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目的:观察雷公藤甲素对血管紧张素Ⅱ(AngⅡ)诱导足细胞损伤的保护作用,并探讨其作用机制。方法:免疫荧光染色分析足细胞骨架肌动蛋白(F-actin)和细胞间连接蛋白1(ZO-1)表达和分布;流式细胞仪分析荧光探针CM-H2DCFDA标记的细胞内活性氧(ROS);Western blot检测p-38,细胞外信号调节激酶1/2(ERK1/2)和C-Jun氨基端激酶(JNK)丝裂原活化蛋白激酶(MAPK)磷酸化水平。结果:AngⅡ呈剂量依赖性破坏足细胞骨架结构和细胞间连接,表现为F-actin在胞质中的应力纤维解聚,F-actin的边聚。细胞间连接的断裂,细胞皱缩。雷公藤甲素呈剂量依赖性的拮抗AngⅡ诱导的骨架蛋白解聚和细胞间连接分子的重排,10ng/ml雷公藤甲素的作用最为明显。雷公藤甲素对足细胞AngⅡ的1型受体(AT1R)和2型受体(AT2R)的表达无明显影响。细胞内ROS及其下游的MAPK信号通路研究显示,AngⅡ(10-7mol/L)诱导足细胞内ROS的明显增加,激活p-38,ERK和JNK三条MAPK信号通路,雷公藤甲素(10ng/ml)或抗氧化剂N乙酰半胱氨酸(NAC)(10mmol/L)预处理细胞后,能够有效遏制AngⅡ诱导的细胞内ROS的产生,抑制MAPK信号通路的活化。结论:雷公藤甲素可稳定足细胞的骨架结构和细胞间连接,拮抗AngⅡ对足细胞的损伤。雷公藤甲素的上述作用与细胞内ROS及下游的p-38、ERKMAPK信号通路密切相关。雷公藤甲素干预AngⅡ诱导的足细胞损伤可能部分参与了其对肾小球疾病的疗效。
Objective: To observe the protective effect of triptolide on angiotensin Ⅱ-induced podocyte injury and to explore its mechanism. Methods: The expression and distribution of actin (F-actin) and intercellular adhesion protein 1 (ZO-1) in podocyte were analyzed by immunofluorescence staining. The intracellular reactive oxygen species (ROS). The phosphorylation levels of p-38, ERK1 / 2 and MAPK were detected by Western blot. RESULTS: AngⅡin a dose-dependent manner destroyed the podocyte cytoskeleton and intercellular junctions. The results showed that stress fibers depolymerized F-actin in the cytoplasm and polygranular F-actin. Breaking of intercellular junctions, cell shrinkage. Triptolide showed a dose-dependent antagonism of AngⅡ-induced depolymerization of the framework protein and rearrangement of intercellular junction molecules, and the effect of triptolide at 10 ng / ml was most pronounced. Triptolide had no significant effect on the expression of Ang Ⅱ type 1 receptor (AT1R) and type 2 receptor (AT2R) in podocytes. The intracellular ROS and its downstream MAPK signaling pathway showed that AngⅡ (10-7mol / L) induced a significant increase of ROS in podocytes, activated three MAPK signaling pathways, p-38, ERK and JNK. Triptolide (10ng / ml or NAC (10 mmol / L) could effectively inhibit the production of intracellular ROS induced by AngⅡand inhibit the activation of MAPK signaling pathway. Conclusion: Triptolide can stabilize the podocyte cytoskeleton and intercellular junctions and antagonize the injury of podocytes induced by AngⅡ. The above effects of triptolide and intracellular ROS and downstream p-38, ERKMAPK signaling pathways are closely related. Triptolide intervention AngⅡ-induced podocyte injury may be partially involved in the efficacy of glomerular diseases.