Protection Effects And Mechanisms Of N-butanol Fraction Of Potentilla Anserine L. Against Hypoxia Injury On EAHY926 Endothelial Cells

Objective: To study the protective effects and mechanisms of n-butanol fraction of Potentilla anserine L. on EAHY926 Endothelial cells in hypoxia injury .Methods:①The human endothelial hybrid cell line Eahy926 were cultured in a modified Eagle's Minimum Essential Medium (acc. to Dulbecco) containing with 10% Fetal calf serum (FCS). Cell cultures were maintained in 5% CO2 atmosphere and saturated humidity at 37°C, for reaching Subconfluent monolayers.②the suitable time of hypoxia was ascertained through MTT, trypan blue staining, and activity assay of LDH, and then the model of hypoxia was established.③The cultured endothelial cell were divided randomly into control group, hypoxia injury model group and intervention group of n-butanol fraction of Potentilla anserine L. at different dose(3mg/ml, 1.5mg/ml, 0.75mg/ml as high, intermediate and low dose). The effect of different doses of Potentilla anserine L. on acute hypoxia injury of endothelial cell were examined through survival rate assay with trypan blue staining, metabolic activity assay with MTT method, and LDH activity assay with colorimetric method.④The effect of different doses of Potentilla anserine L. on acute hypoxia injury of endothelial cell were through cell morphology observation by H.E staining.⑤The possible mechanisms of acute hypoxia injury on endothelial cell were approached by biochemical indicators (SOD, MDA, NOS, NO, ET-1) observation, RT-PCR (HIF-1α, ET-1, eNOS, iNOS), immuno-histochemistry stain and Western Blot (HIF-1α,eNOS).Results: 1 The normal morphology of EAHY926 cells were observed under convert microscope, the cells show productive growth condition, polygonal or spindle epithelial-like shape adhered to the plate, with cobble stone like appearance.2 The effects of n-butanol fraction of Potentilla anserine L. on EAHY926 cells in hypoxia injury: (1)MTT: Compared with model group, each dose of n-butanol fraction of Potentilla anserine L. increase the vitality of endothelial cell subjected to hypoxia(P<0.01). (2)trypan blue staining:Compared with model group,each dose of Potentilla anserine L. increased the survival rate of endothelial cell subjected to hypoxia(p<0.05). (3) H.E staining: After the treatment of n-butanol fraction of Potentilla anserine L., changes of cell morphology, which includ the nuclear chromatin concentration, cell shrinkage, anachromasis, the distance increase and the relationship decrease between cells, were observed. (4)Biochemical Indicators: Compared with control group, the activities of LDH in model group were increased(P<0.01).Compared with model group, each dose of Potentilla anserine L. decreased the activities of LDH (P<0.01). 3 The possible mechanisms of n-butanol fraction of Potentilla anserine L. on EAHY926 cells in hypoxia injury:(1) Biochemical Indicators: Compared with control group, the activities of SOD in model group were increased(P<0.01).Compared with model group, each dose of Potentilla anserine L. decreased the activities of SOD (P<0.05, the high and low doses P<0.01).No significant difference of MDA was observed between control group, model group and intervention group(P>0.05). Compared with control group, the activities of NOS and the content of ET-1 were increased in model group(P<0.01), but the content of NO were decreased (P<0.01).Compared with model group, each dose of Potentilla anserine L. decreased the activities of NOS and the content of ET-1(P<0.01), increased the content of NO(P<0.01). (2)Immuno-histochemistry Stain:Compared with control group, the express levels of HIF-1αincreased(P<0.01) and the express levels of eNOS depressed(P<0.01) in model group. The high and intermediate doses of Potentilla anserine L. depressed the express levels of HIF-1αon hypoxia injury EAHY926 cell in comparsion with model group(P<0.01).Each dose of Potentilla anserine L. increased the express levels of eNOS on hypoxia injury endothelial cell in comparsion with model group (P<0.01).(3)RT-PCR: Compared with control group, the express levels of HIF-1αmRNA,ET-1 mRNA,iNOS mRNA in model group were increased (P<0.01), and the express levels of eNOS depressed(P<0.01) decreased. Each dose of Potentilla anserine L. depressed the express level of HIF-1αmRNA on hypoxia injury endothelial cell in comparsion with model group(P<0.01). The high and intermediate doses of Potentilla anserine L. depressed the express levels of ET-1 mRNA on hypoxia injury endothelial cell compared with model group(P<0.01). The high and intermediate doses of Potentilla anserine L. increased the express levels of eNOS mRNA on hypoxia injury endothelial cell compared with model group(P<0.01).The high dose of Potentilla anserine L. depressed the express levels of iNOS mRNA on hypoxia injury endothelial cell compared with model group (P<0.01).(4)Western Blot:Compared with control group, the express levels of HIF-1αwere increased(P<0.01) and the express levels of eNOS were depressed in model group(P<0.01). Each dose of Potentilla anserine L. decreased the express level of HIF-1αon hypoxia injury endothelial cell compared with model group (P<0.05, the intermediate and low dose p<0.01).The high and intermediate doses of Potentilla anserine L. increased the protein level of eNOS on hypoxia injury endothelial cell compared with model group (P<0.01).Conclusions: That n-butanol fraction of Potentilla anserine L. reduced the release of LDH, increased the survival rate of cell, improved the metabolic activity of EAHY926 cell subjected to hypoxia, indicat its protection effect on endothelial cell in hypoxia injury. The possible mechanisms of this protection might be : elevating the cleaning ability to oxygen free radical, inhibiting the unbalance between NO and ET-1 synthesized, depressing ET-1 level, improving the endothelial function through NO contents increase.