Quercetin Suppresses The Oxidative Stress In Vascular Smooth Muscle Cells

Reactive oxygen species (ROS), such as superoxide (O2?-), hydrogen peroxide (H2O2), hydroxyl radicals (OH?) and hypochlorous acid (HOCL), etc, play important roles in development of cardiovascular diseases. NAD(P)H oxidase, a multi-subunit enzymatic complex, is the major sources of ROS in vascular tissues, which is regulated by cytoplasmic subunits p47phox. Thus, NAD(P)H oxidase is a new antioxidant therapeutic target.Quercetin, as a natural flavonoid, is abundantly present in plants. Now, quercetin is recognized as very potent antioxidants. The present study was designed to investigate crucial mechanisms of quercetin on suppressing the oxidative stress in vascular smooth muscle cells (VSMCs).Methods:The production of O2?- in VSMCs was detected by dihydroethidium (DHE) staining. Malondialdehyde (MDA) content and total superoxide dismutase (SOD) activity assay were performed according to kit manufacturer's protocol. The protein extracts from VSMCs were used to detect the expression and phosphorylation of p47phox by Western blot. RT-PCR was performed to detect the mRNA of p47phox. Results:1 Quercetin inhibits the formation of O2?- induced by H2O2 DHE staining showed that the intensity of red fluorescence increased significantly after treatment with H2O2 (200μmol/L) for 12 h in VSMCs. The preincubation with 50μmol/L quercetin resulted in reduction in O2?- production induced by H2O2. This result suggested that quercetin can inhibit the formation of O2?- induced by H2O2.2 Quercetin decreases MDA content and enhances SOD activityWhen being treated with 200μmol/L H2O2 for 12 h, the level of MDA in culture medium was increased by 2.78-fold compared with control group. However, the amount of MDA was significantly reduced in VSMCs treated by quercetin before H2O2 stimulation, compared to H2O2 group, suggesting that quercetin inhibits the MDA production induced by H2O2.The SOD activity of VSMCs pretreated with quercetin increased by 1.39-fold (P<0.01) compared with H2O2 group, which decreased by 38.2% (P<0.01) compared with control group, suggesting quercetin can elevate the breakdown of O2?- induced by H2O2 via maintenance of SOD activity.3 Effect of H2O2 on the expression of p47phox in VSMCsWestern blot analysis showed that H2O2 triggered p47phox expression. The expression of p47phox increased dramatically in VSMCs treated with H2O2 at different doses (0, 50, 100, 200, 400μmol/L) for 12 h. After treatment by 200μmol/L H2O2 from 0 to 24 h, p47phox protein increased by 1.08-, 1.21-, 1.43-, 1.79- and 1.62-fold compared with control group (P<0.01), respectively. These findings indicated that H2O2 induces the expression of p47phox in a dose- and time-dependent manner.4 Effect of quercetin on the expression of p47phox in VSMCs induced by H2O2RT-PCR and Western blot assays showed that treatment with 200μmol/L H2O2 for 12 h resulted in increased the level of p47phox by 1.75-fold and 1.79-fold compared with control group, respectively. After the cells were preincubated with 50μmol/L quercetin, the gene transcriptional activities and protein expressions of p47phox were significantly reduced, suggesting that quercetin can inhibit the expression of p47phox induced by H2O2. 5 Quercetin inhibits the H2O2-induced phosphorylation of p47phox in VSMCsImmunoprecipitation analysis showed that H2O2 triggered p47phox phosphorylation, which reached to peak at 15 min and kept high level up to 30min. Pretreating of the cells with quercetin, the level of p47phox phosphorylation was significantly reduced by 47.2% (P<0.01). This result suggested that quercetin suppresses the phosphorylation of p47phox induced by H2O2.Conclusions:1 Quercetin suppresses the production of O2?- induced by H2O2 in VSMCs.2 Quercetin plays a protective effect on SOD activity.3 Quercetin decreases the production of O2?- via inhibiting p47phox expression and phosphorylation, and increase in SOD activity.4 Quercetin enhances the antioxidation of VSMCs.