Antagonism Effect Of Genistein On Paraoxon-induced Injury On The Rat Vascular Endothelial Function As Well As Investigation On The Mechanisms

Backgrounds:Vascular endothelial cells （VEC） are a layer of flat epithelial cells,they line internal surface of blood vessels. These cells are involved in various lifeactivities, such as body substance trafficking, blood clotting, immune response,biosynthesize and release many vascular active substances, and help regulate vascularphysiological activities. Several factors including oxidative stress, inflammation,immune response, toxin cause vascular endothelial cell injury and further lead to celldysfunction, blood vessel wall damage, ultimately result in a variety of cardiovasculardiseases. Paraoxon （PO） is the active metabolite of organophosphate insecticideswhich was metabolized in liver by cytochrome P450enzyme. PO enhances bodyoxidative stress, damages vascular endothelial cells, induces cardiovascular diseases.Genistein （GST） is a phytoestrogen with polyphenol hydroxyl group mainly derivedfrom Leguminosae plants. It has direct anti-oxidative effect, and also has indirectanti-oxidative effect mediated by increasing the level and activity of bodyanti-oxidative enzyme and inhibiting NADPH oxidase activity. So it avoids oralleviates vascular endothelial cells oxidative damage and keeps norm function ofVEC. NADPH oxidase was firstly found in macrophage and neutrophil. Nox4andp22phox are the primary form of NADPH oxidase in endothelial cells. The primaryfunction of NADPH oxidase is involved in reactive oxygen species （ROS）biosynthesis and oxidative stress reaction regulation.Objective: To examine the protective effects and potential mechanisms of genisteinon vascular endothelium damage induced by paraoxon.Methods:（1） Rat isolated vascular rings experiment in vitro: Thoracic aortaswere isolated from30male SD rats. The isometric tension of the thoracic aorta rings was measured. The aortic rings were divided into the following groups: control group,the thoracic aorta rings was incubated with0.1%dimethyl sulfoxide （DMSO） for30min. The acetylcholine (Ach,1×10^-9-1×10^-5mol/L)-induced EDR and sodiumnitroprusside (SNP,1×10^-6mol/L)-induced endothelium-independent relaxation weremeasured. Paraoxon groups, the rings were incubated with PO at the concentrationof4.05×10^-9-4.05×10^-5mol/L for30min. The Ach-induced and SNP-inducedrelaxations were measured. PO+GST groups, the rings were incubated with PO(4.05×10^-6mol/L) plus GST(1×10^-9,3×10^-9,1×10^-8,3×10^-8,1×10^-7mol/L) for30min.The Ach-and SNP-induced relaxations and phenyllphrine (PE,1×10^-6mol/L)-induced contraction were measured.（2） Detection of mRNA expression of p22phoxand Nox4by reverse transcription-polymerase chain reaction （RT-PCR）:Thoracic aortas were isolated from the male SD rats and divided into the followinggroups: control group, the thoracic aorta was incubated with0.1%DMSO for30min;GST group, the thoracic aorta was incubated with1×10^-7mol/L GST for30min;Paraoxon group, the thoracic aorta was incubated with PO at the concentration of4.05×10^-5mol/L for30min; PO+GST groups, the rings were incubated with PO（4.05×10－5mol/L） plus GST(1×10^-7mol/L) for30min. The expression of p22phoxand Nox4mRNA was detected by RT-PCR.（3） Detection of protein expression ofp22phox and Nox4by western-blotting: Thoracic aortas were isolated from themale SD rats, the experimental groups and treatments were as （2）. The expression ofp22phox and Nox4protein was detected western-blotting.Results:（1） Rat isolated vascular rings experiment in vitro: PO(4.05×10^-9-4.05×10^-5mol/L) concentration-dependently inhibited Ach-induced EDR, but did notaffect SNP-induced endothelium-independent relaxation of aortic isolated rings. GSTat1×10^-8,3×10^-8,1×10^-7mol/L lessened the inhibition of Ach-induced EDR injuredby PO (4.05×10^-6mol/L) and GST at1×10^-8-3×10^-8mol/L had the concentration-dependent inhibitory effect. But GST did not affect SNP-induced endothelium-independent relaxation of aortic isolated rings. GST also concentration-dependentlyinhibited PE-induced contraction of aortic isolated rings;（2） Detection of mRNA expression of p22phox and Nox4by RT-PCR: treatment with paraoxon resulted inthe mRNA expression of p22phox and Nox4up-regulation compared with the controlgroup; the genistein group, the mRNA expression of p22phox and Nox4wassignificantly repressed compared with the control group; treatment with genisteinplus paraoxon significantly increased the mRNA expression of Nox4（P＜0.05）butthe mRNA expression of p22phox was no significant difference compared with thecontrol group; while compared with the paraoxon group, the PO+GST group, themRNA expression of p22phox was significantly decreased （P＜0.05）but the mRNAexpression of Nox4was no significant difference;（3） Detection of proteinexpression of p22phox and Nox4by western-blotting: treatment with paraoxonresulted in the protein expression of p22phox and Nox4up-regulation compared withthe control group; the genistein group, the protein expression of p22phox and Nox4was significantly repressed compared with the control group; treatment with genisteinplus paraoxon significantly increased the protein expression of Nox4（P＜0.05）butthe protein expression of p22phox was no significant difference compared with thecontrol group; while compared with the paraoxon group, the PO+GST group, theprotein expression of p22phox was significantly decreased （P＜0.05）but the proteinexpression of Nox4was no significant difference.Conclusion:（1）Paraoxon inhibited Ach-induced EDR, genistein could inhibit paraoxon-inducedEDR injury and decrease PE-induced contraction.（2） Paraoxon could up-regulated p22phox and Nox4mRNA and protein expression,injured vascular endothelial function; genistein could down-regulated p22phox andNox4mRNA and protein expression, protected vascular endothelial function.