人参-三七-川芎提取物延缓过氧化氢诱导的内皮细胞衰老中线粒体氧化应激的作用  

Effect of Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma,and Chuanxiong Rhizoma Extract on Mitochondrial Oxidative Stress in Hydrogen Peroxide-induced Endothelial Cell Aging

作　　者：吴烨 王强[2] 修成奎[1] 胡艳红 马琰岩[1] 付莹坤[3] 王雪[1] 雷燕[1] 杨静[1] WU Ye;WANG Qiang;XIU Cheng-kui;HU Yan-hong;MA Yan-yan;FU Ying-kun;WANG Xue;LEI Yan;YANG Jing(Beijing Key Laboratory of Basic Research on Prevention and Treatment of Major Diseases by Traditional Chinese Medicine,Experimental Research Center,China Academy of Chinese Medical Sciences,Beijing 100700,China;Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing 100102,China;Guang'anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100056,China)

机构地区：[1]中国中医科学院医学实验中心,北京市中医药防治重大疾病基础研究重点实验室,北京100700 [2]中国中医科学院望京医院,北京100102 [3]中国中医科学院广安门医院,北京100056

出　　处：《中国实验方剂学杂志》2021年第24期17-24,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81503448,82074260);中国中医科学院自主选题项目(ZZ2018014)。

摘　　要：目的:通过观察人参-三七-川芎提取物(GNC)对过氧化氢(H_(2)O_(2))诱导的人脐静脉内皮细胞(HUVECs)衰老中线粒体氧化应激的影响,探讨GNC对衰老HUVECs的治疗机制。方法:本研究以HUVECs作为研究对象,H_(2)O_(2)诱导内皮细胞衰老作为模型,实验分为空白组,H_(2)O_(2)组,H_(2)O_(2)+DMSO组(DMSO),白藜芦醇组(Resv)及GNC提取物低(GNC-L),中(GNC-M),高(GNC-H)质量浓度组,除空白组,H_(2)O_(2)组外,其余各组分别给予DMSO 1 mL·L^(-1),Resv 8μmol·L^(-1),GNC 400,300,200 mg·L^(-1)药物干预,24 h后除空白组外给予300μmol·L^(-1)H_(2)O_(2)诱导HUVECs衰老,再给予正常培养基培养24 h。采用衰老特异性β-半乳糖苷酶(SA-β-gal)染色法鉴定细胞衰老程度,流式细胞仪分析细胞周期,采用Mito SOX RED荧光染色法测定细胞内线粒体活性氧(mtROS)水平,以JC-10为荧光探针检测细胞内线粒体膜电位变化,蛋白免疫印迹法(Western blot)检测含锰超氧化物歧化酶(MnSOD)与磷酸化(p-)p66的蛋白表达。结果:SA-β-gal染色结果显示,与空白组比较,H_(2)O_(2)组SA-β-gal染色蓝染细胞显著增多(P<0.01);与H_(2)O_(2)组比较,各浓度组SA-β-gal染色蓝染细胞显著减少(P<0.01);细胞周期结果显示,与空白组比较,H_(2)O_(2)组G0/G1期细胞数量比例明显增多(P<0.05),G2/M期细胞比例明显减少(P<0.05);与H_(2)O_(2)组比较,GNC各组及Resv组G0/G1期细胞数量比例明显减少(P<0.05),GNC-H组,Resv组在G2/M期细胞比例明显增加(P<0.05)。线粒体ROS荧光结果显示,与空白组比较,H_(2)O_(2)组强度明显减弱(P<0.05);与H_(2)O_(2)组比较,GNC-H,GNC-M组强度明显增高(P<0.05);线粒体膜电位结果显示,与空白组比较,H_(2)O_(2)组荧光强度显著下降(P<0.01);与H_(2)O_(2)组比较,GNC-H,GNC-M,GNC-L组线粒体膜电位荧光强度显著增高(P<0.01),Resv组明显增高(P<0.05);Western blot结果显示,与空白组比较,H_(2)O_(2)组MnSOD含量明显降低(P<0.05);与H_(2)O_(2)组比较,GNC-H与GNC-M组表Objective:To observe the effect of Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma,and Chuanxiong Rhizoma extract(GNC)on mitochondrial oxidative stress in hydrogen peroxide(H_(2)O_(2))-induced aging of human umbilical vein endothelial cells(HUVECs),and explore the therapeutic mechanism of GNC on aging HUVECs.Method:The HUVECs were classified into the control group(control),H_(2)O_(2) model group(H_(2)O_(2)),H_(2)O_(2)+ DMSO group(DMSO,1 mL·L^(-1)),resveratrol group(Resv,8 μmol·L^(-1)),and low-(200 mg·L^(-1)),medium-(300 mg·L^(-1)),and high-dose(400 mg·L^(-1))GNC(GNC-L,GNC-M,and GNC-H)groups.Except control group and H_(2)O_(2) group,the other groups were intervened with corresponding agents.Subsequently,300 μmol·L^(-1) H_(2)O_(2) was given to other groups except the control group for 4 h to induce aging,and then the cells were cultured in normal media for 24 h.The aging degree,cell cycle,and mitochondrial reactive oxygen species(mtROS)level were determined by SA-β-galactosidase(SA-β-Gal)staining,flow cytometry,and MitoSox red fluorescence staining,respectively.JC-10 was used as a fluorescent probe to detect the changes in mitochondrial membrane potential,and Western blot was performed to detect the expression of manganese superoxide dismutase(MnSOD)and p-p66 proteins.Result:The SA-β-gal staining results showed that H_(2)O_(2) group had increased blue-stained cells compared with other groups(P<0.01).Compared with those in the control group,the ratio of G0/G1 phase cells significantly increased(P<0.05)and that of G2/M phase cells decreased(P<0.05)in the H_(2)O_(2) group.Compared with those in the H_(2)O_(2) group,the proportion of G0/G1 cells decreased(P<0.05)while that of G2/M cells increased(P<0.05)in GNC-H groups and Resv group.The fluorescence staining for determining mitochondrial ROS level showed that the H_(2)O_(2) group had weakened fluorescence intensity than the control,GNC-H,and GNC-M groups(P<0.05).The mitochondrial membrane potential fluorescence intensity of the H_(2)O_(2) group was we

关 键 词：衰老 血管老化 内皮细胞衰老 人参-三七-川芎提取物 氧化应激 

分 类 号：R2-0[医药卫生—中医学] R22