CAJ | 학술논문

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.
목적 탐토양화응격개도적Ras-포외신호조절격매(ERK1/2)신호통로활화재철동동( ALDO)유도적계막세포증식중적작용.방법 체외배양인신소구계막세포,응용3H-흉선밀정(3H-TdR)참입법화세포계수측정계막세포증식；Western인적검측Ki-RasA、c-Raf、MEK1/2화ERK1/2활화.결과 ALDO가현저촉진계막세포증식,항양화제을선반광안산(NAC)、과양화경매(CAT)、초양화물기화매(SOD)현저억제ALDO유도적계막세포증식(균P＜ 0.01).ALDO자격계막세포3h,활화적Ki-RasA、c-Raf、MEK1/2화ERK1/2표체현저증강,분별시대조조적4.05배、3.62배、4.52배화3.40배(균P＜0.01).항양화제NAC궤호완전조단ALDO유도적Ki-RasA、c-Raf、MEK1/2화ERK 1/2활화(균P＜0.01).Ki-RasA siRNA가정농도의뢰성강저계막세포Ki-RasA표체,병현저억제ALDO유도적Ki-RasA활화급계막세포증식(P＜0.01).c-Raf억제제GW5074화MEK1/2억제제PD98059역현저억제ALDO유도적계막세포증식,기억제솔균체도65％(P＜0.01).Ki- RasA siRNA불능강저ALDO유도적린산기순-3격매( PI3K)린산화.연합응용PI3K억제제LY294002화MEKl/2억제제PD98059가완전조단ALDO유도적계막세포증식(P＜0.01).결론 ALDO가통과양화응격활화Ki-RasA-c-Raf-MEK-ERK신호통로.동시조단ERK1/2화PI3K신호통로가완전억제ALDO유도적계막세포증식.
Objective To investigate the role of oxidative stress-dependent Rasextracellular 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.