人参三七川芎提取物延缓内皮细胞复制性衰老的机制研究

Mechanisms of delay endothelial cell replicative senescence by extracts from Panax ginseng,Panax notoginseng and Ligusticum chuanxiong

目的:探讨人参三七川芎提取物对内皮细胞复制性衰老(humanumbilical vein endothelial cells,HUVECs)的影响。方法:体外培养的HUVEC-2C传代至第8代,造成内皮细胞复制性衰老模型。细胞分为4组,8代衰老模型组、维生素E(Vit E)组(50 mg.L-1)、中药高、低(50,20 mg.L-1)剂量组,采用衰老相关β-半乳糖苷酶(SA-β-gal)染色法观察细胞衰老改变,流式细胞仪分析细胞周期,激光共聚焦显微镜观察细胞内活性氧(ROS)含量,硝酸还原酶法检测培养液一氧化氮(NO)和抗超氧阴离子水平,Western-blot法检测各组细胞AngⅡ1型和2型受体(AT1R和AT2R),NAD(P)H氧化酶p47phox的蛋白表达。结果:8代衰老模型组β-gal染色阳性细胞数明显增多,细胞停滞在G0/G1期,激光共聚焦显微镜下ROS荧光强度明显增强,培养液中抗超氧阴离子、NO含量减少,p47phox,AT1R,AT2R蛋白表达上调。给予高、低剂量中药处理后,可明显改善细胞的衰老状态,β-gal染色阳性细胞数减少,G0/G1期细胞减少,G2/M期细胞增加,ROS荧光强度减弱,抗超氧阴离子、NO含量较8代衰老模型组增加,p47phox,AT1R,AT2R蛋白表达下调,差异均有统计学意义(均P<0.05)。结论:人参三七川芎提取物能够延缓内皮细胞复制性衰老,通过活性氧途径下调NADPH氧化酶p47phox的表达,最终使ROS产生减少;同时,中药提取物还能降低衰老内皮细胞AT1R,AT2R的表达,改善内皮功能,这可能是益气活血中药延缓内皮细胞衰老的主要原因之一。

Objective： To explore effect of extracts from Panax ginseng, P. notoginseng and Ligusticum chuanxiong on human umbilical endothelial cells （HUVECs） replicative senescence. Method： HUVECs were induced to aging by generation cultivating to the eighth cells in order to establish a model of endothelial ceils replicative senescence. The cultured HUVECs in vitro were divided into 4 groups, the eighth generation cell-senescence untreated group, Vitamin E group, herbal treated high dose and low dose groups. Changes of HUVECs aging were observed by method of SA-β-gal stained HUVECs and cells cycle were analyzed. Contents of ROS in cells,the levels of anti-superoxide （O^2- ） and nitric oxide （NO） in cell mediums were examined. Western blot were used to analyse protein expression of NADPH oxidase p47phox, angiotensin type 1 and 2 receptor （AT1 R, AT2R）. Result： Compared with Vitamin E group, the positive cell numbers ofβ-gal stained HUVECs were enhanced, cell proliferation was depressed, and the fluorescence intensity of ROS was increased, at the same time, less NO and more O^2- in cells were produced in the eighth generation cell treated group： Protein expression of p47phox, AT1 R and AT2R in cells increased compared with Vit E group. Chinese herbs of high dose and low dose could improve condutions of HUVECs aging. Chinese herbs of high dose and low dose could reduce the positive cell numbers of β-gal stained HUVEC, increase cell proliferation and decrease fluorescence intensity of ROS in cells, at the same time, cells secreting more NO and less O^2-. Protein expression of p47phox, AT1R and AT2R in cells treated with Chinese herbs of high dose and low dose were decreased compared with Vit E group. Conclusion： The study indicated that extracts from P. ginseng, P. notoginseng and L. chuanxiong could delay endothelial cell replicative senescence. Herbal extracts downregulate the expression of NAD （P） H oxidase subunit-p47phox by means of ROS, hence decrease O^2- production and ultimately delay HUVECs in vitro senescence.