葛根素预处理对缺氧复氧损伤后人脐静脉内皮细胞的保护作用-protective effect of puerarin preconditioning on human umbilical vein endothelial cells after hypoxia-reoxygenation injury.docxVIP

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葛根素预处理对缺氧复氧损伤后人脐静脉内皮细胞的保护作用-protective effect of puerarin preconditioning on human umbilical vein endothelial cells after hypoxia-reoxygenation injury.docx

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葛根素预处理对缺氧复氧损伤后人脐静脉内皮细胞的保护作用-protective effect of puerarin preconditioning on human umbilical vein endothelial cells after hypoxia-reoxygenation injury

PAGE VI PAGE VI万方数据培养后可以使HUVECs eNOS总蛋白表达及eNOS Ser633位点磷酸化水平降低、cNOS 酶活力降低，同时iNOS蛋白表达增加、iNOS酶活力增强，内皮细胞功能受损。应用PUE预处理可以上调缺氧/复氧损伤后HUVECs eNOS总蛋白表达及eNOS Ser633位点磷酸化水平，并增加酶活性，并能降低iNOS蛋白表达及酶活性，从而起到保护内皮细胞的作用。【关键词】葛根素；人脐静脉内皮细胞；缺氧/复氧损伤；内皮型一氧化氮合酶Puerarin preconditioning protects human umbilical vein endothelial cells from hypoxia/reoxygenation injuryMajor: Pathology and pathophysiology，Postgraduate: SU Feng，Supervisor: LU De-Qin[Abstract] Objective: To observe effects of puerarin preconditioning on eNOS protein expression, its phosphorylation level at site of Ser633 (p-eNOS Ser633) , iNOS protein expression as well as activities of eNOS and iNOS in human umbilical vein endothelial cells (HUVECs) subjected to hypoxia/reoxygenation-mediated injury, thereby elucidating protective effects of puerarin preconditioning on endothelial cells. Methods: HUVECs were cultured under hypoxic condition for 2, 4 or 8 hours then reoxgenational condition for 4 hours to establish hypoxia/reoxygenation injury cell model. The HUVECs were randomly divided into control group, hypoxia 2h/reoxygenation 4h (H2/R4) group,hypoxia 4h/reoxygenation 4h (H4/R4) group, hypoxia 8h/reoxygenation 4h (H8R4) group, puerarin preconditioning (PUE) group, PUE+H2/R4 group, PUE+H4/R4 group and PUE+H8/R4 group. Puerarin preconditioning group cells were pretreated with DMEM medium containing puerarin at final concentration of 1.0×10-3mol/L for 24 hours. Levels of total eNOS protein, its phosphorylation at site of Ser633 and iNOS protein in eachgroup were measured by western blotting. The activity of NOS was determined via chemical colorimetric methods. Results: Compared with control group, levels of eNOStotal protein expresstion and p-eNOS (Ser633) decreased after H/R injury (P0.05), andgradually reduced with extension of hypoxic time. At the moment, iNOS protein level increased significantly. Compared with same time H/R group, levels of eNOS total protein expresstion and p-eNOS (Ser633) significantly enhanc