川芎嗪对体外血管内皮祖细胞氧化损伤的保护作用

Protective effects of Tetramethylpyrazine on the cultured endothelial progenitor cells following oxidative injury by hydrogen peroxide in vitro

目的:研究川芎嗪对过氧化氢(H2O2)诱导的内皮祖细胞(EPCs)氧化应激损伤的保护作用。方法:采用密度梯度离心法获取兔外周血单个核细胞,EBM-2完全培养基诱导分化,并对培养7 d的细胞进行免疫荧光及免疫细胞化学方法分析。实验分为正常对照组、H2O2组及H2O2川芎嗪预处理组,以600μmol.L-1H2O2诱导EPCs氧化应激损伤,流式细胞仪检测各组细胞凋亡率,测定细胞浆中丙二醛(MDA)含量和细胞培养液上清总超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)活力。结果:兔外周血单个核细胞诱导培养7 d,D il标记的乙酰化低密度脂蛋白(D il-ac-LDL)内吞和荆豆凝集素(FITC-UEA-1)双染色阳性,免疫细胞化学显示培养细胞CD31/CD34/VEGFR-2阳性,证实为EPCs;流式细胞分析显示,H2O2组细胞凋亡率明显高于对照组(P<0.01),川芎嗪预处理组明显抑制H2O2诱导的EPCs凋亡(与H2O2组比较,P<0.05);H2O2可导致EPCs MDA、LDH含量明显增加和SOD活性下降,而用川芎嗪预处理可明显抑制EPCs的氧化应激反应(与H2O2组比较,P<0.05)。结论:川芎嗪对H2O2诱导的EPCs氧化应激损伤有一定的保护作用,其机理可能与其抗凋亡和抗脂质过氧化有关。

Objective To investigate the protective effects of Tetramethylpyrazine on hydrogen peroxide-induced oxidative stress injury in endothelial progenitor cells.Methods Total mononuclear cells were isolated from peripheral blood of rabbits by density gradient centrifugation.The cells were suspended in endothelial basal medium（EBM-2） and plated on fibronectin-coated culture flasks.Immunofluorescence assay and immunocytochemical analysis were performed after seven-day culture.Then the oxidative stress injury in endothelial progenitor cells was induced by hydrogen peroxide.Three groups,i.e.,control group,hydrogen peroxide group（H2O2 600 μmol·L-1） and hydrogen peroxide/Tetramethylpyrazine group（TMP 10 μg·ml-1）,were used to conduct the study.Apoptotic rate of cells was determined by FCM,and content of MDA in cytoplasm and activity of SOD and LDH in cell culture solution were also determined.Results After seven days culture,the adherent cells were reactive to Dil-ac-LDL and FITC-UEA-1.Meanwhile,they showed positive staining for CD31,CD34,VEGFR-2.Compared with the control group,FCM analysis indicated that the apoptotic rate of cells was significantly increased in hydrogen peroxide group[（24.5±1.1）% vs（70.7±1.7）%,P〈0.01].Although the apoptotic rate in TMP group was higher than that in control group[（35.2±0.7）% vs（24.5±1.1）%,P〈0.05],it was lower than that in hydrogen peroxide group[（35.2±0.7）% vs（70.7±1.7）%,P〈0.05].Compared with the control group,MDA and LDH contents were significantly increased in hydrogen peroxide group,but the contents in TMP group were lower than those in hydrogen peroxide group.The activity of SOD was lowest in hydrogen peroxide group.Compared with the hydrogen peroxide group,the activity of SOD was significantly increased in TMP group（P〈0.05）.Conclusion It is suggested that Tetramethylpyrazine may protect endothelial progenitor cells from hydrogen peroxide-induced oxidative stress injury.The mechanism of protection may be associated with anti-lipid peroxidation and anti-apoptosis.