摘要
目的探讨青蒿素(ART)对氧化低密度脂蛋白(ox-LDL)诱导的血管内皮细胞损伤的作用及其潜在的分子机制。方法分别用0、50、100、150、200 mg/ml的ox-LDL及0、1、5、10、20 mmol/L的ART处理对数生长期的人脐静脉内皮细胞EA.hy926,采用MTT法检测细胞活性。为检测ART对细胞的影响,将EA.hy926细胞分为4组:对照组(无处理)、ox-LDL组(100 mg/ml ox-LDL)、ox-LDL+ART组(100 mg/ml ox-LDL+10 mmol/L ART)及ox-LDL+ART+3-甲基腺嘌呤(3-MA)组(100 mg/ml ox-LDL+10 mmol/L ART+5 mmol/L 3-MA);为检测瞬时感受器电位通道香草酸受体亚型Ⅳ(TRPV4)对细胞的影响,将EA.hy926细胞分为4组:对照组、ox-LDL组、ox-LDL+ART组及ox-LDL+ART+钌红(RR)组(100 mg/ml ox-LDL+10 mmol/L ART+10 mmol/L RR)。采用MTT法检测细胞活性,Western blotting检测TRPV4、自噬相关蛋白(LC3-Ⅱ/LC3-Ⅰ、p62)及凋亡相关蛋白(Bcl-2、Bax)的表达,流式细胞仪检测细胞凋亡情况。结果EA.hy926细胞活性随着ox-LDL浓度的升高而降低,而ART作用于ox-LDL诱导的细胞后,细胞活性明显升高(P<0.05)。与对照组比较,ox-LDL组细胞活性,p62、Bcl-2、TRPV4表达水平明显降低,而LC3-Ⅱ/LC3-Ⅰ比值、细胞凋亡率、Bax表达水平明显升高,差异均有统计学意义(P<0.05)。与ox-LDL组比较,ox-LDL+ART组细胞活性、LC3-Ⅱ/LC3-Ⅰ比值、Bcl-2及TRPV4表达水平明显升高,而细胞凋亡率、Bax及p62表达水平明显降低,差异均有统计学意义(P<0.05)。与ox-LDL+ART组比较,ox-LDL+ART+3-MA组及ox-LDL+ART+RR组细胞活性、LC3-Ⅱ/LC3-Ⅰ比值、Bcl-2表达水平明显降低,细胞凋亡率、p62及Bax表达水平明显升高,差异均有统计学意义(P<0.05)。结论ART可通过激活TRPV4促进细胞自噬,从而减轻ox-LDL诱导的血管内皮细胞损伤。
Objective To investigate the effects of artemisinin(ART)on vascular endothelial cell injury induced by oxidized low density lipoprotein(ox-LDL),and explore its potential molecule mechanism.Methods The human umbilical vein endothelial cells EA.hy926 in logarithmic phase were treated respectively with 0,50,100,150 and 200 mg/ml of ox-LDL and 0,1,5,10 and 20 mmol/L of ART.The cell viability were detected by MTT assay.To detected the effect of ART on cells,the EA.hy926 cells were divided into control group(without any treatment),ox-LDL group(treated with 100 mg/ml ox-LDL),ox-LDL+ART group(treated with 100 mg/ml ox-LDL and 10 mmol/L ART)and ox-LDL+ART+3-methyladenine(3-MA)group(treated with 100 mg/ml ox-LDL,10 mmol/L ART and 5 mmol/L 3-MA).To detected the effect of transient receptor potential channel vanillic acid receptor subtypeⅣ(TRPV4)on the cells,the EA.hy926 cells were divided into control group,ox-LDL group,ox-LDL+ART group and ox-LDL+ART+ruthenium red(RR)group(treated with 100 mg/ml ox-LDL,10 mmol/L ART and 10 mmol/L RR).The cell viability were detected by MTT assay.The expressions of TRPV4,autophagy associated proteins(LC3-Ⅱ/LC3-Ⅰand p62)and apoptosis associated protein(Bcl-2,Bax)were detected by Western blotting.Cell apoptosis were detected by flow cytometry.Results The cell viability of EA.hy926 decreased with the increase of ox-LDL concentration.The viability of ox-LDL induced cells was significantly upregulated by ART(P<0.05).Compared with the control group,the viability of cells,and the expression levels of p62,Bcl-2 and TRPV4 decreased significantly in the ox-LDL group(P<0.05),but the LC3-Ⅱ/LC3-Ⅰratio,cell apoptosis rate and expression level of Bax was significantly up-regulated in ox-LDL group(P<0.05).Compared with the ox-LDL group,the cell viability,LC3-Ⅱ/LC3-Ⅰratio,the protein expression levels of Bcl-2 and TRPV4 increased significantly,but the cell apoptosis rate,protein expression levels of p62 and Bax decreased significantly in ox-LDL+ART group(P<0.05).Compared with the ox-LDL+ART group,the cell viability,LC3-Ⅱ/LC3-Ⅰratio,and protein expression level of Bcl-2 decreased significantly(P<0.05),but the cell apoptosis rate,protein expression levels of p62 and Bax were up-regulated significantly in ox-LDL+ART+3-MA group and ox-LDL+ART+RR group(P<0.05).Conclusion ART can promote autophagy by activating TRPV4 to reduce ox-LDL induced vascular endothelial cell injury.
作者
袁向科
江瑞
Yuan Xiang-Ke;Jiang Rui(Department of Peripheral Vascular,Henan Province Hospital of TCM,Zhengzhou 450002,China;Department of Geriatrics,the Third Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450002,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2021年第4期333-339,共7页
Medical Journal of Chinese People's Liberation Army
关键词
青蒿素
外周动脉硬化性疾病
氧化低密度脂蛋白
瞬时感受器电位通道香草酸受体亚型Ⅳ
artemisinin
peripheral arteriosclerotic disease
oxidized low density lipoprotein
transient receptor potential channel vanillic acid receptor subtypeⅣ
