摘要目的 探讨磷酸肌醇3激酶/蛋白激酶B(PI3K/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(HUVEC),在氧化型低密度脂蛋白(ox-LDL,100 mg/L)以及ox-LDL( 100 mg/L)加genistein( 100nmoL/L)分别作用5、10、15、30和60 min的基础上,Greiss反应测定细胞培养上清中一氧化氮(N0)的含量,RT-PCR检测HUVEC eNOS mRNA的表达,Western blot检测HUVEC eNOS蛋白和磷酸化eNOS (Ser1179)的表达水平.Western blot检测PI3K/AKT抑制剂LY294002和NSC154020干预后,genistein 对HUVEC磷酸化eNOS( Ser1179)表达的影响.结果 HUVEC经100 nmol/L genistein(5、10、15、30和60 min)处理后,培养液NO浓度和磷酸化eNOS(Ser1179)表达水平均明显高于ox-LDL组(P均＜0.05),其中genistein 15 min处理组最高,然而eNOS mRNA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PI3K/AKT信号通路抑制剂LY294002和NSC154020干预后磷酸化eNOS( Ser1179)表达明显低于genistein 15 min处理组(P＜0.05).结论 genistein对HUVEC NO的诱导作用与其促进eNOS(Ser1175)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PI3K/AKT抑制剂LY294002和NSC154020阻断,可见genistein在促进eNOS活性过程中通过PI3K/AKT信号通路发挥重要作用.

abstractsObjective 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 ( PI3K 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 cells ( 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.