Protective Effects Of Hesperetin On H₂O₂-induced Cardiomyocytes Oxidative Injury And Possible Mechanisms

In recent years,a large number of domestic and foreign literatures have reported that oxidative stress is the common mechanism of cardiovascular damage caused by many pathological factors.Excess oxygen free radicals?including hydrogen peroxide,superoxide anion,etc.?are produced in the pathological processes of many cardiovascular diseases?such as hypertension,heart failure,ischemic heart disease,hyperlipidemia and so on?,which can induce oxidative stress and lead to myocardial cell death that further aggravate the severity of the disease.Therefore,it is of great significance to reduce oxidative stress damage and reduce myocardial cell apoptosis in the treatment of cardiovascular disease.As an important reactive oxygen species?ROS?source,hydrogen peroxide?H₂O₂?can directly oxidize the lipid and protein on the cell membrane and penetrate the membranes to react with intracellular iron ions generating free radicals with stronger activity,which can induce cell apoptosis and necrosis.H₂O₂ induced oxidative stress is one of the important causes of myocardial cell apoptosis.At present,due to the lack of endogenous antioxidant enzymes and the unstable effects of conventional antioxidants,it is urgent to seek a new breakthrough in the treatment of oxidative stress.Therefore,the present research was performed based on traditional Chinese medicine.Hesperetin?HES?is a kind of natural flavonoid,which widely found in the plant,fruit,food and other plant sources.It has been proved that hesperetin has many biological activities,such as anti-oxidation,anti-inflammatory,anti-aging,reducing blood lipid and anti-tumor.A new study found that hesperetin could protect cardiac function by inhibiting myocardial hypertrophy and fibrosis in mice.However,it is not clear whether it can reduce myocardial apoptosis by regulating the level of oxidative stress.Therefore,the aim of this study is to investigate the protective effect of hesperetin on myocardial oxidative damage and explore the possible mechanisms.A large number of studies have shown that oxidative stress can mediate apoptosis through mitochondria,death receptors,endoplasmic reticulum stress,and so on,when the noxious stimulus breaks the balance of oxidative stress.Mitochondria is a key determinant of cell death and survival.In the process of apoptosis,the structure and function of mitochondria are changed,and the apoptosis related proteins are released into the cytoplasm,which leads to the apoptosis of the downstream executor.The previous study has shown that hesperetin can reduce the apoptosis of H9c2 cells induced by LPS by means of ndogenous mitochondrial dependent apoptotic pathway,but it is not clear whether the protective effect of hesperetin on myocardial oxidative damage is related to mitochondrial dependent endogenous apoptosis.In this study,we further investigate the molecular mechanism of the protective effect of hesperetin on myocardial oxidative damage which might be useful to find a new target for the protection of myocardial injury.Part ? Protective Effect of Hesperetin in the H₂O₂-induced Cardiomyocytes Oxidative Injury Objective:To observe the protective effect of hesperetin pretreatment on oxidative damage induced by H₂O₂ in cardiomyocytes,and further to provide the experimental basis for the following study on the function of hesperidin.Methods:H9c2 cells were cultured to establish the model of myocardial injury induced by H₂O₂,and were randomly divided into four groups: Control,H₂O₂,HES,HES+H₂O₂.In order to determine the optimal damage concentration of H₂O₂ and the optima lprotective concentration of HES,CCK-8 kits were used to detect cell survival rate;to observe the activity of H9c2 myocardial cells viability by detecting the content of MDA and SOD in myocardial cells;to detect the formation of ROS using the DCFH-DA fluorescence probe;to detect the cardiomyocyte apoptosis by Hoechst33258 staining and flow cytometry.Results: 1.Establishment of oxidative damage modeAfter treatment with different concentration of H₂O₂?25-800?M?for 2 hours,the survival rate of myocardial cell decreased gradually with the concentration of H₂O₂.After treatment with 400 ?M H₂O₂ for 2 hours,the cell injury was obviously increased and the cell survival rate was stable and moderate,which confirmed the establishment of oxidative damage model of cardiomyocytes.2.Protective effect of hesperetin on oxidative damage in H9c2 cardiomyocytes 2.1.Determination of drug concentration:The CCK-8 results showed that 10-40?M hesperetin pretreatment for 1 hour in H9c2 cells could significantly reduce the myocardial cell apoptosis rate.And cardiomyocyte apoptosis reached the minimal level at a concentration of 40?M.Therefore,40?M was chosen as the protective concentration for the following experiments.2.2.Detection of MDA and SOD activity:After 1 hour of pretreatment with hesperidin,the production of MDA induced by H₂O₂ was significantly reduced?P<0.05?,and the level of SOD was significantly increased?P <0.05?.2.3.DCFH-DA staining:The levels of ROS in myocardial cells were observed by fluorescence microscope.After H₂O₂ treatment,it was found that the green fluorescence was significantly enhanced,and the level of ROS in cardiomyocytes was significantly increased?P<0.05?.While the level of ROS in myocardial cells was significantly decreased after pretreatment with hesperetin?P<0.05?.2.4.Results of Hoechst 33258 staining,flow cytometry and Caspase-3 activity assay:Compared with the normal control group,the strong blue fluorescence in the injury model group was significantly enhanced,indicating that uptake of Hoechst was increased and the apoptosis rate was also enhanced.The apoptosis rate was about 50% and the activity of Caspase-3 was significantly increased.After 1 hour of pretreatment with hesperidin,the apoptosis rate was decreased.The apoptosis rate was about 30% and the activity of Caspase-3 was decreased obviously.Conclusion:1.Hesperetin pretreatment could protect the H₂O₂-induced cardiomyocytes oxidative injury and achieve the maximum protection at a concentration of 40?M.2.After the hesperetin pretreatment,the reduction of H₂O₂-induced cardiomyocytes oxidative injury might be related to the reduction of myocardial apoptosis.Part ? Study about the mechanism of potective effect of hesperetin in the H₂O₂-induced cardiomyocytes oxidative injury.Objective:To observe the possible protective mechanisms of hesperetin on the oxidative injury in cardiomyocyte.Methods:H9c2 cells were cultured and randomly divided into four groups: Control,H₂O₂,HES,HES+H₂O₂.Apoptosis was detected by Hoechst33258 staining and flow cytometry.The activity of Caspase-8,9,and 12 were detected by extraction protein.Mitochondrial membrane potential was detected by JC-1 staining.The ATP levels were detected by extraction protein.The protein expressions of Bcl-2,Bax and cytochrome c were detected by Western blot.Results:1.Detection results of the activity of Caspase-8,9,12: Compared with the normal control group,the activity of Caspase-8,9,12 in H₂O₂ injury model group was significantly increased(P <0.05.Hesperetin pretreatment could significantly decrease the activity of Caspase-8,9,12?P<0.05?,and the activity of Caspase-9 decreased obviously.2.Results of JC-1 staining : Compared with the normal control group,the red fluorescence reduced and the green fluorescence was strengthened in the H₂O₂ treatment for 2 hours group,indicating that the less of JC-1in mitochondrial matrix and the decline of mitochondrial membrane potential.But the red fluorescence was enhanced and the green fluorescence was decreased after pretreatment with hesperetin,indicating that the mitochondrial membrane potential was reversed.3.Detection results of ATP : Compared with the normal control group,the ATP level of the H₂O₂ damage model group was significantly decreased.But the level of ATP was significantly increased after pretreatment with hesperetin.4.Detection results of Western blot: Compared with the normal group,the expression of Bcl-2 in myocardial cells of H₂O₂ injury model group was significantly decreased,and the expressions of Bax and Cyt-c were significantly increased.But pretreatment with hesperetin could up-regulate the expression of Bcl-2 and down-regulate the expression of Bax and Cyt-c.Conclusion:1.Hesperetin pretreatment could decrease the activity of Caspase-8,9,12,and the activity of Caspase-9 decreased obviously,suggesting that the protective effect of preconditioning with hesperetin on myocardial oxidative damage might be related to the reduction of apoptosis mediated by mitochondrial pathway.2.Hesperetin pretreatment improved mitochondrial dysfunction caused by H₂O₂ damage,and changed the expression of mitochondria related apoptosis protein,suggesting that hesperetin pretreatment might reduce mitochondrial pathway-mediated apoptosis by improving mitochondrial function.