黄芪甲苷对肾纤维化小鼠Toll/MyD88依赖性通路的作用研究

Effect of astragaloside IV based on Toll/MyD88 dependent signaling pathway in treatment of renal fibrosis mice

目的观察黄芪甲苷(astragaloside IV)对单侧输尿管结扎(UUO)小鼠肾组织纤维化程度的影响,探讨其作用机制。方法将雄性C57BL/6小鼠50只随机均分为假手术组,模型组,黄芪甲苷高、中、低剂量(50、30、10 mg/kg)组。除假手术组外,其余各组小鼠均行UUO术,从手术当天黄芪甲苷各组ig给药,连续14 d。检测血清肌酐(Cr)和尿素氮(BUN)水平,HE和Masson染色下光镜观察肾组织病理和胶原的沉积;免疫组化和Western blotting方法从蛋白水平观察各组肾脏中Toll/MyD88依赖信号通路相关分子(TLR4、TLR2、MyD88、TRAF-6、NF-κB、TNF-α、IL-6)的表达。结果模型组小鼠患侧肾脏的纤维化程度、病理损害最明显;Toll/MyD88依赖信号通路相关的分子的表达量最多。而随着黄芪甲苷组药物剂量的增加,小鼠肾脏损伤有明显改善,该信号通路相关因子(TLR4、TLR2、MyD88、TRAF-6、NF-κB)蛋白表达水平也在相应减少,且对该信号通路末端炎症因子TNF-α和IL-6的表达有抑制作用。结论黄芪甲苷可能通过抑制Toll/MyD88依赖信号通路及炎症因子TNF-α和IL-6的释放来改善小鼠UUO所致的肾纤维化。

Objective To study the effect of astragaloside IV on renal fibrosis mice with unilateral ureteral obstruction（UUO） and discuss the mechanism. Methods Male C57BL/6 50 mice were divided into five groups randomly, such as Sham-operated group, model group and high-, medium-, and low-dose astragaloside IV groups. From the day of surgery, the mice in astragaloside IV groups（high-, medium-and low-dose） were treated by gavage of astragaloside IV for 2 weeks in doses of 50, 30, and 10 mg/（kg·d） separately. The mice in Sham-operated group and model group were treated with saline instead of astragaloside IV. Serum creatinine and blood urea nitrogen were detected by chemical methods. Histopathological changes and collagen deposition of affected kidney were observed under optical microscope with HE and MASSON staining. The expression levels of Toll/MyD88 dependent signaling pathway related molecules（TLR4, TLR2, MyD88, TRAF6, NF-Kappa B, TNF-α, and IL-6） in affected kidney were measured by immunohistochemistry and Western blotting methods and observed from protein levels in each group. Results The degree of fibrosis and histopathological damage of affected kidney of mice in model group is the most obvious. And the expression levels of Toll/MyD88 dependent signaling pathway related molecules in affected kidney of mice in model group were the highest. With drug concentration increased in groups of astragaloside IV, in these groups, the injury of affected kidney had been obviously reduced, and the protein expression levels of Toll/MyD88 dependent signaling pathway related molecules（TLR4, TLR2, MyD88, TRAF6, and NF-Kappa B） were also in corresponding reduced, at the same time the expression of terminal inflammatory cytokines（TNF-alpha and IL-6） has been suppressed. Conclusion Astragaloside IV may improve renal interstitial fibrosis in mice after UUO by inhibiting the expression of Toll/MyD88 dependent signaling pathway and release of inflammatory cytokines（TNF-alpha and IL-6）.