Genistein Affect The Synthesis Of Nitric Oxide In Human Umbilical Vein Endothelial Cell By PI3K/AKT Signaling Pathway

Background:Pulmonary hypertension(PH) is a chronic debilitating lung disorder associated with pulmonary vascular remodeling and progressive increase in pulmonary artery(PA) pressure leading to right ventricular(RV) hypertrophy, right heart failure(RHF), and death. In recent years, the use of targeted drugs have improved the survival rate of pulmonary hypertension, but its effect is limited and expensive. Genistein, a natural soybean-derived phytoestrogen, has shown to have vasodilator, cardio-protective, and anti-inflammatory effects, which exerts most of its protective effects may via estrogen receptor-?(ER?) was found in some studies. Previous animal experiments show that genistein can improve rat pulmonary arterial hepertension and survival which induced by monocrotaline, but its mechanism was not clear. Number of studies show that P13K/AKT-e NOS signaling pathway involved in this process. We suspect that genistein up-regulates endothelial nitric oxide synthase(e NOS) activity by PI3K/AKT signaling pathway, increasing endothelium-derived vasodilator nitric oxide(NO) synthesis,which have relaxing blood vessels.Objective:The purpose of this research was to discussion whether genistein would increase the synthesis of NO by up-regulating e NOS activity through PI3K/AKT signaling pathway in human umbilical vein endothelial cells and whether ER? plays a vital part in the process of NO synthesis.Methods:1.Cultured human umbilical vein endothelial cells(HUVEC) in vitro and divided into seven subgroups, ie control group, Gen(100nmol/L) subgroup, PI3 K inhibitor LY2942002(10?M) with Gen(100nmol/L) group, AKT inhibitor MK-22062HCL(5?M) with Gen(100nmol/L) group,e NOS inhibitor L-NAME (10m M) with Gen(100nmol/L) subgroup,ER? inhibitor MPP(1?M) with Ge n(100nmol/L) group and ER? inhibitor PHTPP(1?M) with Gen(100nmol/L) subgroup.2. After cultured 24 hours, nitric oxide levels was measured by nitrate reducates in each group. The expression of proteins, such as total AKT, total e NOS, ER?, ER?, phosphorylation of e NOS(Ser1177) and phosphorylation of AKT(Ser473) were measured by Western-Blots.Results:1. The levels of NO and expression phosphorylation of e NOS(Ser1179) and phosphorylation of AKT(Ser473) were higher in Gen subgroup compared with control(p<0.05).2. While in PI3 K inhibitor LY2942002, AKT inhibitor MK-22062 HCL, e NOS inhibitor L-NAME subgroups, which the expression level of the detected item has declined compared with Gen subgroup(p<0.05), but more higher than control subgroup.3. NO significant difference was found while compared MPP with PHTPP groups in the expression level of the detected item(p>0.05).4. Each group t-AKT, t-e NOS levels have no statistically significant(p>0.05). Conclusion:Gen induce NO synthesis is mainly to promote e NOS(Ser1177) phosphorylation levels increase the role of e NOS activity by PI3K/AKT signaling pathway in human umbilical vein endothelial cells, and estrogen receptor involved in this process, but the ER? and ER? were found no significant difference.