Yang Ru-Qian, Xu Qian, Huangfu Zhi-Min, Zhang Chun-Lai, Yin Yi-Feng, Zhang Jie, Zhao Chun-Ling
Basic Medicine College, Southwest Medical University, Luzhou 646000, China.
Clinical Medical Collage, Southwest Medical University, Luzhou 646000, China.
Zhongguo Zhong Yao Za Zhi. 2018 Sep;43(18):3729-3739. doi: 10.19540/j.cnki.cjcmm.20180426.001.
The aim of this paper is to study the effect of astragaloside Ⅳ on renal fibrosis mice with ischemia-reperfusion injury (IRI) and discuss the mechanism. Male C57BL/6 50 mice were randomly divided into four groups, namely Sham-operated group, model group, AS-Ⅳ prevention group and AS-Ⅳ treatment group. Since the day of surgery, the mice in astragaloside Ⅳ prevention group were treated with astragaloside Ⅳ by gavage for 30 days at the dose of 30 mg·kg⁻¹·d⁻¹. At the 60th day after surgery, the mice in astragaloside Ⅳ treatment group were treated with astragaloside Ⅳ 100 by gavage for 30 days at the dose of 30 mg·kg⁻¹·d⁻¹. The mice in Sham-operated group and model group were treated with double distilled water containing 0.1% ethanol instead of astragaloside Ⅳ. Serum creatinine and blood urea nitrogen were detected by chemical methods. Histopathological changes and collagen deposition of affected kidneys were observed under optical microscope by HE and Masson staining. The expression levels of Toll like receptor pathway related molecules (TLR4,MyD88,TRAF6,TRAM,TRIF,NF-κB,TNF-α,IL-6, IFN-) in affected kidneys were observed by immunohistochemistry, Western blot methods and reverse transcription-PCR atprotein and mRNA levels in each group. The results showed that the degrees of fibrosis and histopathological damage of affected kidneys of mice in model group were the most obvious. And the expression levels of TLR4/MyD88 dependent signaling pathway-related molecules (TLR4 and MyD88, TRAF6 and NF-κB) in affected kidneys of mice in model group were the highest. At the same time, there was no difference in the expression levels of TLR4/MyD88 independent signaling pathway-related molecules（TRAM, TRIF）among sham-operated group, model group, astragaloside IV prevention group and astragaloside Ⅳ treatment group. In astragaloside Ⅳ prevention group and astragaloside Ⅳ treatment group, the injury of affected kidney was obviously reduced, and the protein expression levels of TLR4/MyD88 dependent signaling pathway-related molecules were also correspondingly reduced; at the same time, the expressions of terminal inflammatory cytokines (TNF-α,IL-6, IFN-) were suppressed. Therefore, astragaloside Ⅳ may improve renal interstitial fibrosis in mice after IRI by inhibiting the expression of TLR4/MyD88 dependent signaling pathway and the release of inflammatory cytokines (TNF-α,IL-6, IFN-), while the TLR4/MyD88 independent signaling pathway may not be involved in the process of renal fibrosis after ischemia-reperfusion injury.
本文旨在研究黄芪甲苷Ⅳ对缺血再灌注损伤（IRI）肾纤维化小鼠的影响并探讨其机制。将50只雄性C57BL/6小鼠随机分为四组，即假手术组、模型组、黄芪甲苷Ⅳ预防组和黄芪甲苷Ⅳ治疗组。自手术当天起，黄芪甲苷Ⅳ预防组小鼠以30 mg·kg⁻¹·d⁻¹的剂量通过灌胃给予黄芪甲苷Ⅳ，持续30天。术后第60天，黄芪甲苷Ⅳ治疗组小鼠以30 mg·kg⁻¹·d⁻¹的剂量通过灌胃给予黄芪甲苷Ⅳ 100，持续30天。假手术组和模型组小鼠用含0.1%乙醇的双蒸水代替黄芪甲苷Ⅳ进行处理。采用化学方法检测血清肌酐和血尿素氮。通过HE和Masson染色在光学显微镜下观察受损肾脏的组织病理学变化和胶原沉积。采用免疫组织化学、蛋白质印迹法和逆转录PCR法在蛋白质和mRNA水平观察各组受损肾脏中Toll样受体途径相关分子（TLR4、MyD88、TRAF6、TRAM、TRIF、NF-κB、TNF-α、IL-6、IFN-）的表达水平。结果显示，模型组小鼠受损肾脏的纤维化程度和组织病理学损伤最为明显。模型组小鼠受损肾脏中TLR4/MyD88依赖性信号通路相关分子（TLR4和MyD88、TRAF6和NF-κB）的表达水平最高。同时，假手术组、模型组、黄芪甲苷Ⅳ预防组和黄芪甲苷Ⅳ治疗组之间TLR4/MyD88非依赖性信号通路相关分子（TRAM、TRIF）的表达水平无差异。在黄芪甲苷Ⅳ预防组和黄芪甲苷Ⅳ治疗组中，受损肾脏的损伤明显减轻，TLR4/MyD88依赖性信号通路相关分子的蛋白表达水平也相应降低；同时，终末炎性细胞因子（TNF-α IL-6、IFN-）的表达受到抑制。因此，黄芪甲苷Ⅳ可能通过抑制TLR4/MyD88依赖性信号通路的表达和炎性细胞因子（TNF-α、IL-6、IFN-）的释放来改善IRI后小鼠的肾间质纤维化，而TLR4/MyD88非依赖性信号通路可能不参与缺血再灌注损伤后肾纤维化的过程。
