The Protective Effect Of 7-Difluoromethylyl-5, 4'-Di-methoxyl Isoflavone On Vascular Endothelium Injury By Oxidative Stress Induced With Hydrogen Dioxide

Objective: After the directed reconstitution of genistein in its chemicalconstitution, a series of genistein derivatives were obtained, we established the model of human umbilical vein endothelial cells(HUVEC-12) cultured in vitro and injured by oxidative stress, then sieved an active new chemical entity( 7-Difluoromethylyl-5,4'-Di- methoxyl isoflavone ) from genistein derivatives which has more effective protection of vascular endothelium injury by oxidative stress. The protective effect of active new chemical entity on vascular endothelial cell injury by oxidative stress was studied, and its molecular mechanism of action was also investigated. The objective is to provide the experiment basis for developing a new medicine for cardiovascular disease therapy.Methods: Human umbilical vein endothelial cells incubated with H₂O₂ for 24hin vitro, cell function was measured by LDH assay and cell apoptosis was determined by flow cytometry respectively. Then we established the model of endothelial cells injury by oxidative stress and sieved an active new chemical entity.Experiment groups were divided as follows: (1) blank control group;(2) H₂O)2 damage group;(3) DMSO control group;(4) lead compound group;(5) 7- Difluoromethylyl-5,4'-Di- methoxyl isoflavone group;(5) positive medicine control group. Cell growth and multiplication activity were measured by MTT assay and trypan blue staining and cell counting method. Cell apoptosis was determined by acridine orange staining method and flow cytometry. Reactive oxygen species in endothelial cells was measured by DCFH-DA stimulating fluorescence flow cytometry. The adhesion rate of vascular endothelial cell and mononuclear cell was examined by fluorescent light spectrophotometer. ELISA was used to determine the concentration of E-Selectin, ICAM-1, IL-6 and TNF-alpha in the supernatant of cells. The expression of phosphorylation ERK1/2, NF-kappa B, Caspase-3 in human umbilical vein endothelial cells was examined using Western Blot.Result: Human umbilical vein endothelial cells treated with H₂O₂(1.0 mmol/L) for 24h obviously increased LDH release and apoptotic rate. Cells were incubated with different concentration of genistein derivatives before being injured by H₂O₂(1.0 mmol/L), and we found:(1) the protective effect of 7- Difluoromethylyl-5,4'-Di-hydroxyl isoflavone is higher than lead compound.(2) 7- Difluoromethylyl-5,4'-Di-methoxyl isoflavone is the most effective article among test articles.When human umbilical vein endothelial cells were treated with H₂O₂(1.0 mmol/L) for 24h, the release of LDH was increased; the cell growth and proliferation were suppressed; the apoptosis of endothelial cells was increased; the level of reactive oxygen species in endothelial cells was remarkably elevated; the adhesion rate of vascular endothelial cell and mononuclear cell was rose; the release of E-Selectin, ICAM-1, IL-6 and TNF- alpha were increased; the expression level of phosphorylation ERK1/2, NF-kappa B and Caspase-3 in endothelial cells was up-regulated. After endothelial cells were pretreated with 7-Difluoromethylyl-5,4'-Di- methoxyl isoflavone, experiment results showed:(1) 7-Difluoromethylyl-5,4'-Di- methoxyl isoflavone can improve cell growth and proliferation in a concentration dependent manner, reduce the release of LDH, suppress the H₂O₂-induced apoptosis of endothelial cell and decrease the production of reactive oxygen species.(2) 7- Difluoromethylyl-5,4'-Di- methoxyl isoflavone can effectually inhibit the adhesion of vascular endothelial cell and mononuclear cell induced by oxidation in a concentration dependent manner, and the release of E-Selectin, ICAM-1 was reduced.(3) 7- Difluoromethylyl-5,4'-Di-methoxyl isoflavone can effectively inhibit the release of inflammation factor (IL-6 and TNF-alpha) induced by H₂O₂ from endothelial cell and mononuclear cell.(4) 7- Difluoromethylyl-5,4'-Di-methoxyl isoflavone can suppress H₂O₂-induced expression and activation of phosphorylation ERK1/2, NF- kappa B and Caspase-3 in human umbilical vein endothelial cells.Conclusion:1. 7-Difluoromethylyl-5,4'-Di-methoxyl isoflavone possesses the protective activity of vascular endothelial cell injury by H₂O₂ in vitro.2. 7-Difluoromethylyl-5,4'-Di-methoxyl isoflavone has a remarkable anti-oxidation activity.3. 7-Difluoromethylyl-5,4'-Di-methoxyl isoflavone effectually inhibits the adhesion of vascular endothelial cell and mononuclear cell induced by oxidation, through inhibitting the release of E-Selectin, ICAM-1.4. 7-Difluoromethylyl-5,4'-Di-methoxyl isoflavone inhibits inflammatory reaction of vascular endothelium injury by oxidative stress, through inhibitting the release of IL-6 and TNF-alpha.5. 7-Difluoromethylyl-5,4'-Di-methoxyl isoflavone possesses the protective activity of vascular endothelial cell injury by H₂O₂ in vitro, possibly through down-regulatting the expression of phosphorylation ERK1/2 and suppressing the activation of NF-kappa B and caspase-3.