The Research On The Effects And Mechanism Of Lycopene On Myocardial Ischemia Reperfusion Injury

Objective:The mechanism of myocardial ischemia reperfusion injury（MIRI）may be related to the accumulation of calcium in cells,the interruption of ATP metabolic pathway,the involvement of inflammatory mediators,and the abnormal PH value.Studies have shown that when MIRI occurs,the open abnormality of mitochondrial permeability transition pore（MPTP）usually becomes the final result of MIRI.Studies have confirmed that the opening of mitochondrial permeability transition pore will cause the mitochondrial membrane potential（ΔΨm）of mitochondria,an important organ that provides the necessary energy for organism activity.If this condition has not been improved,the cell apoptosis will be prolonged for a long time.Lycopene,a fat-soluble antioxidant,can alleviate MIRI by inhibiting endoplasmic reticulum stress.Therefore,the aim of this study was to investigate the myocardial protective effect of lycopene and the effect of lycopene on mitochondrial permeability transition channels during myocardial ischemia reperfusion and its possible mechanism.Methods:1.Cardiomyocytes were cultured in a 37℃mixed gas（90%N₂,5%CO₂,5%O₂）three-gas hypoxia incubator for 12h,and then cultured under conventional conditions（37℃、5%CO₂）for 1h.H9c2 cardiomyocytes in control group was cultured under routine conditions;hypoxia and reoxygenation group was cultured under the above conditions;vehicle group was treated with 0.1%DMSO for12h before hypoxia and reoxygenation;lycopene pretreatment group was treated with lycopene（2.5μM,5μM,10μM,20μM,40μM）for 12h after hypoxia and reoxygenation;atractyloside group was pretreated with corresponding concentration of lycopene combined with 20μM concentration of atractyloside for 12h before hypoxia and reoxygenation.2.Langendorff was used to make rat MIRI model.The isolated heart was subjected to artificial intervention for 30 minutes after stopping the flow of K-H solution and 60minutes after reperfusion.In the control group,the hearts were taken out after normal anesthesia;in the reperfusion group,the hearts were taken out after thoracotomy;in the solvent group,the rats in the solvent group were injected with 2 ml of medicinal corn oil daily for 5 consecutive days,followed by ischemia reperfusion;in the lycopene preconditioning group,the rats were injected with the dose of lycopene（10,20,40mg/kg）intraperitoneally,followed by ischemia reperfusion;in the atractyloside group,the corresponding concentration of lycopene after continuous injection,atractyloside 5mg/kg was administered 30 minutes before operation.3.CCK8 assay was used to detect myocardial viability.4.2,3,5-triphenyl tetrazolium chloride assay was used to detect the area of ischemic infarction5.Calcium induction was used to detect MPTP openness in myocardial tissue.6.Fluorescence labeling JC-1 method was used to monitor the changes of mitochondrial membrane potential.7.Flow cytometry was used to detect the apoptotic ratio of myocardial cells.8.The apoptotic ratio of cardiomyocytes was detected by TUNEL.9.Western blot was used to detect the expression of downstream apoptotic proteins cytochrome C,APAF-1,cleaved caspase-9 and cleaved caspase-3 related to MPTP opening;the expression levels of Bax and Bcl₂.11.Cardiomyocytes viability was measured by Trypan blue staining.12.LDH activity was measured to evaluate the changes of enzymology in myocardial injury.13.Statistical analysis.Result:1.Cardiomyocytes activity of lycopene（2.5μM,5μM,10μM,20μM）pretreatment group was higher than that of hypoxia reoxygenation group.2.The infarct size of lycopene（10mg/kg,20mg/kg,40mg/kg）pretreatment group was lower than that of ischemia reperfusion group.3.The sensitivity of mitochondria to calcium induced MPTP opening in myocardial tissue of 40mg/kg lycopene preconditioning group was lower than that of ischemia reperfusion group,and min/max A 540 was increased.Compared with the hypoxia reoxygenation group,theΔΨof H9c2 cardiomyocytes in 10μM lycopene pretreatment group increased;reduce the apoptotic rate of cardiomyocytes.4.The expression levels of cytochrome C,APAF-1,cleaved caspase-9 and cleaved caspase-3 decreased than HR and IR group;the expression level of Bax decreased,the expression level of Bcl₂ increased,and the ratio of the two decreased.5.Compared with lycopene pretreatment,the sensitivity of myocardial mitochondria to calcium induced MPTP opening increased when pretreated with atractyloside,min/max A 540 decreased,and the mitochondrial membrane potential of H9c2 cells decreased.The expression level of above mentioned mitochondrial related apoptotic proteins increased;the apoptotic ratio of cardiomyocytes increased,the survival rate decreased,LDH activity increased.Conclusion:1.Lycopene pretreatment can alleviate MIRI in rats.2.Lycopene pretreatment may reduce MIRI by regulating the expression of Bax and Bcl₂,inhibiting MPTP opening and activation of downstream apoptotic pathway.