Puerarin Preconditioning Protects Human Umbilical Vein Endothelial Cells Against Hypoxia/reoxygenation Injury Through Increasing Endothelial Nitric Oxide Synthase Protein Expression

Objective: To observe effects of puerarin(PUE) preconditioning on endothelial nitric oxide synthase(eNOS) protein expression and its phosphorylation levels, and the signaling pathways may be involved, therefore to explore the mechanism of PUE preconditioning protects the human umbilical vein endothelial cells(HUVECs) from hypoxia(H) /reoxygenation(R) injury.Methods: HUVECs were cultured under hypoxic condition for 2,4 or 8 hours, then normal oxygen condition for 4 hours to establish hypoxia/reoxygenation injury model after the cells were identified by SABC method. HUVECs were randomly divided into normal control group, H/R group, PUE preconditioning group and PUE preconditioning + H/R group(1.0×10-3 mol/L,PUE pretreated the cells for 24 h before H/R).In addition, H4/R4(4 h hypoxia/ 4 h reoxygenation) group cells were treated with ERK1/2 inhibitor U0126(1.0×10-5mol/L) and PKB/Akt inhibitor LY294002(5.0×10-5mol/L) respectively for 1 h before PUE preconditioning, then the cells were cultured under H/R condition. Meanwhile, the simple inhibitor group and solvent control group were established. The levels of eNOS, Akt, ERK1/2 protein expression and p-eNOS(Ser1177, Ser633 and Thr495), p-Akt(Thr308, Ser473), p-ERK were measured by western blot. The activity of constitutive NOS was determined via chemical colorimetric methods. Apoptosis of HUVECs was detected by TUNEL assay. Results: ⑴ Compared with control group, the levels of eNOS protein expression decreased significantly(P<0.05), and p-eNOS(Ser1177 and Ser633) level declined parallel to the decrease of eNOS protein expression; p-Akt(Thr308 and Ser473), p-ERK were significantly reduced(P<0.05), the activity of constitutive NOS(cNOS) decreased as well(P<0.05) after H/R injury. The levels of eNOS Thr495, Akt and ERK1/2 protein expression were unchanged in each group. ⑵ Compared with same time H/R group, PUE preconditioning significantly upregulated the level of eNOS protein expression(P<0.05), and p-eNOS(Ser1177 and Ser633) increased correspondingly; meanwhile p-Akt(Thr308 and Ser473), p-ERK enhanced significantly(P<0.05) and the activity of cNOS increased(P<0.05) after PUE preconditioning. There is no statistical difference in levels of eNOS Thr495, Akt and ERK1/2 protein expression in each group. ⑶ After U0126 treatment, compared with PUE+H4/R4 and PUE group, the levels of eNOS protein expression, p-ERK decreased in U0126+PUE+H4/R4 group and U0126+PUE group(P<0.05). ⑷ After LY294002 treatment, compared with PUE+H4/R4 and PUE group, the levels of eNOS protein expression, p-Akt(Thr308 and Ser473) decreased in LY294002+PUE+H4/R4 group and LY294002+PUE group(P<0.05). ⑸ Compared with control group, the apoptosis index significantly increased in H/R group(P<0.01), PUE preconditioning reduced the apoptosis index(P<0.01). Conclusion: ⑴ Levels of eNOS protein expression and the activity of cNOS significantly decrease in HUVECs after H/R injury, at the same time the apoptosis of cells increases, leading to endothelial damage. ⑵ PUE preconditioning upregulates the levels of eNOS protein expression and the activity of cNOS, reduces the apoptosis of HUVECs which subjected to H/R injury, thus PUE exerts obviously endothelial protective effect. ⑶ The decrease of eNOS protein expression may be related to the inhibition of ERK1/2 and PKB/Akt signaling pathways after H/R injury, while PUE pretreatment can increase the expression of eNOS protein by activating ERK1/2 and PKB/Akt signaling pathways.