Puerarin Preconditioning Protects Human Umbilical Vein Endothelial Cells From Hypoxia/Reoxygenation Injury

Objective: To observe effects of puerarin preconditioning on e NOS protein expression, its phosphorylation level at site of Ser633(p-e NOS Ser633), i NOS protein expression as well as activities of e NOS and i NOS 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 e NOS protein, its phosphorylation at site of Ser633 and i NOS protein in each group were measured by western blotting. The activity of NOS was determined via chemical colorimetric methods. Results: Compared with control group, levels of e NOS total protein expresstion and p-e NOS(Ser633) decreased after H/R injury(P<0.05), and gradually reduced with extension of hypoxic time. At the moment, i NOS protein level increased significantly. Compared with same time H/R group, levels of e NOS total protein expresstion and p-e NOS(Ser633) significantly enhanced, while i NOS protein reduced after PUE preconditioning(P<0.05). Compared with control group, activities of total NOS(t NOS) and induced NOS(i NOS)significantly increased(P<0.01), while activity of constitutive NOS(c NOS) decreased(P<0.05) in each H/R group, and activity of c NOS gradually decreased with prolonged hypoxic time(P<0.05), but activities of t NOS and i NOS were unchanged. Compared with same time H/R group, activity of c NOS significantily increased after PUE preconditioning(P<0.01), on the contrary, activity of i NOS decreased(P<0.05), while activity of t NOS remained unchanged. Conclusion: Levels of total e NOS protein expression and p-e NOS(Ser633) as well as activity of c NOS in HUVECs significantly reduce, meanwhile i NOS protein and its enzyme activity increase after hypoxia/reoxgenation(H/R) injury,leading to endothelial dysfunction. Puerarin preconditioning can up-regulate e NOS protein expression and phosphorylation at Ser633 and its activity, simultaneously down-regulate i NOS protein and its activity in HUVECs which subjected to hypoxia/reoxygenation injury, thus protects endothelial cells from injury. [Key Words] puerarin, hypoxia/ reoxygenation injury, human umbilical vein endothelial...