奈比洛尔对血管内皮细胞胰岛素抵抗的影响

Effect of nebivolol on vascular endothelial cells insulin resistance

目的观察奈比洛尔改善人主动脉内皮细胞(HAEC)胰岛素抵抗(IR)的作用及机制。方法HAEC分为空白组(正常培养,胰岛素不刺激),对照组(正常培养,胰岛素刺激),不同浓度奈比洛尔+对照组(对照组基础上加奈比洛尔0.1,1,10μmol·L^(-1)),IR组(高糖33.3 mmol·L^(-1)+胰岛素1×10^(-7) mol·L^(-1)),不同浓度奈比洛尔+IR组(IR基础上加奈比洛尔0.1,1,10μmol·L^(-1))。48 h后,细胞无血清饥饿处理12 h。以噻唑蓝(MTT)法检测细胞活性,以葡萄糖氧化酶法测HAEC葡萄糖消耗量,以硝基还原酶法检测上清液一氧化氮(NO)水平,以蛋白质印迹法测定蛋白表达。结果与对照组(100%)比较,IR组细胞活性无显著性改变(95.13±2.10)%,且不同浓度奈比洛尔(0.1、1、10μmol·L^(-1))预处理对IR组和对照组细胞活性均无显著性影响。IR组的细胞葡萄糖消耗量和上清液NO水平分别为(0.53±0.17)mmol·L^(-1),(33.11±1.35)μmol·L^(-1),对照组分别为(1.14±0.24)mmol·L^(-1),(38.50±3.43)μmol·L^(-1);1μmol·L^(-1)奈比洛尔+IR组分别为(0.94±0.15)mmol·L^(-1),(39.15±1.44)μmol·L^(-1),10μmol·L^(-1)奈比洛尔+IR组分别为(0.89±0.19)mmol·L^(-1),(37.31±1.90)μmol·L^(-1),对照组与IR组比较,差异有统计学意义(P<0.05);1,10μmol·L^(-1)奈比洛尔+IR组与IR组比较,差异均有统计学意义(P<0.05,P<0.01)。蛋白质印迹显示IR组细胞胰岛素受体底物-1(IRS-1),磷脂酰肌醇-3激酶(PI3K),蛋白激酶B(Akt),磷酸化Akt(p-Akt),内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达均较对照组降低,p-IRS-1蛋白表达升高(P<0.05);1μmol·L^(-1)奈比洛尔+IR组以上蛋白表达与IR组比较,差异均有统计学意义(P<0.05,P<0.01)。结论奈比洛尔调节IRS-1/PI3K/Akt/eNOS改善血管内皮细胞IR。

Objective To observe the effect and mechanism of nebivolol on insulin resistance(IR)in human aortic endothelial cells(HAEC).Methods HAEC were divided into blank group(normally cultured without insulin stimulation),control group(normally cultured stimulated with insulin),different concentration of nebivolol+control group(treated with nebivolol 0.1,1,10μmol·L^(-1) based on control),IR group(treated with high glucose 33.3 mmol·L^(-1) and insulin 1×10^(-7) mol·L^(-1) for 48 h),different concentration of nebivolol+IR group(treated with nebivolol 0.1,1,10μmol·L^(-1) based on IR group).After 48 h,the cells were treated with serum-free starvation for 12 h.Cell activities were measured by MTT assay.Glucose consumption was measured by glucose oxidase method.Nitric oxide(NO)level in superannuate was tested by nitroreductase method.Western blot was used to measure the expressions of different proteins.Results Compared with control group(100%),the cell activity in IR group had no significant change(95.13±2.10)%,and nebirol pretreatment at different concentrations(0.1,1,10μmol·L^(-1))had no significant effect on the cell activity in IR group and control group.The glucose consumption and NO level in IR group were(0.53±0.17)mmol·L^(-1)and(33.11±1.35)μmol·L^(-1),respectively,and in control group were(1.14±0.24)mmol·L^(-1),(38.50±3.43)μmol·L^(-1),1μmol·L^(-1)nebivolol+IR group were(0.94±0.15)mmol·L^(-1),(39.15±1.44)μmol·L^(-1),respectively,10μmol·L^(-1)nebivolol+IR group were(0.89±0.19)mmol·L^(-1)and(37.31±1.90)μmol·L^(-1),respectively,and the difference between control group and IR group was statistically significant(P<0.05).There were statistically significant differences between 1,10μmol·L^(-1)nebivolol+IR group and IR group(P<0.05,P<0.01).Western blot showed that the protein expressions of insulin receptor substrate 1(IRS-1),phosphatidylinositol-3 kinase(PI3K),protein kinase B(Akt),phosphorylated Akt(p-Akt),endothelial nitric oxide synthase(eNOS)and phosphorylated e NOS(p-eNOS)in IR group were lower than those in control group,the expression of p-IRS-1 protein was increased(P<0.05);the expression of above proteins in 1μmol·L^(-1) nebivolol+IR group was significantly different from those in IR group(P<0.05,P<0.01).Conclusion Nebivolol ameliorates vascular endothelial cells IR via IRS-1/PI3K/Akt/eNOS pathway.