| BackgroundAcute myocardial infarction(AMI)is a big threat to global health,myocardial reperfusion therapy by drugs or surgery are widely used to treat the disease.However,the process of reperfusion can itself induce cardiomyocyte death,known as myocardial reperfusion injury,which is an important factor of prognosis.Current studies show that ischemia-reperfusion injury is closely related to reactive oxygen,and local inflammatory responses.In animal experiments,treatment such as reducing the production of ROS,decreasing the local inflammatory response,increasing the expression of heme oxygenase-1(HO-1),or inducing a stress response by ischemic pretreatment reduces the reperfusion injury.However,there is no specific drug that can be used to reduce the ischemia-reperfusion injury in human beings.It’s our urgent to explore the mechanism of ischemia-reperfusion injury and develop novel therapy for this disease.It has been reported that sodium arsenite can induce HO-1 expression and cell stress response,so we speculate that sodium arsenite may have protective effect on myocardial ischemia-reperfusion injury.In this project we try to investigate the effect and mechanism of sodium arsenite on myocardial ischemia-reperfusion injury.ObjectiveTo explore the protective effect and the mechanism of arsenite on myocardial tissue during the first 24 hours using an in vivo myocardial ischemia-reperfusion model in rats.To explore the effect of sodium arsenite on the production of ROS in cardiomyocytes and the protection of myocardiocytes from ROS injury in vitro.We also try to explore the effect of sodium arsenite on the occurrence of cellular stress and the induction of Heme oxygenase-1 and inhibition of NLRP3 inflammasome activation.From these studies,we try to clarify the effect and mechanism of sodium arsenite on myocardial ischemia-reperfusion injury,and to find new strategies for the prevention and treatment for this disease.Methods1.The in vivo model of myocardial ischemia-reperfusion in rats was established to explore the effect of arsenite on myocardial tissue during the first 24 hours.Heart slices were stained with TTC and Evans blue to measure the infarct area and area at risk(AAR),and calculate the ratio of infact size to AAR.2.The evaluation of cardiac function after I/R in rats.After 24 hours of myocardial ischemia-reperfusion recovery,the echocardiography of rat heart in M mode was collected by the VEVO 2100 high-resolution in vivo echocardiography imaging system.The left ventricular diameters at the end of systole or diastole were measured on the long axis or short axis view of the left ventricle beside the sternum.Left ventricular ejection fraction(EF%)and shortening rate of left ventricular short axis(FS%)were calculated.3.In vitro hypoxia re-oxygenation model was used to evaluate the effect of sodium arsenite on the production of ROS in cardiomyocytes.In this model,H9c2 cells were cultured in saturated nitrogen for different time to restore normal ventilation.Superoxide anion was detected by the color change of cytochrome c and adrenaline,and the production of hydrogen peroxide was detected by di-methylphenol orange method.4.To evaluate the effect of sodium arsenite on the survival rate of rat cardiac H9c2 cells in a model of simulated ischemia and reperfusion.H9c2 cells were subjected to metabolic inhibition and recovery.MTT method was used to detect the cell survival rate,and LDH activity test was used to detect the cell damage.5.The protection effect of sodium arsenite on cardiomyocytes from oxidative damage.The cardiomyocytes were treated with sodium arsenite for 1h at different time,and treated with hydrogen peroxide for 8h.The cell survival rates were measured by MTT.6.In vitro respiration experiments were carried out with purified mitochondria.The mitochondria of rat liver were isolated and purified.The mitochondria was re-suspended and adjusted to proper concentration with reaction buffer.The metabolic substrates coupled with respiratory chain complex I or II,ADP and inhibitors were added.The oxygen concentration of the reaction system was measured and recorded in real time by polarography.Sodium arsenite was added before or during the reaction to observe its effect on mitochondrial respiration.7.The phosphorylation of eIF2a and the expression of heme oxygenase-1(HO-1)were detected by Western Blot.The expression of GADD34 and CHOP were detected by Reverse transcription PCR.8.NLRP3 activation experiment.THP-1 cells were first induced to differentiate overnight by phorbol ester(PMA);the differentiated THP-1 cells or primary cultured bone marrow-derived macrophages(BMDM)were pre-treated with LPS for 3 hours,then treated with different activators of NLRP3 inflammasome,such as nigericin and ATP for 30 minutes to 1 hour,and the production of matured IL-1 beta released into medium was detected by ELISA.Results1.The in vivo model of myocardial ischemia-reperfusion in rats showed that pretreatment with sodium arsenite significantly reduced the ratio of myocardial infarction area to AAR(P<0.0001).Echocardiography showed that sodium arsenite increased left ventricular ejection fraction and shortening rate of left ventricular short axis(P<0.01).The data from femoral artery intubation showed that there were no significant changes in heart rate,systolic and diastolic blood pressure between the experimental group and the control group(P>0.05).2.There was no significant effect of sodium arsenite on the production of ROS in cardiomyocytes(P>0.05).3.Sodium arsenite had no significant effect on the survival rate of rat cardiac H9c2 cells in cell model in which H9C2 cells were subjected to metabolic inhibition and recovery(P>0.05).4.Sodium arsenite pretreatment significantly reduced the damage of H9c2 cells caused by hydrogen peroxide.5.Sodium arsenite inhibited oxygen consumption in isolated mitochondria of complex I-linked substrate,but had no significant effect on complex Ⅱ-linked respiration using succinate as the substrate.6.The phosphorylation of eIF-2α and the expression of its downstream genes including GADD34 and CHOP can be induced by the transient treatment of sodium arsenite,and the expression of HO-1 with cardiovascular protection can also be induced by sodium arsenite.7.The activation of NLRP3 inflammasome can be inhibited by sodium arsenite which plays an important role at the priming stage of inflammasome activation.Conclusions1.Pretreatment with sodium arsenite significantly reduces the ratio of myocardial infarction area to AAR,increases left ventricular ejection fraction and shortening rate of left ventricular short axis.Sodium arsenite attenuates myocardial ischemia-reperfusion injury in rats.2.The mechanisms by which sodium arsenite pretreatment protect heart from ischemia-reperfusion injury include:reducing the oxidative damage of cells by hydrogen peroxide;inducing the phosphorylation of eIF-2α and the expression of heme oxygenase-1;inhibiting the activation of NLRP3 inflammasome. |
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