The Research On The Effects And Mechanisms Of Morin On Myocardial Ischemia Reperfusion Injury

Objective:Acute myocardial infarction(AMI)is one of the most serious and fatal diseases in the world.Timely reperfusion therapy to restore the blood supply of the ischemia site is currently considered to be the most effective treatment to prevent myocardial injury.However,reperfusion therapy itself may lead to an increased myocardial injury,which is called myocardial ischemia reperfusion injury(MIRI).This injury mainly manifests as myocardial stunning,severe lethal ventricular arrhythmia,coronary artery no-reflow and even increased myocardial infarction areas,threatening patients' lives.The process of MIRI is complex and may involve a variety of mechanisms,such as oxidative stress,calcium overload,apoptosis and energy metabolism disorders.Mitochondrial permeability transition pore(MPTP)is a transmembrane structure in mitochondrion.The irreversible MPTP opening leads to the decrease of mitochondrial membrane potential(??m),activating the mitochondrial apoptotic pathway and leading to cell death.Studies have found that MPTP remains closed during ischemia,but opens in the early phase of reperfusion,which is one of the important mechanisms of the occurrence of MIRI.Pretreatment or posttreatment of cyclosporin A(CsA),a specific inhibitor of MPTP opening,has been confirmed to reduce MIRI by inhibiting the MPTP opening in many animal experiments and clinical studies.Therefore,exploring effective drugs and interventions to inhibit the MPTP opening has become an important target for the prevention and treatment of MIRI.Morin is a natural flavonoid with a variety of pharmacological effects such as anti-oxidation,anti-inflammatory and anti-apoptosis.It can scavenge reactive oxygen species(ROS),inhibit lipid peroxidation and improve the activity of various antioxidant enzymes.As morin can scavenge ROS and reduce its level,we infer that morin can reduce MIRI by inhibiting the MPTP opening.In summary,the aim of this study was to identify the protective effect of morin on MIRI.The research objects of this study were rat H9c2 cardiomyocytes and isolated rat hearts.By establishing H9c2 cardiomyocyte model of HR injury and rat model of IR,this study aimed at the following two parts:(1)observing and comparing the differences of HR injury and MIRI among HR/IR group and morin pretreatment groups to identify the protective effect of morin on MIRI;(2)observing and comparing the differences of MPTP opening,the activation of mitochondrial downstream apoptotic pathway and the expression of regulation factors of MPTP opening among HR/IR group and morin pretreatment groups.In addition,atractyloside(ATR),a specific activator of MPTP opening,was used to identify whether it can abrogate the protective effect of morin on MIRI to reveal the possible mechanism of the cardioprotection of morin and its acting target.Methods:1.H9c2 cells were subjected to cardiomyocyte model of HR injury.Culture medium was removed and changed to Earle's medium without glucose and FBS,and cells were cultured in tri-gas incubator with 90%N2,5%CO2 and 5%O2 at 37? for 12 h to induce hypoxia.After that,Earle's medium was removed and the cells were cultured with normal medium in incubators with 5%CO2 at 37? to reoxygenate for 1 h.Cardiomyocytes of control group and IR group were not given medication.Cardiomyocytes of Vehicle group were treated with 0.1%DMSO for 12h followed by H12h/Rlh.Cardiomyocytes of morin pretreatment groups were treated with morin at doses of 12.5?M,25?M and 50?M for 12h followed by H12h/R1h.ATR at a dose of 20?M was given to cardiomyocytes in optimum dose of morin pretreatment group for 12h followed by H12h/R1h.2.Rat model of MIRI was established by using Langendorff perfusion apparatus.All isolated rat hearts were subjected to global ischemia for 30 min followed by 60 min reperfusion.Rats in IR group were not given medication.Rats in Vehicle group were treated with 10%PEG400 by intraperitoneal injection once per day for a total of 5 days before surgery.Rats in morin pretreatment groups were treated with morin at doses of 10 mg/kg,20 mg/kg and 40 mg/kg by intraperitoneal injection once per day for a total of 5 days before surgery.ATR at a dose of 5mg/kg was given to rats in optimum dose of morin pretreatment group 30 min before surgery.3.CCK8 was used to measure the cell viability.4.Typan blue staining was used to measure the cell viability,and the cell survival rate was calculated.5.LDH activity was measured to assess the cell injury.6.Flow cytometry was used to measure the cell apoptosis rate.7.Heart rate and coronary flow were measured at stabilization,30 min and 60 min of reperfusion.8.HE staining was used to measure the changes of myocardial microstructure.9.TTC staining was used to measure the myocardial infarction areas,and the myocardial infarction percentage was calculated.10.The MPTP opening was measured by calcium-induced method,and min/max A540 was calculated.11.JC-1 staining was used to measure the changes of mitochondrial membrane potential.12.The mRNA expressions of mitochondrial downstream apoptotic pathway and the regulating factors of MPTP opening were measured by using Real Time PCR.13.The protein expressions of mitochondrial downstream apoptotic pathway and the regulating factors of MPTP opening were measured by using Western blot.14.TUNEL was used to measure the myocardial apoptosis,and the apoptosis rate was calculated.15.Statistical analysis:Differences between two groups were evaluated by using independent sample T test.Differences among three or more groups were firstly evaluated by using one-way analysis of variance(ANOVA),and if the differences were significant,multiple comparison analysis was further performed by using Fisher's Least Significant Difference(LSD)test.All P values less than 0.05 were considered statistically significant.All P values less than 0.01 were considered significantly statistically significant.Results:1.The cell viability in morin pretreatment groups was significantly higher than that in HR group.The cell survival rates in morin pretreatment groups were significantly higher than that in HR group.The LDH activity in morin pretreatment groups was significantly lower than that in HR group.The cell apoptosis rates in morin pretreatment groups were significantly lower than that in HR group.2.Compared with IR group,heart rate and coronary flow measured at 30 min and 60 min of reperfusion in morin pretreatment groups were significantly recovered.The damages of myocardial microstructure in morin pretreatment groups were reduced to some extent compared with that in IR group to some extent.The myocardial infarction percentages in morin pretreatment groups were significantly lower than that in IR group.3.The sensitivity of MPTP opening induced by calcium in morin20 group was significantly lower than that in HR group.The value of min/max A540 in morin20 group was significantly higher than that in HR group.The decrease of mitochondrial membrane potential was significantly inhibited by morin pretreatment at the dose of 25?M.4.The mRNA and protein expressions of Cytochrome C,apoptotic protease activating factor-1(APAF1),Caspase9 and Caspase3 in morin25 group were significantly decreased compared with those in HR group.Compared with IR group,the results in morin20 group also showed the same trend.5.The mRNA and protein expressions of Bax and the ratio of Bax to Bcl2 in morin25 group were significantly decreased compared with those in HR group,and the mRNA and protein expressions of Bcl2 in morin25 group were significantly increased compared with those in HR group.Compared with IR group,the results in morin20 group also showed the same trend.6.MPTP opening,min/max A540 and the decrease of ??m were reversed by ATR in cardiomyocyte and myocardium.The mRNA and protein expressions of mitochondrial downstream apoptotic pathway and the regulating factors of MPTP opening were reversed by ATR in cardiomyocyte and myocardium.The beneficial effects of morin on cell injury,including the cell viability,the survival rate,the LDH activity and the cell apoptosis rate were reversed by ATR in cardiomyocyte.The beneficial effects of morin on myocardial injury,including myocardial microstructure,the myocardial infarction percentages and the apoptosis rate were reversed by ATR in myocardium.Conclusion:1.Morin can effectively reduce rat MIRI.2.The protective effect of morin on MIRI may be involved with the regulation of Bax and Bcl2,and the inhibition of MPTP opening and mitochondrial downstream apoptotic pathway.