氧化应激介导的Ras-ERK信号通路活化参与醛固酮诱导的肾小球系膜细胞增殖

Oxidative stress-dependent Ras-ERK activation involves in aldosterone-induced mesangial cellproliferation

目的探讨氧化应激介导的Ras-胞外信号调节激酶（ERKl／2）信号通路活化在醛固酮（ALDO）诱导的系膜细胞增殖中的作用。方法体外培养人肾小球系膜细胞，应用^3H-胸腺嘧啶（^3H-TdR）掺人法和细胞计数测定系膜细胞增殖；Western印迹检测Ki．RasA、c-Raf、MEKl／2和ERKI／2活化。结果ALDO可显著促进系膜细胞增殖，抗氧化剂乙酰半胱氨酸（NAC）、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）显著抑制ALDO诱导的系膜细胞增殖（均P〈0．01）。ALDO刺激系膜细胞3h，活化的Ki-RasA、c．Raf、MEKl／2和ERKl／2表达显著增强，分别是对照组的4．05倍、3．62倍、4．52倍和3．40倍（均P〈0．01）。抗氧化剂NAC几乎完全阻断ALDO诱导的Ki．RasA、c-Raf、MEKl／2和ERKl／2活化（均P〈0．01）。Ki．RasAsiRNA可呈浓度依赖性降低系膜细胞Ki．RasA表达，并显著抑制ALDO诱导的Ki-RasA活化及系膜细胞增殖（P〈0．01）。c-Raf抑制剂GW5074和MEKl／2抑制剂PD98059亦显著抑制ALDO诱导的系膜细胞增殖，其抑制率均达到65％（P〈O．01）。Ki-RasAsiRNA不能降低ALDO诱导的磷酸肌醇．3激酶（P13K）磷酸化。联合应用P13K抑制剂LY294002和MEKl／2抑制剂PD98059可完全阻断ALDO诱导的系膜细胞增殖（P〈O．01）。结论ALDO可通过氧化应激活化Ki-RasA-C-Raf-MEK-ERK信号通路。同时阻断ERKl／2和P13K信号通路可完全抑制ALDO诱导的系膜细胞增殖。

Objective To investigate the role of oxidative stress-dependent Ras- extracellular signal-regulated kinase （ERK1/2） signaling in aldosterone （ALDO）-induced mesangial cell proliferation. Methods The incorporation of 3H-thymidine （3H-TdR） and cell count were used as the measure of mesangial cell （MC） proliferation. Western blotting was used to detect the activation of Ki-RasA, c-Raf, MEK1/2, ERK1/2 and PI3K. Results Aldosterone significantly induced human mesangial cell proliferation, and anti-oxidant N-Acetylcysteine （NAC）, catalase, and super oxide dismutase （SOD） significantly inhibited ALDO-induced mesangial cell proliferation （P〈 0.01, respectively）. Stimulation by ALDO for 3 h, Ki-RasA, c-Raf, MEK1/2, and ERK1/2 activity increased by 4.05-, 3.62-, 4.52-, and 3.40-fold compared with control group （P 〈0.01, respectively）. NAC almost completely blocked ALDO-induced Ki-RasA, c-Raf, MEK1/2, andERK1/2 activation （P〈0.01, respectively）. Ki-RasA siRNA dose-dependently inhibited Ki-RasA expression, ALDO-induced Ki-RasA activation, and mesangial cell proliferation （P 〈0.01, respectively）, c-Raf inhibitor GW5074 and MEK1/2 inhibitor PD98059 also reduced ALDO-induced mesangial cell proliferation by 65% respectvely （P〈0.01）. Ki-RasA siRNA had no effect on ALDO- induced PI3K phosphorylation. Combining LY294002 and PD98059 completely blocked ALDO- induced mesangial cell proliferation （P〈0.01）. Conclusions ALDO-induced Ki-RasA-c-Raf- MEK-ERK signaling activation is dependent on reactive oxygen species （ROS） production, which mediates ALDO-induced mesangial cell proliferation. Inhibition of both ERK1/2 and PI3K signaling simultaneously completely blocks ALDO-induced mesangial cell proliferation.