α-硫辛酸通过mTOR信号通路调节高糖诱导的大鼠肾小球系膜细胞增殖

Alpha lipoic acid modulates high glucose-induced rat mesangial cell proliferation via mTOR signaling pathway

目的观察α-硫辛酸（α lipoicacid，LA）是否通过哺乳动物雷帕霉素靶蛋白（mTOR）信号通路调节高糖诱导的肾小球系膜细胞增殖。方法应用四甲基偶氮唑盐（MTT）法检测细胞增殖，流式细胞术检测细胞周期，实时定量PCR检测AMP活化的蛋白激酶（AMPK）mRNA的表达，Western印迹法检测AMPK、蛋白激酶B（Akt）、mTOR、核糖体40S小亚基s6蛋白激酶（p70S6K）、真核细胞翻译起始因子4E结合蛋白1（4EBP1）的磷酸化水平。结果0．25mmol／LLA促进高糖培养的大鼠肾小球系膜细胞增殖（P〈0．01），增加S期细胞数量（P〈0．01），并增加肾小球系膜细胞Akt、mTOR、p70S6K、4EBP1磷酸化水平（均P〈0．05）。而抑制Akt活性后，0．25mmol／LLA的上述作用消失。相反，1．0mmol／LLA则抑制高糖培养的大鼠肾小球系膜细胞增殖（P〈0．01），并降低S期细胞比例（P〈0．01），还增加AMPK的磷酸化（P〈0．01），降低mTOR、p70S6K、4EBPl的蛋白表达活性（P〈0．05）。抑制AMPK活性可逆转1．0mmol／LLA的上述作用。结论LA可通过调控roTOR信号通路，对高糖诱导的肾小球系膜细胞增殖呈浓度依赖性的调节作用。

Objective To examine whether alpha lipoic acid（LA） regulates high glucose-induced mesangial cell proliferation via mammalian target of rapamycin （mTOR） signaling. Methods The cell proliferation and cycle were determined by methylthiazoletetrazolium（MTT） assay and flow cytometry, respectively. The mRNA expression of AMP-activated protein kinase（AMPK） was detected by realtimc PCR. The phosphorylation levels of protein kinase B （ Akt）, mTOR, eukaryotic translation initiation factor 4E binding protein 1 （4EBP1）, and 70S6 kinase （p70S6K） were measured by Western blot. Results 0.25 mmol/L LA promoted high glucose-sitmulated rat mesangial cell proliferation（ P〈0.01 ） and entry of cell cycle into S phase（ P〈0.01 ）, along with increased phosphorylation levels of Akt, mTOR, p70S6K, and 4EBP1 （P〈0.05）. These effects of 0.25 mmol/L LA disappeared when Akt activity was inhibited. On the contrary, 1.0 mmol/L LA inhibited high glucose-induced cell proliferation（ P〈0.01 ） and entry of cell cycle into S phase （ P〈0.01 ）, with the decreased phosphorylation levels of roTOR, p70S6K, and 4EBP1 （ P〈 0.05 ） and the enhanced activity of AMPK（ P〈0.01 ）. These effects of 1.0 mmol/L LA were prevented when AMPK activity was inhibited. Conclusions LA dose-dependently regulates mesangial cell proliferation induced by glucose via mTOR signaling pathway.