHY19-006)。

摘　　要：目的:观察三石汤对P2X7R/PKR信号通路介导的巨噬细胞NLRP3炎症小体活化的影响,从而明确三石汤治疗痛风性关节炎的分子机制。方法:将THP-1巨噬细胞分为空白组、模型组、三石汤低剂量、中剂量、高剂量和抑制剂组。除空白组外,其余各组用尿酸单钠结晶诱导构建痛风性关节炎细胞模型。采用流式细胞术检测各组巨噬细胞ROS水平,以ELISA法检测各组MDA含量及SOD和GSH-PX的活性,以Western blot检测P2X7R/PKR信号通路和NLRP3炎症小体相关蛋白的表达。在此基础上,构建巨噬细胞与滑膜细胞共培养体系,用CCK8和流式细胞术分别检测滑膜细胞的活性及凋亡水平。结果:与空白组相比,模型组巨噬细胞ROS水平、MDA的含量、SOD和GSH-PX的活性显著增加,NLRP3、Mature IL-1β、Pro IL-1β、Mature IL-18、Pro IL-18、Mature Caspase-1、GSDMD-NT、P2X7R和p-PKR蛋白的表达水平显著上调,GSDMD-FL蛋白的表达显著下调,差异均具有统计学意义(P<0.05和P<0.01)。与模型组比较,三石汤能够降低巨噬细胞ROS水平、MDA的含量、SOD和GSH-PX的活性,下调巨噬细胞NLRP3、Mature IL-1β、Pro IL-1β、Mature IL-18、Pro IL-18、Mature Caspase-1、GSDMD-NT、P2X7R和p-PKR蛋白的表达,上调GSDMD-FL蛋白的表达,差异均具有统计学意义(P<0.05和P<0.01)。此外,与空白组相对比,模型组中滑膜细胞的活性降低,其凋亡的水平上升,组间比较差异均具有统计学意义(P<0.05)。而与模型组相对比,三石汤能够显著地增加滑膜细胞的活性,抑制细胞的凋亡水平,各组间比较差异均具有统计学意义(P<0.05和P<0.01)。结论:三石汤能够通过抑制P2X7R/PKR信号通路的激活从而降低NLRP3活化水平,达到治疗痛风性关节炎的作用。Objective:To observe the effect of Sanshi decoction on P2X7R/PKR pathway⁃mediated activation of macro⁃phage NLRP3 inflammasome in order to elucidate the molecular mechanism of Sanshi decoction in the treatment of gouty arthritis.Methods:THP⁃1 macrophages were divided into control group,model group,low dose group,medium dose group,high dose group of Sanshi decoction and inhibitor group.The remaining groups were induced with monosodium urate crystals to establish a gouty arthritis cell model except the control group.Flow cytometry was used to detect macrophage ROS levels in each group,ELI⁃SA to detect MDA levels and SOD and GSH⁃PX activities in each group,and Western blot to detect P2X7R/PKR pathway and NLRP3 inflammasome⁃associated protein expression.We also used CCK⁃8 and flow cytometry to measure MH7A activity and apoptotic levels. Results: Compared with the control group, the ROS level, the content of MDA, the activities of SOD andGSH⁃PX were significantly increased, and the expression levels of NLRP3, full⁃length IL⁃1β, pro⁃IL⁃1β, full⁃length IL⁃18,pro⁃IL⁃18, full⁃length caspase⁃1, GSDMD⁃NT, P2X7R and p⁃PKR protein expression were significantly upregulated, andGSDMD⁃FL protein expression was significantly downregulated in the model group, and that the differences between them werestatistically significant( P<0.05 and P<0.01). Compared with the model group, Sanshi decoction could reduce macrophage ROSlevels, MDA content, SOD and GSH⁃PX activities, and downregulate macrophage NLRP3, mature IL⁃1β, pro IL⁃1β, matureIL⁃18, pro IL⁃18, mature caspase⁃1, GSDMD⁃NT, P2X7R and p⁃PKR protein expression, and upregulate GSDMD⁃FL proteinexpression, with statistically significant differences (P<0.05 and P<0.01). In addition, MH7A activity was downregulated, andapoptosis level was upregulated in the model group in comparison with the control group, and differences were all significantly dif⁃ferent( P<0.05). As compared to the model group, Sanshi decoction could

关 键 词：痛风性关节炎 三石汤 NLRP3炎症小体 P2X7R/PKR信号通路 巨噬细胞 

分 类 号：R285[医药卫生—中药学]