Effects Of Quercetin On Homocysteine-Induced Apoptosis Of H9C2 Cardiomyocytes

Objective:In the present stuy,we aim to To investigate the effect of quercetin（Que）on homocysteine-induced apoptosis of H9C2 cardiomyocytes and its possible mechanism,And provide the experimental basis for the clinical application of quercetin.Methods:1、H9C2 cardiomyocytes were cultured in vitro。Using MTT assay to detect the effects of different concentration of quercetin（10umol/L,20umol/L,30umol/L,40umol/L,50umol/L）on the viability of H9C2 myocardial cells in basic state,and choose the concentration that had no influence on H9C2 cardiomyocyte viability for next step experiment。2、Using MTT assay to detect the effects of different concentration of homocysteine（0.1mmol/L,0.5mmol/L,1mmol/L,2.5mmol/L,5mmol/L）on the viability of H9C2 myocardial cells,and choose the viability rate at about 50-60%for next step experiment。3、To observe the protective effect of quercetin on homocysteine-induced H9C2 cardiomyocyte injury。The experiment was divided into the following groups:normal control group,2.5mmol/L homocysteine group,quercetin（10umol/L,20umol/L,30umol/L）+homocysteine（2.5mmol/L）group,Using MTT assay to detect the protective effect of quercetin on homocysteine-induced apoptosis of H9C2 cardiomyocytes。The results showed that quercetin could protect homocysteine-induced cardiomyocyte viability decrease and 20umol/L quercetin had the most protective effect,so we choose the 20umol/L of quercetin concentration for next step experiment。4、The apoptotic cells were observed by Hoechst3342 staining with fluorescence microscopy.The experiment was divided into the following groups:normal control group,2.5mmol/L homocysteine group,20umol/Lquercetin+2.5mmol/L homocysteine group,and the cells in each group were cultured for 12 h。5、The expression of caspase-3 was detected by Western blot。The experiment group is divided the same as above。6、（2’, 7-Dichlorodihydrofluorescein diacetate,H2DCFDA）probes were used to detect the reactive oxygen species（ROS）in the H9C2 cardiomyocytes by fluorescence spectrophotometer.The experiment group is divided the same as above.Results:1、Low concentration of quercetin has no obvious effect on the viability of H9C2 cardiomyocytes in the basic state after 12 h stimulation。Compared with normal control group,Quercetin concentration group（10umol/L,20umol/L,30umol/L）,the cells A₄₉₀values has no statistical difference。While the higher concentration of quercetin will decrease the H9C2 cardiomyocyte viability,compared with normal control group,the A₄₉₀90 values of different concentration Quercetin（40umol/L,50umol/L）groups were sigificantly lower,the difference was statistical significance（P<0.05）。2、The concentration of homocysteine in 0.1-5mmol/L decreased the viability of H9C2 cells in a concentration dependence。Compared with normal control group,the A₄₉₀values of different homocysteine concentration groups were sigificantly lower,the difference was statistical significance（P<0.05）。3、Quercetin play a protective role on 2.5mmol/L homocysteine-induced apoptosis of H9C2 cardiomyocytes。Compared with normal control group,the A₄₉₀values of 2.5mmol/L homocysteine group were sigificantly lower,the difference was statistical significance（P<0.05）。Compared with homocysteine group,the A490 values of different concentration Quercetin（10umol/L,20umol/L,30umol/L）and homocysteine group were sigificantly higher,the difference was statistical significance（P<0.05）。4、Hoechst3342 staining show that homocysteine group induce more H9C2 myocardial cells apoptosis compared with normal control group,while the Quercetin paly a protective role compared with homocysteine group。5、western blot show that compared with normal control group,the relative expression of Caspase3 in homocysteine group was significantly increased（P<0.05）.Compared with homocysteine group,the relative expression of Caspase3 in quercetin and homocysteine group was significantly decreased（P<0.05）.6、Compared with normal control group,the homocysteine group increase the production of ROS（P<0.05）,while the quercetin and homocysteine group decrease the production of ROS compared with homocysteine group（P<0.05）。Conclusion:1.Homocysteine can induce apoptosis of H9C2cardiomyocytes。2.Quercetin can protect homocysteine-induced cardiomyocyte apoptosis。3.Protective effects of quercetin on homocysteine induced cardiomyocyte apoptosis may be related to the reduction of oxidative stress。...