切应力通过Pim1/eNOS途径调节人脐静脉内皮细胞NO分泌

Shear stress regulates NO production in vascular endothelial cells through Pim1/eNOS signaling pathway

目的:探讨层流切应力是否可通过Pim1调节内皮型一氧化氮合酶(eNOS)活性,从而调节血管内皮细胞一氧化氮(NO)分泌。方法:体外原代培养人脐静脉内皮细胞(HUVECs),运用平行平板流动腔系统给HUVECs加载层流切应力(15 dyn/cm^2)。采用Western blot法检测Pim1蛋白表达及eNOS-Ser1177磷酸化水平;硝酸还原酶法检测NO分泌量;利用特异性小干扰RNA(siRNA)转染技术沉默Pim1基因后再检测上述指标的变化。结果:切应力作用HUVECs 15 min,可以显著上调Pim1蛋白表达(P<0.05),同时显著增强eNOS-Ser1177磷酸化水平(P<0.05),伴随HUVECs NO分泌显著增多(P<0.05)。转染siPim1可以抑制切应力诱导的Pim1表达(P<0.05),同时抑制eNOS-Ser1177磷酸化(P<0.05),NO分泌随之显著降低(P<0.05)。结论:流体切应力可能通过Pim1/eNOS途径调节血管内皮细胞NO分泌。

AIM:To determine whether laminar shear stress regulates nitric oxide(NO)production in vascular endothelial cells through Pim1/endothelial nitric oxide synthase(eNOS)signaling pathway.METHODS:Human umbilical vein endothelial cells(HUVECs)were exposed to laminar shear stress using a parallel-plate flow system.NO production is evaluated by NO assay kit.Pim1 protein expression and eNOS phosphorylation were determined by Western blot.A specific small interfering RNA was used to knock down Pim1 gene expression,and then the changes of above indicators were detected.RESULTS:After 15-min exposure of HUVECs to laminar shear stress(15 dyn/cm^2),rapid increases in Pim1 protein expression and NO production were observed(P<0.05).Shear stress also caused time-dependent stimulation of eNOS phosphorylation(P<0.05).The shear-induced Pim1 expression and NO production were abrogated in the HUVECs transfected with siPim1(P<0.05).Pim1 silencing also prevented shear-induced rise of eNOS-Ser1177 phosphorylation(P<0.05).CONCLUSION:Pim1 may account for shear-induced NO production in endothelial cells due to phosphorylation activation of eNOS.