| Objective:Cardiovascular diseases are responsible for a third of all death and among them,ischemic heart disease is the leading cause.Coronary artery obstruction is the main cause of myocardial ischemia.Although restoring ischemic myocardial blood supply in the shortest time after myocardial ischemia is currently an effective clinical treatment,the recovery of blood and oxygen supply after blood reperfusion may be the direct cause of increased inflammation and apoptotic response,which may lead to cardiomyocytes further injury.Therefore,an effective way to reduce myocardial cell injury after myocardial ischemia/reperfusion and improve cardiac function,may provide a new therapeutic strategy for clinical treatment of coronary artery interventional surgery.It has been reported that Withaferin A has anti-inflammatory and anti-oxidation effects.However,the relationship between Withaferin A and ischemic heart disease is still unclear.The research aims to explore the protective effect of Withaferin A on myocardial ischemia/reperfusion injury.Animal models,in vitro methods and various molecular biological techniques were used to elucidate the molecular regulatory mechanism of Withaferin A during myocardial protect function.This project will provide experimental fundamental for improving the therapeutic effect of interventional surgery in patients with myocardial infarction.Methods:1.Myocardial ischemia/reperfusion animal model was used to mimic the coronary intervention after myocardial infarction in human.Left anterior descending coronary artery ligation was performed in anesthetized mice.The ligation line will be loosened in 30 minutes after ischemia to restore blood supply.Myocardial tissue in ischemia/reperfusion area of left ventricle was isolated 3 hours after reperfusion for signal analysis.Cardiac function,TUNEL staining and myocardial risk area staining were measured 24 hours after reperfusion.2.Grouping of experiment AnimalsSham group: without ligation of left anterior descending branch of coronary artery,other operations were performed same with MI/R group.Myocardial infarction/reperfusion group: Left anterior descending coronary artery ischemia/reperfusion was performed under inhalation anesthesia in mice.Low concentration of WFA pretreatment group: mice were intraperitoneally injected with 1 mg/kg WFA 1.5 hour before operation.High concentration of WFA pretreatment group: mice were intraperitoneally injected with 5 mg/kg WFA 1.5 hour before operation.3.Measurement of cardiac function in miceUltrasound measurement of cardiac function: Under inhalation anesthesia,the probe was adjusted to the short axis panel of bilateral papillary muscles.Left ventricular Doppler images of three systolic cycles were recorded continuously under M mode,then calculated cardiac function indexes.The left ventricular function was measured by carotid artery intubation: Under inhalation anesthesia,the right common carotid artery was separated and a probe with a guide wire was slowly fed into the left ventricle from the arterial micro-incision.The left ventricular pressure,left ventricular end-diastolic pressure and heart rate were recorded and the cardiac function indexes were calculated.4.Measurement of myocardial infarction area: 24 hours after ischemic myocardial reperfusion in mice,the heart sections were stained by Evans Blue-TTC staining,then calculate the percentage of cardiac infarction risk area.5.Myocardial apoptosis was detected by TUNEL staining.Myocardial ischemia reperfusion was performed for 24 hours in mice.Paraffin sections were made after formalin fixation.Myocardial apoptosis rate was calculated by TUNEL staining.6.The changes of mitochondrial apoptosis-related proteins were detected by Real Time PCR.Collect and purified the total RNA of cardiac tissue.The changes of mitochondrial apoptosis-related genes were detected by Real Time PCR after reverse transcription.7.Western Blot assay was used to detect protein expression: myocardial ischemia reperfusion for 3 hours in mice,myocardial tissue protein was extracted from ischemic area,and Western Blot assay was used to detect protein expression.Results:1.Low concentration WFA significantly improves myocardial ischemia/reperfusion injury1.1 Doppler echocardiography was used to detect the establishment of myocardial ischemia/reperfusion injury model in mice.The establishment of myocardial ischemia/reperfusion injury model was determined by ultrasound image and electrocardiogram.1.2 Left ventricular ejection fraction(LVEF)and left ventricular shortening fraction(LVFS)of mice in myocardial ischemia/reperfusion(MI/R)group were significantly lower than those in sham group,while LVEF and LVFS in 1mg/kg WFA preconditioning group were significantly higher than those in MI/R group.There was no statistical difference of LVEF and LVFS between 5mg/kg WFA preconditioning group and MI/R group.1.3 The results of hemodynamic test showed that the maximum rate of increase of ventricular pressure(+d P/dt max)and the maximum rate of decrease of ventricular pressure(-d P/dt max)in MI/R group were significantly lower than those in Sham group,while the +d P/dt Max and-d P/dt Max in 1mg/kg WFA group were significantly higher than those in MI/R group.There was no significant difference of the + d P/dt Max and-d P/dt Max in 5mg/kg WFA group and MI/R group.1.4 Evans Blue-TTC staining showed that myocardial infarction area of 1mg/kg WFA pretreatment group was significantly lower than that of MI/R group,and there was no significant difference between 5mg/kg WFA pretreatment group and MI/R group.2.WFA inhibits mitochondrial apoptosis and reduces myocardial ischemia/reperfusion injury2.1 TUNEL staining showed that the apoptotic rate of myocardial cells in 1mg/kg WFA pretreatment group was significantly lower than that in MI/R group,and the expression of Cleaved caspase-3 protein and caspase-3 activity in WFA group were also significantly lower than those in MI/R group,suggesting that the myocardial protective effect of WFA relies on the inhibition of myocardial apoptosis during myocardiac ischemia/reperfusion injury.2.2 WFA significantly decreased the expression of Cleaved caspase-9,a marker of mitochondrial apoptotic pathway,while the expression of Cleaved caspase-8,an exogenous apoptotic protein in WFA group,was not significantly different from that in MI/R group,suggesting that mitochondrial apoptosis is the key signaling pathway of myocardial protection in WFA,WFA plays an anti-apoptotic role by promoting the increase of Bcl-2 activity.2.3 AMPK is the key regulatory protein in the inhibition of mitochondrial apoptosis by WFA.3.The protective effect of WFA on myocardial ischemia/reperfusion injury depends on AMPK activation.Myocardial ischemia/reperfusion injury model was constructed by using AMPK alpha 2 myocardial specific inactivation mice.Doppler echocardiography showed that there was no difference in LVEF and LVFS between WFA group and MI/R group.Hemodynamic tests and Evans Blue staining showed that WFA group+d P/dt max,-d P/dt Max and myocardial infarction area had no significant difference with MI/R group,suggesting that the myocardial protection of WFA depended on AMPK alpha 2 activation.4.WFA promotes autophagic degradation,maintains autophagic balance and reduces myocardial ischemia/reperfusion injury4.1 Compared with Sham group,LC3 II/I ratio in MI/R group increased,while LC3 II/I ratio in WFA group decreased significantly compared with MI/R group,while Lamp-2 protein expression increased and p62 protein expression decreased in WFA group compared with MI/R group,suggesting that WFA maintains autophagy balance by promoting autophagic degradation.4.2 After inhibiting lysosomal activity by chloroquine,the LC3 II/I ratio and p62 protein expression in WFA group increased,and the apoptotic level of myocardial cells increased.It is suggested that autophagy disorder caused by abnormal autophagy degradation promotes apoptosis of cardiomyocytes,while WFA reduces apoptosis of cardiac myocytes and alleviates myocardial ischemia/reperfusion injury by promoting autophagy degradation and maintaining autophagy balance.4.3 Compared with MI/R group of AMPK DN mice,the ratio of LC3 II/I in WFA group increased significantly,suggesting that AMPK alpha 2 is a key regulatory protein for WFA to promote autophagy degradation.Conclusion:1.Low concentration(1mg/kg)of WFA significantly improve the cardiac function and reduce the area of necrosis myocardial necrosis during ischemia/reperfusion injury.2.WFA inhibited Bcl-2-mediated mitochondria apoptosis,decreased apoptosis rate of cardiomyocytes,protect cardiac from ischemia/reperfusion injury.3.AMPK plays a key role during myocardial ischemia/reperfusion injury by inhibition of myocardial mitochondrial apoptosis.4.,Withaferin A can promote degradation of autophagy inhibited by myocardial ischemia/reperfusion injury,decrease lc3 ii / I ratio,maintaining the balance of autophagy formation and degradation. |
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