Protective Effects And Mechanisms Of Breviscapine On Vascular Endothelial Cells

Objective: To observe the protective effects of Breviscapine hydrogen peroxide impaired HUVECs and to explore its possible mechanism.Isolating and getting BMECs and building OGD model and To discuss the protective effect and mechanism study of breviscapine on BMECs with OGD injury.Methods:?1?Cultivating HUVECs cells in vitro.The HUVECs was modeled by 500?mol/L H₂O₂ injured for 39 h and inducated by breviscapine?20,100,200,400 mg/L?.The drug toxicity of breviscapine,dosage relations of hydrogen peroxide impaire and cells survival rate were evaluated by MTT method.The content of nitric oxide?NO?and the activity of glutathione-s-transferase?GST?in cell culture supernates,the content of malondialdehyde?MDA?and the activity of superoxide dismutase?SOD?in cells were examined by kits.?2?Isolating the original endothelial cells from rat's cerebral micro-vascular by sieve filtration method and enzyme digestion and the BMECs were cultured by ? factor immunohistochemistry.Building oxygen-glucose deprivation?OGD?model by hypoxia chamber and the sugar-free DMEM-F12 Co-culture 6h.?3?The protective effects of breviscapine and the protrction of BMECs cells after OGD injury were verified by MTT method.The apoptosis was tested by flow cytometry,the expression of Bax and Bcl-2 were tested by real-time PCR,the activity of tumor necrosis factor-??TNF-??,Interleeukelin-6?IL-6?and endothelial nitric oxide synthase?eNOS?in BMECs cultural supernatants,the activity of endothelin-1?ET-1?,tissue-type plasminogen activator?t-PA?,Interleeukelin-1??IL-1??,plasminogen activator inhibitor 1?PAI-1?,human Prostaeycline 2?PGI2?and interferon-??INF-??in BMECs were determined by ELISA kits and the content of catalase?CAT?,nitric oxide synthase?NOS?,xanthine oxidase from buttermilk?XOD?,superoxide dismutase?SOD?and lactate dehydrogenase?LDH?in BMECs cultural supernatants were determined by kits.Results:?1?Breviscapine produces the less toxicity to HUVECs.The different doses of breviscapine can effectively reduce MDA and NO content,increase SOD and GST activity and significantly improve the HUVECs injury by H₂O₂.?2?The BMECs were cultured by ?factor immunohistochemistry and cell morphology.?3?The OGD model had significant inhibition effects on the activity of cells.The longer the OGD administrated,the higher the inhibition ratio of cells.It is believed that the 50% inhibition ratio is the best modeling time points when 6h later in OGD model.?4?There were slight toxic effects of breviscapine on BMECs.The different concentrations of breviscapine could protect the injured BMECs,effectively inhibited apoptosis,up-regulate Bcl-2 expression and down-regulate Bax expression,reduce ET-1 secretion to increase PGI2 secretion,reduce TNF-?,IL-6,IL-1?,INF-? and LDH content and PAI-1,NOS and eNOS activity and increase SOD,XOD and t-PA activity.Conclusions:?1?The breviscapine had no toxic effects on HUVECs and BMECs.?2?Breviscapine showed the significantly protective effects on HUVECs injured by H₂O₂.Its mechanism might relate to inhibit lipid peroxidation,increase antioxidase activity of body and facilitate synthesis of NO.?3?There were significantly protective effects of breviscapine on BMECs with OGD injury.Its protective mechanisms might relate to inflammatory response and apoptosis and increase anti-oxidative enzyme activity and antioxidant capacity of body and coagulant and fibrinolytic regulating capacity of BMECs.