Wang Shuo, Wu Di, Liu Lei, Cui Jie, Qiao Wei-Li, Sun Hong, Yan Chang-Dong
Department of Physiology; Institute of Cardiovascular Disease; Jiangsu Provincial Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221004, China.
Sheng Li Xue Bao. 2014 Oct 25;66(5):575-82.
The present study was aimed to investigate the effect of pretreatment with Danshensu (DSS) on rat aortic endothelial cells (RAECs) senescence and the underlying mechanisms. Cultured RAECs at fourth and twelfth passages were taken as young and old groups, respectively. DSS and DSS+nicotinamide (DSS+N) groups were incubated with DSS and DSS in combination with nicotinamide, an inhibitor of silent information regulator 1 (SIRT1), from the fourth to twelfth passage, respectively. The cell status of senescence was determined by the senescence-associated β-galactosidase (SA β-gal) staining, and 4,6-diamino-2-phenyl indole (DAPI) fluorescent dye was used to detect senescence associated heterochromatin foci (SAHF) formation; Thiobarbituric acid (TBA) and colorimetric methods were used to evaluate malondialdehyde (MDA) and H₂O₂contents; Western blot was employed to analysis the expressions of xanthine oxidase (XOD), SIRT1 and superoxide dismutase 2 (SOD₂) in the RAECs. The results showed that, in comparison with young group, the old group exhibited higher SA β-gal positive and SAHF formation rates, as well as higher MDA and H₂O₂levels (P < 0.05 or P < 0.01), whereas DSS pretreatment reduced SA β-gal positive and SAHF formation rates, decreased MDA and H2O2 contents (P < 0.05 or P < 0.01). The protection of DSS was reversed by nicotinamide. Compared with the young group, the old group showed higher expression levels of XOD, but lower SIRT1 and SOD₂expression levels (P < 0.05 or P < 0.01). With the pretreatment of DSS, the expression of XOD was declined, and the expression levels of SIRT1 and SOD₂were elevated, while nicotinamide reversed the effects of DSS. These results suggest that DSS delays senescence of RAECs via up-regulation of SIRT1.
本研究旨在探讨丹参素（DSS）预处理对大鼠主动脉内皮细胞（RAECs）衰老的影响及其潜在机制。将培养至第4代和第12代的RAECs分别作为年轻组和老年组。DSS组和DSS +烟酰胺（DSS + N）组分别在第4代至第12代期间用DSS以及DSS与沉默信息调节因子1（SIRT1）抑制剂烟酰胺联合孵育。通过衰老相关β-半乳糖苷酶（SA β-gal）染色确定细胞衰老状态，并用4,6-二氨基-2-苯基吲哚（DAPI）荧光染料检测衰老相关异染色质灶（SAHF）的形成；采用硫代巴比妥酸（TBA）法和比色法评估丙二醛（MDA）和H₂O₂含量；采用蛋白质免疫印迹法分析RAECs中黄嘌呤氧化酶（XOD）、SIRT1和超氧化物歧化酶2（SOD₂）的表达。结果显示，与年轻组相比，老年组SA β-gal阳性率和SAHF形成率更高，MDA和H₂O₂水平也更高（P < 0.05或P < 0.01），而DSS预处理可降低SA β-gal阳性率和SAHF形成率，降低MDA和H₂O₂含量（P < 0.05或P < 0.01）。烟酰胺可逆转DSS的保护作用。与年轻组相比，老年组XOD表达水平更高，但SIRT1和SOD₂表达水平更低（P < 0.05或P < 0.01）。经DSS预处理后，XOD表达下降，SIRT1和SOD₂表达水平升高，而烟酰胺可逆转DSS的作用。这些结果表明，DSS通过上调SIRT1延缓RAECs衰老。
