Xiao Xinhuai, Xu Miqing, Fang Yanling
Department of Geriatrics, Second Affiliated Hospital, Guangzhou Medical University, Guangzhou 510260, China. *Corresponding author, E-mail:
Department of Geriatrics, Second Affiliated Hospital, Guangzhou Medical University, Guangzhou 510260, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2019 Sep;35(9):806-811.
Objective To explore the effect of tanshinone IIA (TSA) on hydrogen peroxide (HO)-induced senescence of human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Methods HUVECs were cultured in vitro and divided into the control group, model group and TSA group. The cells in the TSA group were pre-treated with TSA for 24 hours. HO was used to induce cell senescence in the model and TSA groups. Transfection with SIRT1 siRNA was used for the knockdown of SIRT1 in HUVECs. CCK-8 assay was performed to detect cell viability. The expression levels of senescence-related proteins (P21 and P26), SIRT1, phosphorylated endothelial nitric oxide synthase (p-eNOS), and eNOS were detected by Western blot analysis. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to evaluate cell senescence. Results Pretreatment with TSA at low concentrations (10, 20 and 40 μg/mL) for 24 hours did not affect cell viability, while high concentrations (80, 160 and 320 μg/mL) decreased cell viability significantly. In addition, 10, 20 and 40 μg/mL of TSA promoted HO-mediated cell viability of HUVECs in a concentration-dependent manner. Compared with the control group, the positive rate of SA-β-gal staining in the model group increased, while the positive rate in the TSA group was significantly lower than that in the model group. The expression levels of P21 and P16 protein in the model group were higher than those in the control group, while SIRT1 and p-eNOS/eNOS were lower than those in the control group. Conversely, the expression of P21 and P16 proteins in the TSA group were lower than those in the model group, and SIRT1 and p-eNOS/eNOS were higher in the TSA group than those in the model group. Transfected with SIRT1 siRNA significantly down-regulated the expression of SIRT1 in HUVECs and the positive rate of SA-β-gal staining was notably raised when SIRT1 was silenced in TSA-treated HUVECs. Conclusion TSA attenuates HO-induced endothelial cell senescence by activating SIRT1/eNOS signaling pathway.
目的 探讨丹参酮IIA（TSA）对过氧化氢（HO）诱导的人脐静脉内皮细胞（HUVECs）衰老的影响及其潜在机制。方法 将HUVECs体外培养，分为对照组、模型组和TSA组。TSA组细胞先用TSA预处理24小时。模型组和TSA组用HO诱导细胞衰老。采用SIRT1 siRNA转染敲低HUVECs中的SIRT1。采用CCK-8法检测细胞活力。通过蛋白质免疫印迹分析检测衰老相关蛋白（P21和P26）、SIRT1、磷酸化内皮型一氧化氮合酶（p-eNOS）和eNOS的表达水平。进行衰老相关β-半乳糖苷酶（SA-β-gal）染色以评估细胞衰老。结果 低浓度（10、20和40μg/mL）的TSA预处理24小时不影响细胞活力，而高浓度（80、160和320μg/mL）显著降低细胞活力。此外，10、20和40μg/mL的TSA以浓度依赖的方式促进HO介导的HUVECs细胞活力。与对照组相比，模型组SA-β-gal染色阳性率升高，而TSA组阳性率明显低于模型组。模型组P21和P16蛋白表达水平高于对照组，而SIRT1和p-eNOS/eNOS低于对照组。相反，TSA组P21和P16蛋白表达低于模型组，TSA组SIRT1和p-eNOS/eNOS高于模型组。用SIRT1 siRNA转染显著下调HUVECs中SIRT1的表达，在TSA处理的HUVECs中沉默SIRT1时，SA-β-gal染色阳性率显著升高。结论 TSA通过激活SIRT1/eNOS信号通路减轻HO诱导的内皮细胞衰老。
