醛固酮通过PI3K-Akt-mTOR-p70S6K1信号通路促进系膜细胞增殖

目的 探讨氧化应激依赖的表皮生长因子受体（EGFR）活化在醛固酮（ALDO）诱导的磷酸肌醇-3激酶-蛋白激酶B（PI3K-Akt）信号通路活化及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；荧光探针2，7-二氯二氢荧光素乙酰乙酸检测细胞内活性氧（ROS）的产生；Western印迹法检测EGFR、PI3K、Akt、雷帕霉素靶蛋白（mTOR）和核糖体蛋白p70S6激酶1（p70S6K1）磷酸化。 结果 （1）ALDO可呈时间依赖性和剂量依赖性的方式促进肾小球系膜细胞ROS产生，100 nmol/L ALDO刺激3 min，ROS产生即显著增加，刺激60 min达到高峰；1、10和100 nmol/L ALDO刺激60 min，ROS产生分别是对照组的1.50、1.70和2.14倍。抗氧化剂乙酰半胱氨酸（NAC）能阻断ALDO诱导的系膜细胞增殖。（2）ALDO可呈时间依赖性和剂量依赖性的方式诱导肾小球系膜细胞EGFR磷酸化，100 nmol/L ALDO刺激5 min，EGFR磷酸化显著增强，刺激60 min达到高峰；1、10和100 nmol/L ALDO刺激60 min，EGFR磷酸化分别是对照组的2.29、3.55和5.25倍。NAC和盐皮质激素受体（MR）阻断剂依普利酮能显著抑制ALDO诱导的EGFR磷酸化，而EGFR特异性抑制剂AG1478能完全阻断ALDO诱导的系膜细胞增殖。（3）100 nmol/L ALDO刺激60 min能显著增加PI3K、Akt、mTOR和p70S6K1磷酸化，其增加倍数分别为对照组的4.35、5.38、3.85和3.57倍，应用NAC和AG1478能阻断ALDO诱导的PI3K、Akt、mTOR和p70S6K1磷酸化。（4）PI3K抑制剂LY294002、Akt抑制剂以及mTOR抑制剂雷帕霉素能显著抑制ALDO诱导的系膜细胞增殖，其抑制率达到50%~55%。 结论 ALDO通过氧化应激依赖的EGFR磷酸化促进肾小球系膜细胞PI3K-Akt-mTOR-p70S6K1信号通路活化及细胞增殖。

Aldosterone induces mesangial cell proliferation via PI3K-Akt-mTOR-p70S6K1 activation

Objective To elucidate the role of oxidative stress-dependent epidermal growth factor receptor (EGFR) trans-activation in the aldosterone (ALDO)-induced phosphatidyl inositol 3-kinase (PI3K)-Akt activation and mesangial cells (MCs) proliferation. Methods The incorporation of 3H-thymidine (3H-TdR) and cell count were used to measure MCs proliferation. Reactive oxygen species (ROS) production was determined by DCFDA fluorescence. EGFR, PI3K, Akt, mammalian target of rapamycin (mTOR), and p70S6K1 phosphorylation were assayed by Western blotting. Results (1) ALDO induced ROS production in time- and dose-dependent manner in cultured human MCs. ROS generation was significantly increased by incubation with 100 nmol/L ALDO for 3 min, and peaked at 60 min. ROS production was increased by 1.50-, 1.70-, and 2.14-fold after stimulation by 100 nmol/L ALDO for 60 min. Antioxidant N-acetylcysteine (NAC) almost completely blocked ALDO-induced MCs proliferation. (2) ALDO induced EGFR phosphorylation in time- and dose-dependent manner in cultured human MCs. EGFR phosphorylation was significantly increased by incubation with 100 nmol/L ALDO for 5 min, and peaked at 60 min. EGFR trans-activation was increased by 2.29-, 3.55-, and 5.25-fold after stimulation by 100 nmol/L ALDO for 60 min. NAC and mineralocorticoid receptor (MR) antagonist eplerenone significantly inhibited ALDO-induced EGFR phosphorylation, and EGFR antagonist AG1478 blocked ALDO-induced MCs proliferation. (3) ALDO significantly stimulated the phosphorylation of PI3K, Akt, mTOR and p70S6K1 by 4.35-, 5.38-, 3.85-, and 3.57-fold. PI3K inhibitor LY 294002, Akt inhibitor, and mTOR inhibitor rapamycin reduced ALDO-induced MCs proliferation by 50%-55%. Conclusion Oxidative stress-dependent EGFR trans-activation mediates ALDO-induced PI3K-Akt-mTOR-p70S6K1 signal pathway activation and MCs proliferation.