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目的:研究载脂蛋白A-I模拟肽L-4F是否减轻过氧化氢(hydrogen peroxide,H2O2)诱导的小鼠骨髓来源内皮祖细胞(endothelial progenitor cells,EPCs)氧化应激损伤及其机制。方法:通过密度梯度离心法分离小鼠骨髓单个核细胞,条件培养基EGM-2MV诱导分化培养EPCs。对培养EPCs先以PI3K/AKT抑制剂LY294002(30μmol/L)干预2 h后加入L-4F(75 mg/L)4 h,再加入100μmol/L H2O224 h。MTT法检测细胞活力,试剂盒检测各实验组培养基超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)含量,以评价细胞氧化与抗氧化平衡及脂质过氧化程度;流式细胞术Annexin V/PI双染法检测细胞凋亡;免疫印迹法检测p-AKT蛋白水平。结果:L-4F预处理显著抑制了H2O2诱导的细胞活力降低、SOD活性下降、MDA含量增加及细胞凋亡。H2O2下调p-AKT的蛋白水平,L-4F呈浓度依赖性地上调p-AKT的蛋白水平。LY294002抑制了L-4F减轻EPCs损伤的作用。结论:载脂蛋白A-I模拟肽L-4F部分通过PI3K/AKT通路减轻H2O2诱导的EPCs氧化应激损伤。
Objective: To investigate whether L-4F, an apolipoprotein A-I mimetic peptide, can reduce oxidative stress injury induced by hydrogen peroxide (H2O2) in mouse bone marrow-derived endothelial progenitor cells (EPCs) and its mechanism. Methods: Murine bone marrow mononuclear cells were isolated by density gradient centrifugation. EPCs were induced by conditioned medium EGM-2MV. EPCs were treated with LY294002 (30μmol / L) for 2 h and then cultured in EPCs containing L-4F (75 mg / L) for 4 h followed by 100 μmol / L H2O2 for 24 h. Cell viability was detected by MTT assay. The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in the medium of each experimental group were detected by kit to evaluate the balance between cell oxidation and anti-oxidation and lipid peroxidation ; Apoptosis was detected by flow cytometry Annexin V / PI double staining; p-AKT protein level was detected by Western blot. Results: L-4F pretreatment significantly inhibited H2O2-induced cell viability, decreased SOD activity, increased MDA content and apoptosis. H2O2 down-regulated the protein level of p-AKT, and L-4F up-regulated the protein level of p-AKT in a concentration-dependent manner. LY294002 inhibited the effect of L-4F on EPCs injury. CONCLUSION: L-4F part of apolipoprotein A-I mimetic peptide alleviates H2O2-induced oxidative stress injury in EPCs via PI3K / AKT pathway.