松萝酸对类风湿关节炎成纤维样滑膜细胞HMGB1/RAGE炎症通路及VEGF、IL-6、IL-1β表达的影响

Effect of usnic acid on HMGB1/RAGE inflammatory pathway in fibroblast-like synoviocytes of RA and VEGF,IL-6 and IL-1βexpression levels

目的构建类风湿关节炎(RA)成纤维样滑膜细胞(RAFLS)模型,研究松萝酸对其高迁移率族蛋白B1(HMGB1)/晚期糖基化终产物受体(RAGE)炎症通路及血管内皮生长因子(VEGF)、白细胞介素(IL)-6、IL-1β表达的影响。方法选取贵州中医药大学第二附属医院骨科行关节置换治疗的RA患者,于术中获取滑膜组织。将细胞分为空白对照组(胎牛血清)、溶剂对照组(胎牛血清+10%羟丙基倍他环糊精)、松萝酸高剂量组(松萝酸50μg/mL)、松萝酸中剂量组(松萝酸10μg/mL)、松萝酸低剂量组(松萝酸2.5μg/mL)和羟氯喹组(羟氯喹50 mmol/L)。MTT检测不同浓度松萝酸处理后细胞增殖情况,Western blot检测各组细胞HMGB1、RAGE蛋白表达,ELISA检测各组细胞HMGB1、VEGF、IL-6、IL-1β表达。结果与空白对照组比较,松萝酸高、中、低剂量组及羟氯喹组细胞凋亡比例增加(P<0.05),松萝酸高、中剂量组和羟氯喹组HMGB1、RAGE蛋白表达明显降低(P<0.05),松萝酸高、中、低剂量组和羟氯喹组HMGB1、VEGF、IL-6、IL-1β表达均降低(P<0.05);与羟氯喹组比较,松萝酸中、低剂量组细胞凋亡比例减少(P<0.05),松萝酸低剂量组HMGB1蛋白表达明显升高(P<0.05),松萝酸中、低剂量组RAGE蛋白表达明显升高(P<0.05)。结论松萝酸可能通过调控HMGB1/RAGE炎症通路及VEGF、IL-6、IL-1β等炎症因子表达,减轻滑膜细胞的炎症反应。

Objective To construct the rheumatoid arthritis fibroblast-like synoviocytes(RAFLS)model,and to investigate the effects of usnic acid on the inflammatory pathway of high-mobility group protein B1(HMGB1)/receptor for activated glycation end products(RAGE)and the expression levels of vascular endothelial growth factor(VEGF),interleukin(IL)-6 and IL-1β.Methods The patients with RA undergoing joint replacement surgery in the orthopedic department of the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine were selected and the synovial tissues were obtained during operation.The cells were divided into the blank control group(fetal calf serum),solvent control group(fetal calf serum+10%hydroxypropyl betacyclodextrin),usnic acid high dose group(usnic acid 50μg/mL),usnic acid middle dose group(usnic acid 10μg/mL),usnic acid low dose group(usnic acid 2.5μg/mL)and hydroxychloroquine group(hydroxychloroquine 50 mmol/L).The cell proliferation after different concentrations of usnic acid treatment was detected by MTT,the HMGB1 and RAGE protein expression in cells of each group was detected by Western blot,and the expression levels of HMGB1,VEGF,IL-6 and IL-1βwere detected by ELISA.Results Compared with the blank control group,the proportions of cellular apoptosis in the usnic acid high,middle and low doses groups and hydroxychloroquine group were increased(P<0.05);the expression levels of HMGB1 and RAGE protein in the usnic acid high,middle and low doses groups and hydroxychloroquine group were significantly decreased(P<0.05),the expression levels of HMGB1,VEGF,IL-6 and IL-1βwere decreased(P<0.05);compared with the hydroxychloroquine group,the cellular apoptosis proportions of the usnic acid high,middle and low doses groups were decreased(P<0.05),the HMGB1 protein expression level in the usnic acid low dose group was significantly increased(P<005),the RAGE protein expression level in the the usnic acid middle amd low doses groups was significantly increased(P<0.05).Conclusion Usnic acid alleviates the inflammatory reaction of synoviocytes possibly by regulating HMGB1/RAGE inflammatory pathway and VEGF,IL-6 and IL-1βinflammatory factors expression levels.