小窝在G蛋白偶联雌激素受体调控小鼠主动脉内皮细胞一氧化氮合酶活性中的作用

Effect of caveolae on G protein coupled estrogen receptor-regulated nitric oxide synthase activity in mouse aortic endothelial cells

目的探讨小窝(caveolae)对雌激素通过G蛋白偶联雌激素受体(GPER)调控小鼠主动脉内皮型一氧化氮合酶(eNOS)活性的影响。方法当细胞融合度达到80%~90%时,选取内皮细胞分为对照1组、DMSO组、雌二醇1组、雌二醇2组、雌二醇3组(n=5),其中雌二醇1组、雌二醇2组、雌二醇3组按浓度梯度加入雌二醇(10、50、100 nmol/L),对照1组不予任何处理,DMSO组加入50μl DMSO培养。另选取内皮细胞分为对照2组(未作任何处理)、阴性对照1组[加入小窝蛋白1(Cav-1)阴性对照干扰RNA]、Cav-1敲减组(加入Cav-1干扰RNA);选取部分内皮细胞设对照3组(未作任何处理)、阴性对照2组(加入GPER阴性对照干扰RNA)、GPER敲减组(加入GPER干扰RNA)。检测Cav-1、GPER、eNOS、磷酸化eNOS(p-eNOS)蛋白表达。结果与对照1组比较,雌二醇3组p-eNOS蛋白表达明显增加(0.47±0.07 vs 0.20±0.04,P<0.05)。对照2组、阴性对照1组、Cav-1敲减组GPER、eNOS、Cav-1蛋白表达比较,差异有统计学意义(P<0.05,P<0.01)。与对照2组比较,Cav-1敲减组GPER、eNOS、Cav-1蛋白表达明显降低(P<0.05,P<0.01)。对照3组、阴性对照2组、GPER敲减组eNOS、p-eNOS表达比较,无统计学差异(P>0.05)。与对照3组比较,GPER敲减组GPER蛋白表达明显降低(0.56±0.22 vs 2.40±0.65,P<0.05)。结论小窝参与了雌激素通过GPER调控小鼠主动脉eNOS活性的过程。

Objective To study the effect of caveolae on GPER-regulated eNOS activity in mouse aortic endothelial cells.Methods The mouse aortic endothelial cells at a fusin level of 80%-90%were divided into control group 1,DMSO group,estradiol group 1,estradiol group 2,estradiol group 3 with 10 nmol/L estradiol,50 nmol/L estradiol and 100 nmol/L estradiol added into estradiol group 1,estradiol group 2 and estradiol group 3 respectively.No estradiol was added into control group and 50μl DMSO was added into DMSO group and cultured.The mouse aortic endothelial cells were further divided into control group 2 into which no Cav-1 was added,negative control group 1 into which Cav-1 was added for negative control of siRNA,Cav-1 knockdown group into which Cav-1 siRNA was added,control group 3 into which no GPER was added,negative control group 2 into which GPER was added for negative control of siRNA,GPER knockdown group into which GPER siRNA was added.The expressions of Cav-1,GPER,eNOS and p-eNOS protein were detected.Results The expression level of eNOS protein was significantly higher in estradiol group 3 than in control group 1(0.47±0.07 vs 0.20±0.04,P<0.05).The expression levels of GPER,eNOS and Cav-1 protein were significantly different in control group 2,negative control group 1 and Cav-1 knockdown group(P<0.05,P<0.01)and significantly lower in Cav-1 knockdown group than in control group 2(P<0.05,P<0.01).No significant difference in expressions of eNOS and p-eNOS was detected in control group 3,negative control group 2 and GPER knockdown group(P>0.05).The expression level of GPER protein was significantly lower in GPER knockdown group than in control group 3(0.56±0.22 vs 2.40±0.65,P<0.05).Conclusion Caveolae is involved in GPER-regulated eNOS activity in mice aortic endothelial cells.