Study On Mechanisms Underlying The Cardiomyocytes Protective Effects Of Quercetin Against Ischemia/Reperfusion Injury

The morbidity and mortality of cardiovascular diseases increased significantlyduring recent years. Finding the nutrients and phytochemicals which havecardiovascular protective effects and exploring its molecular mechanism havebecome one of the important assignments of nutrition research. Epidemiologicalsurvey found, the daily intake of flavonoids is significantly negative related to thedevelopment of the incidence of cardiovascular disease. Quercetin (3,3’,4’,5,7-pentahydroxy-flavone), is a plant derived flavonoids compounds widely found infruits, vegetables and grains. Ischemia/reperfusion injury refers that when ischemictissue is reperfused, the tissue and organ function further damaged.Ischemia/reperfusion injury is the main reason of ominous prognosis and death ofcardiovascular diseases. Researches showed that quercetin may be effective againstmyocardial ischemia/reperfusion injury, but the mechanism is not clear.The aim of this study was to explore the molecular mechanism of quercetin onmyocardial ischemia/reperfusion injury. An anoxia/reoxygenation (A/R) injurymodel of primary cultured neonatal rat cardiomyocytes was conducts to imitate invivo ischemia/reperfusion injury.Firstly, cardiomyocytes were pretreated with10μ M,20μ M,40μ M and80μM quercetin24hours before A/R respectively, and then detected the cell survival rateand14-3-3protein expression level of each group, to selected best dosage ofquercetin to upregulate14-3-3η protein expression and the play the cardio protectiveeffects simultaneously. Then cardiomyocytes were pretreated with20μM quercetin at24hours,48hours,72hours before the A/R respectively, to select the bestpretreatment time for quercetin to upregulate the14-3-3ηin cardiomyocytes sufferedA/R. The results revealed that20μ M quercetin pretreated at72h before A/Rsignificantly increased the expression of14-3-3η protein in primary cultured neonatalrat cardiomyocytes and played a cardio protective role simultaneously, compared withthe control group and A/R group, p<001. Next, neonatal rat primary cardiomyocytes infected by14-3-3η RNAiadenovirus, and pretreated with quercetin, detected the cell survival rate, activity ofCPK and LDH activity, content of ROS and MDA, activity of antioxidases SOD andGSH-Px, mitochondrial membrane potential, mPTP opening, apoptosis after A/Rexperiment, compared the result with the group did not infected14-3-3η RNAiadenovirus to investigate the relationship between the cardio protective effect ofquercetin and its ability to upregulated the14-3-3η expression. The resultsconfirmed that cardio protective effects from A/R injury of quercetin, includingoxidative stress, mitochondrial membrane potential, stability, inhibiting the openingof mPTP and reduce the cell apoptosis were dependent on its ability to upregulating14-3-3η expression.14-3-3η is an important regulating protein in cells.14-3-3ηactualize itsbiological function through combining with different target proteins, then activate thetarget protein, change the conformational of target protein, or help target proteinstranslocate to the specific place in cells. PKC ε is the critical endogenous protectiveprotein in cardiomyocytes against ischemia/reperfusion injury. Studies suggested thatPKCε should translocate from cytoplasm to mitochondria during the cardioprotectionmediated by it. But how the translocation happened is still not know.To investigate the interaction between14-3-3and PKC ε, we use GST-pulldown,Co-IP, immunofluorescence double staining and laser scanning confocal microscopy.Results confirmed that14-3-3η combined with PKC ε in neonatal rat primarycardiomyocytes suffered A/R. Result of immunofluorescence staining andmitochondrial tracer showed that,14-3-3η and PKCε can both located tomitochondria in cultured neonatal rat primary cardiomyocytes with A/R damage.To further explore the effect of14-3-3η in PKCε mediated myocardial againstischemia/reperfusion injury, we constructed the recombinant adenovirusADeasy14-3-3η and ADeasyPKCε. Neonatal rat primary cardiomyocytes wereinfected by ADeasyPKCε, co-infected by ADeasyPKCε and ADeasy14-3-3η,co-infected by ADeasyPKCε and AD14-3-3ηRNAi, infected by ADeasyPKCε plusquercetin pretreated respectively. Then detected the of cell survival rate, CPK andLDH activity, and oxidative stress related to intracellular ROS and MDA content antioxidant activity of GSH-Px and SOD, isolated the mitochondria of each group,western blot detected the mitochondrial distribution of PKCε, detected the indexassociated with mitochondria function including mitochondrial membrane potentialand mPTP opening, and the apoptosis associated Caspase3activity and cell apoptosisrate. To investigate the effects of downregulation of14-3-3η, exogenous or quercetinpretreatment induced14-3-3η upregulation on the mitochondrial distribution ofPKCε and the cardio protective effects mediated by it. Results showed that14-3-3ηprotein overexpression in cardiomyocytes no matter induced by quercetin orrecombinant adenovirus, could promote the migration of PKCε from cytosol tomitochondria, and enhanced the A/R injury resistance of primary neonatal ratcardiomyocytes mediated by PKCε. The influences of14-3-3η RNAi adenoviruswere just reverse.In summary, we confirmed that the mechanisms underlying the anti-A/R injurycardio protective effect of quercetin involved in upregulating the expression of14-3-3η protein, then promote the PKCε trans locating to mitochondria, enhance theeffect of PKCε in maintenance mitochondrial membrane potential, closing mPTP,decreasing apoptosis.This study revealed the molecular mechanism underlying the cardio protectiveeffects of quercetin against A/R damage, which provided a new theoretical foundationand basis for application of quercetin in cardiovascular protection. Opened up theideas to research mechanism of plant chemical and develop the plant nutrients withthe cardiovascular protective effects.