| OBJECTIVE:This study explored the protective mechanism of Tongxinluo on cardiomyocytes from the perspective of autophagy and screened for potential target microRNAs in order to provide new drug targets for clinical treatment.METHODS:Experiment 1:Seventy Sprague-Dawley(SD)male adult rats underwent coronary artery ligation surgery from one hundred and five rats were randomly divided into 7 groups:sham operation group,model group,Tongxinlu low dose group(0.5g/kg/d).Tongxinluo high-dose group(1g/kg/d),positive drug atorvastatin group(7.2mg/kg/d),chloroquine(10mg/kg/d)group,Tongxinluo high dose+chloroquine(10mg/Group of kg/d),10rats per group.After 7 days of pre-administration,the left anterior descending coronary artery ligation was performed for 50 min and reperfusion for 4h to prepare an animal model of myocardial ischemia-reperfusion injury(the sham operation group was only threaded without ligation),and the electrocardiogram,myocardial infarct size,myocardial apoptosis index,The protective effect of Tongxinluo on myocardial ischemia-reperfusion injury in rats was determined by myocardial enzymes.The microvascular endothelial morphology and autophagosomes in the ischemic myocardial tissue were observed by transmission electron microscopy.The autophagy proteins LC3,p62,Beclinl and mitochondrial autophagy proteins PINK1,Parkin and Ubiquitin were detected by Western blot.The effect of phagocytosis and the mitochondrial autophagy pathway.Experiment 2:MicroRNA microarray technique was used to screen microRNA expression changes in the experimental rat model group and the Tongxinlu high dose group,and differentially expressed microRNAs were verified by qPCR.Rat H9c2 cardiomyocytes were transfected by microRNA mimics overexpression,and the model of in vivo ischemia-reperfusion injury was simulated by hypoxia 4h/reoxygenation for 4h at the cell level.The purpose of overexpression was detected by CCK8,LDH and flow cytometry.The phenotypic effect of microRNA on cardiomyocytes;the effects of laser confocal microscopy and transmission electron microscopy on the autophagic flow and autophagosomes of cardiomyocytes,and the reported genes of target microRNAs were predicted by bioinformatics methods.Result:Experiment 11.The model group of rat myocardial ischemia-reperfusion injury was successful.The electrocardiogram of each group had obvious ST segment changes after ligature,and the heart rate increased.The ST segment changed slightly after reperfusion,which may be accompanied by T wave.Inversion and other performance;after 4 hours of reperfusion,the ST segment of the electrocardiogram of each group had different degrees of fall.2.There was no myocardial infarction and obvious cardiomyocyte apoptosis in the sham operation group,and the expression of myocardial enzyme cTnI was normal.The model group had obvious myocardial infarction area and the infarct size was significantly different from that of the sham operation group(P<0.01),the apoptotic index of cardiomyocytes increased significantly(P<0.01),and the expression of myocardial enzyme cTnI increased(P<0.01).Compared with the model group,the myocardial infarct size was significantly decreased(P<0.05),myocardial apoptosis index and myocardial enzyme cTnI expression were decreased(P<0.05);atorvastatin Compared with the model group,the myocardial infarct size,myocardial apoptosis index and myocardial enzyme cTnI expression were significantly different(P<0.05),and the effect was comparable to the Tongxinluo high dose group;the chloroquine group was compared with the model group.Myocardial infarct size,myocardial apoptosis index and myocardial enzyme cTnI expression were significantly increased(P<0.05).Myocardial infarct size and myocardial apoptosis index were significantly increased in Tongxinluo high dose+chloroquine group compared with Tongxinluo high dose group.(P<0.05),myocardial enzyme cTnl expression increased(P>0.05)3.There was no obvious abnormality in the myocardial cell microstructure of the sham operation group.Compared with the sham operation group,the myocardial cells and myocardial microvascular endothelial cells were disordered in microstructure,mitochondria swelling and division,mitochondrial inner and outer membrane gap widening,autophagosomes.More and more obvious(P<0.05);Tongxinluo high-dose group and atorvastatin group can improve myocardial mitochondrial structure and myocardial microvascular endothelial cell microstructure,and the number of autophagosomes is higher than that of model group.(P<0.05);compared with the model group,the chloroquine group had obvious myocardial cell structure,mitochondria disintegration and division;Tongxinluo high dose + chloroquine group and Tongxinluo high dose group,the myocardial cell microstructure was not See obvious changes.4.The expression levels of autophagy proteins LC3,p62 and Beclin1 were lower in the sham operation group.Compared with the expression,the expression of autophagy proteins LC3 and p62 was significantly increased(P<0.05),and Beclinl protein was up-regulated.The difference was(P>0.05);Tongxinluo could increase the expression of LC3(P<0.05),p62(P<0.05)and Beclinl(P>0.05)in a dose-dependent manner,and its effect was comparable to that of atorvastatin.Compared with the model group,autophagy protein was significantly down-regulated in the chloroquine group(Beclin1(P>0.05)).The expression of LC3,p62 and Beclinl was higher in the high-dose+chloroquine group and the high-dose group.Decline,but no statistical difference(P>0.05).5.The PINK1,Parkin protein and Ubiquitin protein in the sham operation group were slightly expressed.Compared with the sham operation group,the model group had a significant increase(P<0.05).Compared with the model group,Tongxinluo Both high and low dose groups increased PINK1 and Parkin protein expression(P<0.05)and decreased Ubiquitin protein(P<0.05).Among them,Tongxinluo high dose group and atorvastatin could compare PINK1 and Parkin proteins.There was a significant increase in the model group(P<0.05),and there was no significant difference in Ubiquitin protein(P>0.05).6.The expression of VEGF in the model group was significantly higher than that in the sham operation group(P<0.05).The expression level of VEGF in the high dose group was significantly higher than that in the model group(P<0.05).The atorvastatin group was higher.There was no significant increase compared with the model group(P>0.05).Experiment 21.By microRNA chip screening and qPCR verification,miR-450a-5p,miR-532-5p and miR-193a-3p were significantly up-regulated in the high dose group of Tongxinluo compared with the model group(P<0.05),and the expression of the three microRNA in qPCR verification share the same trend with the chip.2.Hypoxia 4h/reoxygenation for 4h of rats induced a decrease in cell viability(P<0.05),increased LDH release(P<0.05),increased apoptosis(P<0.05),and transfection of miR-450a-5p,miR-532-5p and miR-193a-3p increased cell viability,decreased LDH release and apoptosis,and had statistically significant differences(P<0.05).3.Transfected with miR-450a-5p,miR-532-5p mimics increased its expression and increased autophagic flux in cells compared with the model group(P<0.05).4.Combined with bioinformatics prediction and previous literature reports,miR-532-5p and miR-450a-5p are associated with myocardial ischemia and hypoxia.PDCD4 and TP53 may be the target gene of miR-532-5p or miR-450a-5p through which Tongxinluo protects cardiomyocytes.Conclusion:1.Myocardial ischemia-reperfusion injury model group caused obvious myocardial injury,high-dose Tongxinluo pre-administration can alleviate myocardial ischemia-reperfusion injury and protect cardiomyocytes;2.Tongxinluo is closely related to alleviating myocardial ischemia-reperfusion injury and regulating the level of autophagy in cardiomyocytes;3.Tongxinluo cleared the damaged mitochondria through the PINK1/Parkin mitochondrial autophagy pathway,and cooperated with the ubiquitin proteasome system to treat the degradation of misfolded proteins,maintain intracellular balance,and protect cardiomyocytes;4.miR-450a-5p and miR-532-5p may be the target microRNAs that protect the ischemic myocardium by Tongxinluo.PDCD4 and P53 may be the target genes of miR-532-5p or miR-450a-5p by which Tongxinluo alleviate myocardial ischemia-reperfusion injury. |
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