Effects Of Genistein On The ENOS Expression Of HUVECs

Objective: To explore the effect of genistein on the eNOS expression of HUVECs induced byoxidized low density lipoprotein.Methods:1. To explore the effect of genistein on endothelial nitric oxide synthase (eNOS) expression fora long time intervention：(1) Human umbilical vein endothelial cells (HUVECs) were incubated with ox-LDL(100mg/L)for24h, then treated with different concentrations of genistein(10nmol/L，50nmol/L，100nmol/L)for24h. Cell viability was measured by MTT method; the mRNA expression of eNOS wasdetected by real-time RT-PCR, the protein expression of eNOS was determined by Western blot.(2) HUVECs were exposed to ox-LDL (100mg/L) in the presense or absence of Estrogenantagonists ICI182780(1μM) or/and actinomycin D (5mg/L) for30min, then were treated withgenistein (100nmol/L) for24h. The mRNA expression of eNOS was detected by real-timeRT-PCR, and the protein expression of eNOS was determined by Western blot.2. To observe the effect of genistein on short time intervention:HUVECs were incubated with ox-LDL (100mg/L), then were stimulated with genistein(100nmol/L) for5min、10min、15min、30min and60min. The production of NO was assessedby Griess reaction in cell culture supernatant. The expressions of eNOS mRNA were measuredwith RT-PCR. The expressions of eNOS protein and phosphorylation eNOS (Ser1177) proteinwere measured with western blot. Meanwhile, the effect of genistein was observed withLY294002or NSC154020(PI3K and AKT inhibitors) through measuring phosphorylationeNOS(Ser1177) level. Results:1. The effect of genistein on eNOS expression for a long time intervention:(1) MTT showed: cell viability of HUVECs treated with ox-LDL (100mg/L) was significantlyinhibited (P<0.05), while genistein significantly enhanced cell viability. With the increase ofgenistein concentration (10nmol/L,50nmol/L,100nmol/L), OD value was increased moresignificantly (P<0.05).(2) RT-PCR and Western blot showed: ox-LDL down-regulated the expression of eNOS mRNAand protein (P<0.05vs that of basal level); genistein up-regulated the expression of eNOSmRNA and protein (P<0.05vs that of ox-LDL-treated group). Furthermore, the increase bygenistein can be inverted by estrogen antagonists ICI182780or actinomycin D(P<0.05).2. The effect of genistein on eNOS expression for a short time intervention:Compared with the ox-LDL-treated group, the concentrations of NO and the expression level ofphosphorylation eNOS(Ser1177) increased (P<0.05) in genistein-treated group，with the peak atthe point of15min (P<0.05)，however，the expression of eNOS mRNA and non-phosphorylatedeNOS protein did not change (P>0.05). Furthermore，the expression level of phosphorylationeNOS(Ser1177) was inhibited by PI3K/AKT inhibitors LY294002and NSC154020(P<0.05).Conclusion:(1) The effect of genistein on eNOS expression for a long time intervention：genistein couldup-regulate the expression of eNOS, which is associated with genes transcription regulated byestrogen receptor.(2) The effect of genistein on eNOS expression for a short time intervention：genisteinpromoted the activity of eNOS through increasing phosphorylation eNOS(Ser1177) level throughPI3K/AKT signaling pathway.