调脂颗粒干预血管紧张素Ⅱ诱导内皮细胞自噬水平的实验研究

摘要

Objective:To investigate the role of Tiaozhi Granule on Ang Ⅱ-induced autophagy in HUVECs.Methods:HUVECs were incubated by Ang Ⅱ with or without pretreatment with Tiaozhi Granule.MTT method was applied to detect cell viability.Expression of LC3-Ⅱ,beclin-1 and SQSTM1/p62,which were markers of cell autophagy levels,was determined by western blot.Immunofluorescence was used to analyze the change of autophagosome number.NO content was measured by NO analysis kit.Results:Ang Ⅱ treatment for 24 hours did not affect the cell survival,and Tiaozhi Granule with Ang Ⅱ also had no effect on cell viability.Ang Ⅱ stimulation increased the ratio of LC3-Ⅱ to LC3-Ⅰ and beclin-1 expression,and decreased SQSTM1/p62 expression and the levels of NO in HUVECs.In addition,the number of autophagosome was markedly increased.Tiaozhi Granule pretreatment inhibited the effects of Ang Ⅱ on the ratio of LC3-Ⅱ to LC3-Ⅰ,beclin-1 and SQSTM1/p62 expression,and also reversed the decrease of NO content induced by Ang Ⅱ (P＜0.05).Conclusions:Tiaozhi Granule inhibits Ang Ⅱ-induced autophagy in endothelial cells,which may be mediated by NO production.%目的 研究调脂颗粒对血管紧张素Ⅱ(AngⅡ)诱导人脐静脉内皮细胞(HUVECs)自噬水平的影响及其机制.方法 采用中药血清药理学方法,以调脂颗粒含药血清和AngⅡ处理HUVECs,用MTT比色法检测血管内皮细胞生长的影响,Western blot检测各组自噬相关蛋白LC3-Ⅱ、beclin-1、SQSTM1/p62的相对表达量变化,免疫荧光法检测自噬小体的数量变化,用NO试剂盒检测各组NO含量的变化.结果 AngⅡ处理HUVECs对细胞活力无改变,调脂颗粒含药血清联合AngⅡ对细胞活力也无明显影响.AngⅡ刺激内皮细胞后LC3-Ⅱ/LC3-Ⅰ 、beclin-1表达量增加,SQSTM1/p62表达量降低,自噬小体数量显著增加,NO含量下降.调脂颗粒含药血清预处理后逆转了AngⅡ诱导的LC3-Ⅱ/LC3-Ⅰ、beclin-1表达量的增多、SQSTM1/p62表达量的下降、自噬小体数量的减少及NO含量的降低(P＜0.05).结论 调脂颗粒对AngⅡ诱导的细胞自噬具有抑制作用,其机制可能与调节细胞内NO生成量有关.