摘要
目的探讨磷酸肌醇3激酶／蛋白激酶B（P13K／AKT）信号通路在植物雌激素染料木素（genistein）促进内皮型一氧化氮合酶（eNOS）活性过程中的作用。方法体外培养人脐静脉内皮细胞（14UVEC），在氧化型低密度脂蛋白（OX—LDL，100ms／L）以及0x—LDL（100ms／L）加genistein（10（3nmol／L）分别作用5、10、15、30和60min的基础上，Greiss反应测定细胞培养上清中一氧化氮（NO）的含量，RT-PCR检测HUVECeNOSmRNA的表达，Westernblot检测HUVECeNOS蛋白和磷酸化eNOS（Ser1179）的表达水平。Westernblot检测P13K／AKT抑制剂LY294002和NSCl54020干预后，genistein对HUVEC磷酸化eNOS（Ser1179）表达的影响。结果HUVEC经100nmol／Lgenistein（5、10、15、30和60rain）处理后，培养液NO浓度和磷酸化eNOS（Ser1179）表达水平均明显高于lOX—LDL组（P均〈0．05），其中genistein15min处理组最高，然而eNOSmRNA和非磷酸化eNOS蛋白表达水平与OX-LDL组比较差异无统计学意义（P〉0．05）。HUVEC经P13K／AKT信号通路抑制剂LY294002和NSCl54020干预后磷酸化eNOS（Ser1179）表达明显低于genistein15min处理组（P〈0．05）。结论genistein对HUVECNO的诱导作用与其促进eNOS（Ser1179）磷酸化进而提高eNOS活性密切相关，而genistein对磷酸化eNOS（Ser1179）的诱导作用被P13K／AKT抑制剂LY294002和NSCl54020阻断，可见genistein在促进eNOS活性过程中通过P13K／AKT信号通路发挥重要作用。
Objective Genistein could inhibit the development of atherosclerosis. This study explored the role of PI3K/AKT signaling during genistein promoted eNOS activation. Methods Human umbilical vein endothelial cells （HUVECs） were incubated with ox-LDL （ 100 mg/L）, then treated with genistein （ 100 nmol/L） for 5, 10, 15, 30 and 60 min. The production of NO was assessed by Griess reaction in cell culture supernatant. The mRNA expression of endothelial nitric oxide synthase （eNOS） was detected by reverse transcription-polymerase chain reaction （RT-PCR）. The protein expression of eNOS and phosphorylation eNOS（Ser1179 ）were determined by Western blot. The effect of genistein on phosphorylation eNOS（ Ser1179） level was also observed in the presence of LY294002 or NSC154020 （ PDK and AKT inhibitors）. Results The concentration of NO and the expression level of phosphorylation eNOS （ Ser1179 ） were significantly increased in ox-LDL ＋ genistein treated cells than ox-LDL treated oells （ all P 〈 0. 05 ）, and the peak effects were observed at 15 min, however, eNOS mRNA and non-phosphorylated eNOS protein expression were similar between the two groups （ P 〉 0. 05 ） . Furthermore, the expression level of phosphorylation eNOS（ Ser1179） was significantly lower in PIK3/AKT inhibitors LY294002 and NSC154020 treated cells compared with ox-LDL ＋ genistein treated cells （all P 〈 0. 05 ）. Conclusion Genistein could promote the activity of eNOS through increasing phosphorylation eNOS （Ser1179） level through PI3K/AKT pathway.
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2012年第4期327-331,共5页
Chinese Journal of Cardiology
基金
山西省青年科技研究基金项目（2007021046）
