Inhibitory Effect Of Ginkgo Biloba Extract On The Expression Of Inducible Nitric Oxide Synthase In Endothelial Cells

Aims:To investigate the inhibitory effect of Ginkgo biloba extract on the expression of inducible nitric oxide synthase in endothelial cells and its mechanism.Methods:The human endothelial cell line ECV304 cells were cultured in 1 mL of RPMI-1640 medium in the absence or presence of EGb. Then, Cells were cultured without or with EGbfor various periods of time. After cells were incubated with EGb for 2 or 4 hr, the amounts of NO metabolites released by the cells were quantitated. NOS protein expression was determined by western immunoblotting analysis. mRNA levels were examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis.Results:(1) the effect of EGb on endothelial cells was not dependent on changes in intracellular calcium concentration. Pretreatment of cells with EGb(50 μg/mL) for 2 hr did not have a significant effect on the amount of NO metabolites released by ECV304 cells. After cells were incubated with EGb for 4 hr, the amount of NO metabolites released into the culture medium was reduced significantly (to 70% of control).Cells then were incubated with various concentrations of EGb for 4 hr, and the amount of NO released into the culture medium was determined. EGb inhibited the release of NO metabolites by endothelial cells in a concentration-dependent manner. inclusion of the calcium ionophore in the culture medium did not change the inhibitory effect of EGb on NO release from endothelial cells. (2) Decreased NOS activities in EGb-treated cells might be due to a reduction in the levels of enzyme proteins. 50μg and 100 μg/mL of EGb treatment caused a 27%and 43% reduction in the levels of iNOS protein, respectively. (3) A reduction in iNOS protein levels in EGb-treated cells might be a result of changes in the expression of iNOS mRNA in these cells. EGb inhibited the expression of iNOS mRNA in a concentration-dependent manner. After cells were treated with EGb at concentrations of 50μg/mL and 100μg/mL, the ratio of iNOS to P-actin was reduced from 0.22(control) to 0.16 and 0.14, respectively, reflecting a significant decrease in levels of mRNA as compared with the control.Conclusions:1. The effect of EGb on endothelial cells was not dependent on changes in intracellular calcium concentration.2. Decreased NOS activities in EGb-treated cells might be due to a reduction in the levels of enzyme proteins.3. Decreased production of NO in endothelial cells was a result of reduced levels of iNOS mRNA in EGb-treated cells.