血塞通调节miRNA-146影响内皮细胞凋亡的干预作用  

Intervention Effect of Xuesaitong on Apoptosis of Endothelial Cells by miRNA-146

作　　者：王阶[1] 滕菲[2] 段练[1,3] 刘咏梅[1] 陈光[1,3] 

机构地区：[1]中国中医科学院广安门医院,北京100053 [2]首都医科大学附属北京中医医院,北京100010 [3]北京中医药大学,北京100029

出　　处：《中国实验方剂学杂志》2017年第19期6-10,共5页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81473561;81173116)

摘　　要：目的:探讨血塞通(XST)对过氧化氢诱导人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)凋亡的影响及作用机制。方法:体外培养HUVEC,H_2O_2作为氧化剂作用于HUVEC制作氧化应激模型。通过血塞通干预,观察H_2O_2诱导的HUVEC细胞凋亡的变化,从表观遗传学微小RNA(microRNA,miRNA)角度探讨血塞通对HUVEC的保护作用及其作用机制。将体外培养的HUVEC分为空白组,H_2O_2模型组,H_2O_2模型+XST(30 mg·L^(-1),处理24 h)组。通过细胞计数试剂盒-8(CCK8)法检测细胞活力,Annexin V/碘化丙啶(PI)测定细胞凋亡,实时荧光定量核酸扩增检测系统(Real-time PCR detecting system,Real-time PCR)检测miRNA-146和miRNA-199表达水平。结果:HUVEC经过100μmol·L^(-1)H_2O_2处理之后,细胞晚期凋亡和早期凋亡都有明显升高(P<0.05)。用质量浓度从1~100 mg·L^(-1)XST HUVEC细胞进行预处理后,发现用10 mg·L^(-1)XST预处理可以明显地改善细胞增殖情况。Annexin V/PI双染细胞凋亡检测发现HUVEC经过100μmol·L^(-1)H_2O_2处理之后,细胞PI阳性率R3(晚期凋亡)和Annexin V阳性率R5(早期凋亡)都有明显升高(P<0.05)。H_2O_2模型经过30 mg·L^(-1)XST处理之后,可以明显降低细胞PI阳性率R3(晚期凋亡)和Annexin V阳性率R5(早期凋亡)(P<0.05)。100μmol·L^(-1)H_2O_2处理可以明显升高hsa-miR-146b-5p和hsa-miR-199a-5p表达(P<0.05)。与单纯的H_2O_2处理比较,经过30 mg·L^(-1)的血塞通处理之后,可以明显降低hsa-miR-146b-5p表达(P<0.05)。结论:血塞通可以明显抵抗H_2O_2诱导的HUVEC细胞氧化损伤,可能是通过抑制hsa-miR-146b-5p的升高发挥保护作用。Objective： To investigate the effect and mechanism of Xuesaitong（XST） on the apoptosis of human umbilical vein endothelial cells （HUVEC） induced by hydrogen peroxide. Method： HUVECs were cultured in vitro and H2O2 was used as oxidants to establish oxidative stress model in HUVEC. The changes of apoptosis induced by H2O2 in HUVEC cells were observed by XST intervention, and the protective effect of XST on HUVEC was discussed from the perspective of epigenetic miRNA. The cultured HUVECs were divided into blank control group, H2O2 model group, and H2O2 model＋XST （30 mg·L^-1, 24 h） group. The cell viability was detected by cell counting kit8 （CCK8） method; apoptosis was detected by Annexin V/Propidium iodide （PI）; and the expression of miRNA-146 and miRNA-199 was detected by Real-time PCR. Result： After 100 μmol·L^-1 H2O2 treatment, the late apoptosis and early apoptosis of HUVECs were significantly increased （P〈0.05）. Pretreatment of HUVEC cells with a dose of 1 mg·L-1 to 100 mg·L^-1 showed that a significant improvement in cell proliferation was present in 10 mg·L^-1 preconditioning. Apoptotic detection of Annexin V/PI double staining showed that the PI positive rate R3 （late apoptotic） and Annexin V positive rate R5 （early apoptosis） were significantly increased after HUVEC treatment with 100 μmol·L^-1 H2O2 significantly （P〈0.05）. While the cell PI positive rate R3 （late apoptosis） and Annexin V positive rate R5 （early apoptosis） were significantly reduced after 30 mg·L^-1 XST treatment in H2O2 models （P〈0.05）. The expression levels of hsa-miR-146b-5p and hsa-miR-199a-5p were significantly increased by H2O2 treatment at 100 μmol·L^-1. As compared with H2O2 group, the expression of hsa-miR-146b-5p was significantly decreased after treatment with 30 mg·L^-1 XST （P〈0.05）. Conclusion： XST can significantly resist H2O2-induced oxidative damage in HUVEC cells, which may play a protective role by inhibiting the increase of hsa-miR-146b-5p.

关 键 词：人脐静脉内皮细胞 血塞通 氧化损伤 细胞凋亡 微小RNA 

分 类 号：R287[医药卫生—中药学] R259[医药卫生—中医学]