葡萄籽原花青素通过下调miR-34a减轻过氧化氢诱导的心肌细胞损伤研究

Study of the grape seed proanthocyanidin reducing hydrogen peroxide-induced cardiac muscle cell injury by down-regulating miR-34a

目的探讨葡萄籽原花青素(GSP)通过下调微小RNA-34a(miR-34a)对过氧化氢(H2O2)诱导的心肌细胞损伤的影响。方法体外培养心肌细胞H9c2,建立H2O2诱导心肌细胞损伤模型。用MTT法检测心肌细胞存活情况并筛选H2O2最适作用浓度。将细胞分为7组:空白对照组(无任何处理的心肌细胞),模型组(300μmol·L-1H2O2的无血清培养液)和低、中、高3个剂量实验组(GSP:10,20,40μmol·L-1)以及阴性转染组(将miR-34a阴性对照质粒转染至模型组心肌细胞)、转染组(将miR-34a mimics质粒转染至高剂量实验组心肌细胞),均培养12 h。用流式细胞仪检测心肌细胞凋亡率;用实时荧光定量PCR法检测miR-34a表达水平。结果培养12 h后,空白对照组、模型组和高剂量实验组的细胞凋亡率分别为(3. 72±0. 62)%,(52. 03±2. 67)%和(15. 21±1. 52)%;空白对照组、模型组和高剂量实验组的miR-34a水平(2-ΔΔCt值)分别为1. 01±0. 12,3. 16±0. 52和1. 52±0. 25,模型组与空白对照组比较,或高剂量实验组与模型组比较,上述指标的差异均有统计学意义(均P <0. 05)。模型组、阴性转染组、高剂量实验组和转染组的心肌细胞存活率分别为(50. 13±3. 35)%,(49. 02±3. 21)%,(86. 37±4. 39)%和(68. 16±2. 36)%,高剂量实验组和转染组与模型组比较,细胞存活率均显著升高,差异均有统计学意义(均P <0. 05)。结论 GSP可减轻H2O2诱导的心肌细胞损伤,其可能通过下调miR-34a表达进而保护心肌细胞。

Objective To investigate the effect of grape seed proanthocyanidin(GSP)on hydrogen peroxide(H2O2)-induced cardiac muscle cell injury by down-regulating micro RNA-34a(miR-34a).Methods H9c2 cardiac muscle cells was cultured in vitro to establish a model of cardiac muscle cell injury induced by H2O2.The survival of cardiac muscle cells was detected by MTT method and the optimal concentration of H2O2 was screened.The cells were divided into seven groups:blank control group(cardiac muscle cells without any treatment),model group(serum-free culture medium with 300μmol·L-1 H2O2),experimental-L,-M,-H groups(GSP:10,20,40μmol·L-1),negative transfection group(miR-34a negative control plasmid was transfected into cardiac muscle cells of model group),transfection group(miR-34a mimics plasmid was transfected into cardiac muscle cells of experimental-H group),and all cultured for 12 h.The apoptotic rate of cardiac muscle cells was detected by flow cytometry.The expression of miR-34a was detected by real-time fluorescence quantitative PCR.Results After 12 h of culture,the apoptotic rates of blank control group,model group and experimental-H group were(3.72±0.62)%,(52.03±2.67)%,and(15.21±1.52)%,respectively;the levels(2-ΔΔCt value)of miR-34a of blank control group,model group and experimental-H group were 1.01±0.12,3.16±0.52,and 1.52±0.25,respectively.There were significant differences of the factors between model group and blank control group,or the experimental-H group and model group(all P<0.05).The survival rates of cardiac muscle cells in model group,negative transfection group,experimental-H group,and transfection group were(50.13±3.35)%,(49.02±3.21)%,(86.37±4.39)%,and(68.16±2.36)%,there were significant differences between the experimental-H group,transfection group and model group(all P<0.05).Conclusion GSP can alleviate the cardiac muscle cell injury induced by H2O2,which may protect cardiac muscle cells by down-regulating the expression of miR-34a.