摘要
心血管疾病严重危害人类健康,其致病机制主要是氧化应激诱导的内皮细胞凋亡,保护内皮细胞免受氧化应激损伤是防治心血管疾病的关键。为了探讨罗布麻总黄酮(total flavonoids of Folium Apocyni Veneti,TFF)抑制过氧化氢(hydrogen peroxide,H_2O_2)诱导的EA.hy926细胞凋亡的机制,采用不同浓度TFF处理EA.hy926细胞,一定浓度的H_2O_2造模,通过MTT法、Hoechst33342/PI荧光双染、流式细胞术检测各组的细胞凋亡情况,并用Western-blot和荧光定量PCR检测相关蛋白及m RNA的表达水平。研究发现,H_2O_2能明显诱导EA.hy926细胞凋亡,而TFF可降低H_2O_2引起的内皮细胞凋亡,表现为凋亡率降低、线粒体膜电位(mitochondrial membrane potential,MMP)增加、裂解的半胱天冬酶-3,-9(cleaved caspase-3,-9)表达减少、髓细胞白血病因子-1(myeloid cell leukemia-1,Mcl-1)表达增加、磷脂酰肌醇-3-激酶/蛋白激酶B/糖原合成酶激酶-3β(phosphatidylinositol-3-kinase/protein kinase B/glycogen synthase kinase-3β,PI3K/Akt/GSK3β)通路中Akt、GSK3β蛋白的磷酸化水平升高。上述结果表明,TFF主要是通过激活PI3K/Akt/GSK3β通路,调节Mcl-1的表达,保护MMP,进而抑制caspase蛋白的剪切活化,抑制H_2O_2诱导的EA.hy926凋亡。
Human health is threatened by cardiovascular disease seriously, and the pathogenic mechanism is mainly considered to be the endothelial cell apoptosis induced by oxidative stress. Therefore, protection of endothelial cells from oxidative damage is the key prevention of cardiovascular disease. To investigate the effects of total flavonoids of Folium Apocyni Veneti （TFF） on hydrogen peroxide （HRO2）-induced EA.hy926 cell apoptosis, EA.hy926 cells were treated with different concentrations of TFF or H2O2 to select the best concentration. The rate of apoptosis was detected by MTT assay, Hoechst33342/PI double staining and flow cytometric analysis. Meanwhile, the expressions of mitochondrial apoptosis-related proteins and PI3K/Akt/ GSK3β pathway related proteins induced by TFF were detected using Western-blot. Furthermore, the mRNA expression levels of corresponding proteins were detected by fluorescence quantitative PCR. The results showed that H2O2-induced EA.hy926 cell apoptosis was inhibited by TFF, with decreased apoptosis rate, increased MMP, down-regulated expression of cleaved caspase-9, -3, up-regulated expression of Mcl-1 and increased phosphorylation of Akt and GSK3β. These results indicated that TFF reduced H2O2-induced cell apoptosis mainly through activating PI3K/Akt/GSK3fl pathway, increasing Mcl-1 expression, protecting mitochondrial membrane potential and inhibiting caspase cleavage and activation.
出处
《生命科学研究》
CAS
CSCD
2016年第2期145-152,共8页
Life Science Research
基金
国家自然基金资助项目(81274132)
