SIRT1/eNOS/NO通路在人参皂苷Rb1抗内皮细胞复制性衰老中的作用

Role of SIRT1/eNOS/NO signaling pathway in protective effect of ginsenoside Rb1 against replicative senescence of endothelial cells

目的:观察人参皂苷Rb1延缓人脐静脉内皮细胞(HUVECs)复制性衰老的作用,并探讨SIRT1/e NOS/NO通路在其中的作用机制。方法:建立原代HUVECs复制性衰老模型,根据细胞形态的变化、衰老相关β-半乳糖苷酶(SA-β-Gal)染色阳性率和纤溶酶原激活物抑制剂1(PAI-1)的表达水平评估HUVECs衰老情况;采用real-time PCR方法和Western blot法检测沉默SIRT1前后衰老细胞中e NOS和PAI-1的mRNA和蛋白表达,并检测细胞上清NO的含量。结果:累积细胞群体倍增水平(CPDL)为16的HUVECs可作为复制性衰老模型; 80μmol/L人参皂苷Rb1处理后衰老细胞内SIRT1和eNOS的mRNA及蛋白表达水平增加,NO含量增加(P <0. 05),而PAI-1的mRNA和蛋白表达水平下降(P <0. 05);沉默SIRT1后,衰老细胞内e NOS的mRNA及蛋白表达减少,PAI-1的mRNA及蛋白表达增加,NO含量减少(P <0. 05);与SIRT1沉默组比较,在沉默SIRT1基础上加用人参皂苷Rb1后,e NOS和PAI-1表达水平及NO的含量未见明显变化。结论:人参皂苷Rb1可通过调控SIRT1/e NOS/NO通路延缓HUVECs复制性衰老。

AIM:To explore the effect of ginsenoside Rb1 on replicative senescence of endothelial cells and the role of SIRT1/eNOS/NO signaling pathway in this process.METHODS:The replicative senescence model of primary human umbilical vein endothelial cells(HUVECs)was established.The morphological change of the cells,the proportion of senescence-associatedβ-galactosidase(SA-β-Gal)positive cells and the plasminogen activator inhibitor 1(PAI-1)expression were detected to assess the senescence model.The expression of eNOS and PAI-1 at mRNA and protein levels in the aging cells was determined by real-time PCR and Western blot before and after silencing of SIRT1 was performed.The NO concentration in the cell culture supernatant was measured by nitrate reductase assay.RESULTS:HUVECs with cumulative population-doubling level(CPDL)at 16 were chosen as the replicative senescence model in this research.Ginsenoside Rb1 at 80μmol/L significantly reduced the expression of PAI-1 at mRNA and protein levels.Furthermore,ginsenoside Rb1 increased the expression of SIRT1 and eNOS at mRNA and protein levels,and increased the NO content.SIRT1 silencing inhibited the expression of eNOS at mRNA and protein levels and reduced NO generation,leading to an increase in the expression of PAI-1 at mRNA and protein levels.Upon intervention of ginsenoside Rb1,the eNOS and PAI-1 expression and the level of NO were not reversed.CONCLUSION:Ginsenoside Rb1 modulates SIRT1/eNOS/NO signaling pathway to prevent the replicative senescence of HUVECs.