丹参素通过SIRT1-SOD信号通路延缓大鼠主动脉内皮细胞老化

Danshensu delays the senescence of rat aortic endothelial cells via activation of SIRT1-SOD pathway

本研究旨在观察丹参素(Danshensu,DSS)预处理对大鼠主动脉内皮细胞(rat aortic endothelial cells,RAECs)老化的影响及机制。原代RAECs株传代培养到第四代为年轻组细胞(Young group),传代至第十二代为老年组细胞(Old group);自第四代起,给予DSS孵育到第十二代,即为DSS组细胞;自第四代起,联合给予DSS和细胞沉默信息调节因子1(SIRT1)抑制剂尼克酰胺(NAM)孵育到第十二代,即为DSS+N组细胞。用细胞衰老β-半乳糖苷酶(SAβ-gal)染色法鉴定细胞老化程度,DAPI荧光染色检测老化相关的异染色质位点(senescence associated heterochromatin foci,SAHF)的表达变化,硫代巴比妥酸(TBA)法和化学比色法检测RAECs中丙二醛(MDA)和过氧化氢(H2O2)含量,免疫印迹法测定细胞SIRT1、黄嘌呤氧化酶(XOD)和超氧化物歧化酶2(SOD2)的表达。结果显示,与年轻组相比,老年组细胞发生明显衰老,细胞SAβ-gal阳性率、SAHF形成率、MDA及H2O2含量显著增加(均P<0.01);DSS处理能明显减少老年组细胞SAβ-gal阳性率、SAHF形成率、MDA及H2O2含量(P<0.05或P<0.01),SIRT1阻断剂可以逆转DSS对细胞的保护作用;免疫印迹结果显示,与年轻组相比,老年组细胞XOD蛋白表达增加,SIRT1和SOD2蛋白表达减少(P<0.05或P<0.01),DSS可以使老年组细胞XOD蛋白表达减少,SIRT1和SOD2蛋白表达增加(P<0.05);而给予SIRT1阻断剂逆转DSS的作用。以上结果提示,DSS预处理能减轻RAECs的氧化应激水平,延缓细胞的衰老进程,其机制可能与SIRT1蛋白表达的上调有关。

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 H2O2 contents; 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 H2O2 levels（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 SOD2 expression 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 SOD2 were elevated, while nicotinamide reversed the effects of DSS. These results suggest that DSS delays senescence of RAECs via up-regulation of SIRT1.