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

目的 探讨氧化应激介导的Ras-胞外信号调节激酶(ERK1/2)信号通路活化在醛同酮( 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)掺入法和细胞计数测定系膜细胞增殖；Western印迹检测Ki-RasA、c-Raf、MEK1/2和ERK1/2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、MEK1/2和ERK1/2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、MEK1/2和ERK 1/2活化(均P＜0.01).Ki-RasA siRNA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MEK1/2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＜0.01).Ki- RasA siRNA不能降低ALDO诱导的磷酸肌醇-3激酶( PI3K)磷酸化.联合应用PI3K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＜0.01).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ERK1/2和PI3K信号通路可完全抑制ALDO诱导的系膜细胞增殖.

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

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, and ERK1/2 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.