Total Flavonoids Of Folium Apocyni Veneti And Isoquercitrin Inhibits Hydrogen Peroxide-induced Apoptosis Of Ea.hy926 Cells

Objective: The aim of study is to research the effects of Total Flavonoids of Folium Apocyni veneti(TFF) and isoquercitrin(ISO) on H2O2-induced EA.hy926 cells apoptosis and to explore the possible mechanism.Methods: 1. MTT assay was used to detect the protective effects of TFF and ISO against H2O2-induced loss of viability in EA.hy926 cells; 2. Hoechst33342 / PI double staining, Annexin V-FITC/PI double staining by flow cytometry, mitochondrial membrane potential assay were used to detect the effects of TFF and ISO on H2O2-induced EA.hy926 cells apoptosis; 3. The effects of TFF and ISO on the expression of cleaved caspase-3,-9 and Mcl-1 were detected by western-blot and the m RNA expression level of caspase-3,-9, and Mcl-1 were detected by fluorescence quantitative PCR. 4. The phosphorylation level of Akt and GSK3β induced by TFF, ISO, LY294002 and SB216373 respectively in EA.hy926 cells was detected by western-blot.Results: 1. TFF and ISO at concentrations of 4.5-144 mg/L and 2.5-80 μmol/L respectively had no cytotoxity on EA.hy926 cells and the cell viability was decreased after exposure to 200 μmol/L H2O2 for 4 h. TFF(9, 18, 36 mg/L) and ISO(5, 10, 20 μmol /L) inhibited significantly the decrease of EA.hy926 cells viability induced by H2O2. 2. Hoechst33342 / PI double staining and Annexin V-FITC / PI double staining assay demonstrated that H2O2 significantly increased the cells apoptosis rate(P<0.01), TFF and ISO could induced the apoptosis rate decreased in a dose-dependent manner. The mitochondrial membrane potential assay showed that H2O2 significantly decreased EA.hy926 cells mitochondrial membrane potential, while TFF and ISO reversed the effects. 3. Western-blot showed that H2O2 treatment significantly increased cleavage of caspase-3 and caspase-9 in comparison with that of the control group(P<0.01). In contrast, pretreatment with TFF and ISO dose-dependently decreased the amounts of cleaved caspase-3 and caspase-9 in H2O2-treated cells in comparison with those of cells treated with H2O2 only(P<0.01 vs. the H2O2-treated group). FQ-PCR showed that caspase-3,-9 m RNA levels of the H2O2-treated group were increased in comparison with those of the control group(P<0.01), while pretreatment with TFF and ISO dose-dependently decreased the amounts of caspase-3,-9 in H2O2-treated cells incomparison with H2O2-treated group(P<0.01 vs. the H2O2-treated group). Western-blot and FQ-PCR showed that the Mcl-1 protein and m RNA levels of the group treated with H2O2 were reduced compared with those of the control group(P<0.01). However, H2O2-treated EA.hy926 cells pretreated with TFF and ISO showed significantly increased Mcl-1 protein and m RNA expression levels in comparison with those of the H2O2-treated group(P<0.01). 4. Western-blot showed that TFF and ISO could induce p-Akt, p-GSK3β, Mcl-1 expression increase compared with the control group, however cells pretreated with PI3K/Akt inhibitor LY294002 showed significantly reduced levels of Akt and GSK-3β phosphorylation and Mcl-1 expression in comparison with those of cells treated with TFF or ISO. Additionally, p-GSK3β, Mcl-1 expression was increased in GSK3β inhibitor SB216373 group compared with control group, cells pretreated with TFF and ISO had no significant difference in p-GSK3β and Mcl-1 expression in comparison with SB216763 group.Conclusion : The present study provided evidence that TFF and ISO mediated anti-apoptotic responses in H2O2-treated EA.hy926 cells by activating PI3K/Akt/GSK3β, inducing phosphorylation of GSK3β, decreasing Mcl-1 degration, inhibiting activation of caspase-3 and caspase-9.