雷公藤甲素对血管紧张素Ⅱ诱导足细胞损伤的保护作用及其机制研究

Triptolide protects podocytes from angiotensin Ⅱ induced injury in vitro

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 investigate whether tripotolide have direct effect on podocyte injured by Ang II .9 If so, how does it work？ Methodology：The heat-sensitive mouse podocytes were divided into three groups： negative control, positive control treated with Ang I1 （10^-6, 10^-7, 10^-8mol/L） and pretrcated with triptolide （1, 3, 10 ng/ml） for 2 hours. F-actin and zonula oeeludens-1 （ZO-1） were observed by fluorescence microscopy. To explore the underlying mechanism, we observed reactive oxygen species （ROS） generation and the subsequent MAPK activation by flow cytometry and western blot. Results：Ang II induced actin cytoskeleton reorganization and ZO-1 redistribution in a dose-dependent manner. In untreated podocytes, the ZO-1 staining was peripherally distributed at contacts of adjacent cells as fine segments, while it appeared markedly fragmented in response Ang II, indicating tight junction loose and cell ruffling. Triptolide stabilized actin filaments and improved ZO-1 distribution in a dose dependent manner. At the dose of 10 ng/ml, triptolide almost completely restored the normal cytoskeleton and cell contact in podocyte without affecting cell survival. Ang II （10^-7 mol/L） significantly increased ROS generation in podocyte. This effect was observed rapidly at 10 min and maintained for 30 rain. Pretreatment of podocyte with triptolide （ 10 ng/ml） or antioxidant NAC （ 10 mmoL/L） before Ang II exposure led to a significant reduction in the cellular ROS level. In addition, antioxidant NAC successfully reduced Ang II-induced podocyte damage as showed by cytoskeleton staining, indicating that ROS generation mediates Ang-induced podocyte injury. Furthermore, triptolide effectively inhibited Ang II-induced p-38, ERK1/2 and JNK MAPK activation, which are the downstream signaling molecules of ROS. SB203580 and U0126, which are specific kinase inhibitor for p-38 and ERK MAPK, can block Ang 1I induced podocyte injury. Conelusion：Triptolide showed dramatic protective effect in Ang II induced podocyte injury. The protective effect of triptolide might partially contribute to the inhibition of ROS generation and the subsequent MAPK activation.