Protection Of Farrerol Against Oxidative Stress-Induced Damage Of Endothelial Cells And Its Underlying Mechanisms

Objective:Farrerol, a major flavanone present in plants, is the main active substance of "Man-shan-hong"(Rhododendron dauricum L.) for antibechic. Modern pharmacological studies indicate that farrerol also has antibacterial, anti-inflammatory, immune suppression and inhibition of vascular smooth muscle cells. Hesperetin and naringenin are main flavanone present in citrus fruits, which display predominantly antioxidative and cytoprotective activities. However, researches on antioxidative and cytoprotective effects of farrerol are very limited. Therefore this paper was designed to research the antioxidative and cytoprotective effects of farrerol. The first part of this paper demonstrated the antioxidative and cytoprotective effects of farrerol on hydrogen peroxide (H2O2)-induced EA.hy926cell lines. The second part of the paper demonstrated the regulation of farrerol on hydrogen peroxide (H2O2)-induced MAPK activation in EA.hy926cell lines. In the third part, the preliminary antiothrombosis of farrerol in vivo were studied on FeCl3-induce carotid artery thrombosis in rats.Methods:(1) MTT assay was used to detect the inhibition of farrerol on H2O2-induce decrease of EA.hy926cells viability.(2) The intracellular and serum MDA content and SOD, GSH-Px enzymatic activities were detected by UV spectrophotometry to evaluate the regulation of farrerol on H2O2-induced cellular redoxidative level.(3) The regulation of farrerol on H2O2-induced cellular reactive oxygen species (ROS) generation was measured using flow cytometry.(4) PI/Annexin V-FITC double staining assay by flow cytometry was used to detect the effect of farrerol on the cell apoptotic ratio in H2O2-induced EA.hy926cells.(5) The regulation of farrerol on apoptosis-related Bcl-2and Bax genes expressions were obtained by RT-PCR.(6) Western blot method was used to detect the protein expressions of Bcl-2, Bax, cleaved caspase-3, occludin and MAPK activation.(7) Immunofluorescent staining was used to detect the expression of occludin.(8) HE staining was used to detect antithrombosis of farrerol in FeCl3-induce carotid artery thrombosis in rats.Results:(1) Farrerol at the concentration of less60μmol/L had little cytotoxity on EA.hy926cells.(2) Farrerol (15,30, and60μmol/L) inhibited significantly the decreases of cell viability and cellular enzymatic activities of SOD and GSH-Px and the increase of ROS generation in H2O2-induced EA.hy926cells in a dose-dependent manner (p<0.05).(3) PI/Annexin V-FITC double staining assay demonstrated that farrerol at the concentrations of15,30, and60μmol/L attenuated significantly H2O2-induced EA.hy926cells apoptotic ratio from39.1%to32.35%,21.9%, and15.4%, respectively (p<0.05).(4) Western blot and RT-PCR analysis showed that farrerol of15,30, and60μmol/L inhibited significantly H2O2-induced increase of Bax mRNA and protein expression and the decrease of Bcl-2mRNA and protein expression in a dose-dependent manner (p<0.05).(5) Western blot demonstrated that farrerol showed the obvious regulation on the expression of cleaved caspase-3in H2O2-induced EA.hy926cells in a dose-dependent manner (p<0.05).(6) Western blot and Immuno fluorescent staining demonstrated that farrerol showed the obvious regulation on the expression of occludin in H2O2-induced EA.hy926cells in a dose-dependent manner (p<0.05).(7) Western blot demonstrated that farrerol possessed the obvious regulation on the activiation of protein kinases ERK1/2, p38MAPK and c-Src and protein phosphase SHP-2(p<0.05), which were likely associated with the regulation of apoptosis induced by H2O2in EA.hy926cells.(8) In FeCl3-induced rat carotid arterial thrombosis model, compared with the model, farrerol treatment (20,40, and80mg/kg, i.g.) for14days prevented the the increase of MDA content and the decrease of SOD and GSH-Px activity in Spregue Dawey rats serum in a dose-dependent manner (p<0.05).(9) Hematoxylin and eosin (HE) staining indicated that farrerol with high dose (80mg/kg) prevented the thrombosis in FeCl3-induced rat carotid arterial thrombosis model.Conclusions:(1) Farrerol had a inhibitive effect on H2O2-induced apoptosis in EA.hy926cells, which is likely associated with the regulation of intracellular MDA and ROS levels, the activities of SOD and GSH-Px, and modulation of the expression of Bax, Bcl-2, cleavedcaspase-3, as well as the phosphorylation of ERX1/2and p38.(2) The regulation of farrerol on the phosphorylation of ERX1/2was likely associated with the modulation on Src and SHP-2.(3) It was demonstrated preliminarily that farrerol displayed antiathrombosis in FeCl3-induced rat carotid arterial thrombosis model.(4) It was indicated preliminarily that farrerol possessed antioxidative and cytoprotective activities.