| Objective:To observe the protective effect of the ultra-filtration extract from angelicu sinensis,hedysarum ploybotrys and the role of endothelial cell apoptosis related gene in the process to human umbilical vein endothelial cells(HUVECs) injure induced by hydrogen dioxide(H2O2);to discuss the mechanism of its therapeutic action on cardiac and encephalic diseases.Methods:HUVECs were injured by 200μmol·L-1 H2O2 then the ultra-filtration extract from angelicu sinensis and hedysarum ploybotrys of different final concentrations with different final molecular weight(50000,100000,200000)were added HUVECs.The HUVECs were divided into normal control group,simple drug group,H2O2 damage group and drug intervention group.The extent of injured cell and the effect of the ultra-filtration extract from angelicu sinensis and hedysarum ploybotrys were evaluated by following means:Cell viability was measured by MTT assay,the activity of superoxide dismutase(SOD) were measured by xanthine oxidase, the activity of malondialdehyde(MDA) were measured by Thibabituric Acid(TBA), using flow cytometry(FCM)to analyze the cell cycle and apoptosis;Chemical method to detect intracellular Ca2+ concentration and cell culture on the concentration of NO in the supernatant;Using RT-PCR and microarray experiment of combining new technology "PCR Array" detect each group of molecules that are associated with apoptosis differentially expressed;RT-PCR technology in the m RNA level detection between groups and apoptosis related gene expression and Western Blot technique in the level of protein detection between groups the expression of apoptosis related gene expression.Results:1. MTT results indicated :100, 200, 300μmol·L-1H2O2 separately act on ECV 304 cells, 6 h, 12 h, 24 h, 48 h after cell activity decreased obviously, compared with normal control group with significant difference(P﹤0.05), 200μmol·L-1H2O2 incubation of 6 h, cell activity was the half of the value for the normal control group cell activity(LD50),cell damage is moderate.2. Different concentrations of ultra-filtration extract from angelicu sinensis and hedysarum ploybotrys mixture by MTT value between treatment group and control group with significant difference(P﹤0.05); ultra-filtration extract from angelicu sinensis and hedysarum ploybotrys mixture act on ECV 304 cells 32 h after, in1.5 g/L ~ 21.0 g/L concentration range has significant effect on promoting proliferation. Especially with 6.0 g/L ~ 15.0 g/L proliferation effect of peak concentration range.3.H2O2+ ultra-filtration extract from angelicu sinensis and hedysarum ploybotrys mixture molecular weight(50000,100000,200000) 6.0 g/L, compared with the control group with significant difference(P﹤0.05);compared with H2O2 damage group have significant difference(P﹤0.05),And in 100000 the strongest effect on promoting proliferation of molecular weight of 6.0 g/L.4.200μmol·L-1H2O2 act on ECV- 304 cells can significantly reduce the vitality of SOD after 6 h, enhance the content of MDA, compared with normal control group with significant difference(P﹤0.05),the drug intervention group compared with H2O2 damage group had significant difference(P﹤0.05).Compared with normal control group,H2O2 damage group of intracellular Ca2+concentration significantly increased(P ﹤ 0.05),compared with H2O2 damage group,drug intervention group of intracellular Ca2+ concentration significantly decreased(P﹤0.05).5. Compared with normal control group,H2O2 damage cell apoptosis rate increased significantly(P ﹤ 0.05), Cells significantly rasied HSP70 gene expression(P ﹤ 0.05), e NOSm RNA express down regulation(P ﹤ 0.05), The NO content in the cell culture supernatant on reducing(P﹤0.05).Compared with H2O2 damage group, drugs intervention group cell apoptosis rate reduced significantly(P ﹤ 0.05), cells raising HSP70 gene expression(P﹤0.05), e NOSm RNA express rised(P﹤0.05), the NO content in the cell culture supernatant on elevated(P﹤0.05).Conclusion:1. Hydrogen peroxide can cause endothelial cell survival rate decrease, of which 200μmol·L-1H2O2 act on 6 h cell damage is moderate.2. The ultra-filtration extract from angelicu sinensis and hedysarum ploybotrys mixture act on ECV 304 cells 32 h after, in1.5 g/L ~ 21.0 g/L concentration range has significant effect on promoting proliferation. Especially with 6.0 g/L ~ 15.0 g/L proliferation effect of peak concentration range.3.The ultra-filtration extract from angelicu sinensis and hedysarum ploybotrys could markly protect HUVECs that were injured by H2O2, With molecular weight 100000, 6.0 g/L protection effect is the most value one, And its protective effect may be by adjusting the participated in the impact on the proliferation of ECV304 cell cycle.4. The ultra-filtration extract from angelicu sinensis and hedysarum ploybotrys could remarkable protect HUVECs that were injured by H2O2, its effect is increasing the vitality of SOD, reduce the vitality of MDA, reduce the release of intracellular Ca2+, as well as reduce the endothelial cell apoptosis mechanism.5. The ultra-filtration extract from angelicu sinensis and hedysarum ploybotrys could remarkable antagonism HUVECs that were injured by H2O2, with H2O2 resistance to the effect of human umbilical vein endothelial cells apoptosis, can promote the expression of HSP70 gene, maintain normal endothelial cell function, raised e NOSm RNA expression, to maintain the NO synthesis and secretion. |
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