| Coronary microembolization(CME)is a common complication in patients with acute coronary syndromes(ACS)during percutaneous coronary intervention(PCI)which could be caused by microvascular obstruction with atherosclerotic plaque,microthrombus or neutrophils-platelet aggregates.CME can lead to no-reflow or slow reflow,which is one of the important reasons for the lack of clinical benefit in the myocardial reperfusion therapy.CME may result in myocardial contractile dysfunction and notable arrhythmias which are strongly associated with cardiac function and clinical follow-ups.How to accurately assess and effectively prevent CME has been a complicated problem for cardiovascular intervention physicians.In our previous studies,we found that myocardial apoptosis and inflammation may play important roles in the cardiac dysfunction after CME.But a recent study found that micro RNAs(miRs)and myocardial autophagy may also play a vital role in CME.However,the specific molecular mechanism is still unclear.MiRs are endogenous non-coding small RNA molecules that regulate the expression of target genes at the post-transcriptional level,by pair-bonding with 3’-untranslated region(3’-UTR)of target m RNA.These m RNAs play an important role in a variety of pathophysiological process including growth,development,inflammation and apoptosis.Profound researchers have demonstrated that miRs take part in occurrence and development of almost all cardiovascular diseases.However,the role of miRs in the regulation of myocardial autophagy after CME remains elusive.Autophagy is a widely existing normal physiological process,as well as a defense mechanism of cells against the adverse environment.It is involved in the pathological process of many diseases.Normal levels of autophagy protect cells from adverse environmental stimuli,but excessive autophagy and inadequate autophagy may lead to disease.The high expression of miR-30 family in myocardial has been found to play an important role in a series of cardiac dysfunction such as myocardial hypertrophy,myocardial infarction,heart failure and myocardial fibrosis.In addition,recent advances have been made in the regulation of autophagy by the miR-30 family.However,the specific role of miR-30e-3p in autophagy after CME is unclear.In this study,we constructed a CME model in the rat to observe the changes of myocardial autophagy after CME in organ and molecular level,to investigate the relationship between myocardial injury induced by CME,cardiac dysfunction and myocardial autophagy level.Furthermore,we also want to reveal the physiological and pathological functions of miR-30e-3p and autophagy in myocardial injury induced by CME and to clarify their specific effects on the myocardial injury.Part 1 Changes of myocardial autophagy and miR-30e-3p expression in rats with coronary microembolizationObjective: To observe the changes of myocardial autophagy and miR-30e-3p expression in rats with coronary microembolization.Methods: Thirty-six SD rats were randomly divided into sham group(n = 6)and CME group(n = 30).CME group were randomly divided into 1h,3h,6h,9h and 12 h subgroup(n = 6 per subgroup).CME model was constructed by injecting polystyrene microspheres into the left ventricle and the sham group was injected with normal saline.Cardiac function was evaluated by echocardiography and serum cardiac troponin I(c Tn I)was examined by ELISA.HE staining and HBFP staining were used to observe and evaluate the myocardial micro-infarction area.RT-q PCR,western blot and transmission electron microscope(TEM)were used to detect the expression of miR-30e-3p,autophagy related protein LC3 and p62 and autophagic vacuoles respectively.Results: 1.Compared with the sham group,the cardiac function at 6h,9h and 12 h in the CME group were significantly decreased.The specific performances were left ventricular systolic dysfunction and left ventricular dilation: left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS)and cardiac output(CO)decreased,while left ventricular internal diameter at end-diastole(LVIDd)and left ventricular internal diameter at end-systole(LVIDs)increased.2.Compared with the sham group,the c Tn I level of the CME group was increased significantly except for the 1h subgroup and 9h reached the peak value.3.Compared with the sham group,HE staining of myocardial tissue in the CME group showed that myocardial nuclear dissolved or disappeared around microembolism ball,accompanied by a large number of inflammatory cell infiltration;HBFP staining showed that myocardial micro-infarct zone was red and the normal part was yellow.The nucleus was stained blue and many micro-infarction focus were focally distributed.4.Compared with the sham group,the expression of miR-30e-3p in 6h,9h and 12 h subgroups were decreased significantly after CME in rats.5.Compared with the sham group,the expression of LC3 II protein was increased significantly and the expression of p62 protein was decreased significantly in the 1h,3h,6h subgroup of CME;while the expression of LC3 II protein was decreased significantly and the expression of p62 protein was increased significantly in the 9h,12 h subgroup.6.TEM observation indicated that myofibrillar structure was normal and mitochondrial membrane was integral in the sham group;while myofibril fragmentation,mitochondrial swelling,and double-layer membrane structure of typical autophagic vacuole were seen in the CME group.Conclusion: In the CME model of rat,the expression of miR-30e-3p in myocardial was down regulated and accompanied with inhibited autophagy and decreased cardiac function.Part 2 MiR-30e-3p targeting Egr-1 regulates autophagy and mediates myocardial injury induced by coronary microembolizationObjective: To investigate the relationship between myocardial autophagy level and miR-30e-3p expression level after CME,and to elucidate whether miR-30e-3p targeting Egr-1 in myocardium regulates autophagy-mediated CME-induced myocardial injury.Methods: Thirty SD rats were randomly divided into sham group,CME group,CME+miR-30e-3p group,CME+miR-NC group,CME+miR-30e-3p+3-MA group(n=6 per group).Sham group and CME group were injected 0.5ml physiological saline through tail vein,and the other interventions were the same as in part one experiment.CME+miR-30e-3p group and CME+miR-NC group were transfected with miR-30e-3p and miR-NC respectively by recombinant adeno-associated virus serotype 9(r AAV9)through tail vein for two weeks,and then CME model was established.CME+miR-30e-3p+3-MA group received intraperitoneal injection of 3-MA(30mg/kg)30 minutes before establishing CME model,and the other interventions were the same with the CME+miR-30e-3p group.Double luciferase reporter gene system was used to verify whether Egr-1 was the target gene of miR-30e-3p.Cardiac function was evaluated by echocardiography and serum cardiac troponin I(c Tn I)was examined by ELISA.HE staining and HBFP staining were used to observe and evaluate the myocardial micro-infarction area.RT-q PCR,western blot and transmission electron microscope(TEM)were used to detect the expression of miR-30e-3p and Egr-1 m RNA,autophagy related protein LC3,p62,Beclin-1 and target protein Egr-1,and autophagic vacuoles respectively.Results: 1.The results of the bioinformatics database prediction showed that Egr-1 may be a potential target gene for miR-30e-3p.The results of luciferase activity test showed that miR-30e-3p could bind directly to Egr-1 3’UTR sequence.The Egr-1 3’UTR mutation binding site,however,had no effect on luciferase activity.2.Compared with the sham group,the expression of miR-30e-3p was significantly decreased in the CME group.Compared with the CME group,the expression of miR-30e-3p was significantly increased in the CME+MIR group,but not in the CME+NC group.Compared with the sham group,the expression of Egr-1 m RNA in the CME group was significantly increased,and the other three groups were also increased.3.Compared with the sham group,the cardiac function of the rats in the CME group was significantly decreased.Compared with the CME group,the cardiac function of the rats transfected with miR-30e-3p by r AAV9 was significantly improved,but not in the CME+NC group.The specific performances were increase of LVEF,LVFS,CO and decrease of LVIDd and LVIDs.Compared with the CME+MIR group,the cardiac function of 3-MA pretreatment group was significantly decreased.4.Compared with the sham group,the level of c Tn I in the CME group was increased significantly.The level of c Tn I was decreased significantly in the CME+MIR group compared with the CME group.The level of c Tn I in the 3-MA pretreatment group was increased significantly compared with the CME+MIR group.5.Except for the sham group,HE staining of myocardial tissue in the other groups showed that myocardial nuclear dissolved or disappeared around microembolism ball and myocardial tissue was edema degeneration accompanied by a large number of inflammatory cell infiltration.HBFP staining showed that myocardial micro-infarct zone was red and the normal part was yellow.The nucleus was stained blue and many micro-infarction focuses were focally distributed.The micro-infarction size of CME group,CME+MIR group,CME+NC group,and CME+MIR+3-MA group were(15.11±2.72)%,(7.43±2.01)%,(14.66±2.37)% and(12.34±2.55)% respectively.Compared with the CME group,the myocardial micro-infarction size in the CME+MIR group was decreased significantly(P < 0.05).Compared with the CME+MIR group,the myocardial micro-infarction size in the CME+NC group and CME+MIR+3-MA group were increased significantly(P < 0.05).6.Compared with the sham group,the expression of LC3Ⅱ and Beclin-1 protein in the CME group was significantly decreased,and the expression of p62 and Egr-1 protein was significantly increased.Compared with the CME group,the expression of LC3Ⅱ and Beclin-1 protein in the CME+MIR group was significantly increased,and the expression of p62 and Egr-1 protein was significantly decreased.Compared with the CME+MIR group,the expression of LC3Ⅱ and Beclin-1 protein in the CME+NC group and CME+MIR+3-MA group was significantly decreased,and the expression of p62 protein was significantly increased.The expression of Egr-1 protein in the CME+NC group was significantly increased,but not in the CME+MIR+3-MA group. 7.TEM observation indicated that myofibrillar structure was normal and mitochondrial membrane was integral in the sham group,while myofibril fragmentation and mitochondrial swelling were seen in the CME group.Compared with the CME group,the number of autophagic vacuoles was increased in the CME+MIR group.The number of autophagic vacuoles was decreased in the CME+MIR+3-MA group compared with the CME+MIR group.Conclusion: 1.Egr-1 is one of the target genes of miR-30e-3p.2.The level of myocardial autophagy after CME was closely related to the expression of miR-30e-3p.3.MiR-30e-3p partially targeting Egr-1 regulates autophagy-mediated CME-induced myocardial injury.Part 3 Mechanism of Egr-1/Bim/Beclin-1 signaling pathway in myocardial injury induced by coronary microembolizationObjective: To investigate the activation of Egr-1/Bim/Beclin-1 signaling pathway after CME and its specific mechanism in mediating myocardial injury.Methods: Thirty SD rats were randomly divided into Sham group,CME group,CME+Egr-1 sh RNA group,CME+Control sh RNA group,CME+ Egr-1 sh RNA+3-MA group(n=6 per group).Sham group and CME group were injected 0.5ml physiological saline through tail vein,and the other interventions were the same as in part one experiment.CME+Egr-1 sh RNA and CME+Control sh RNA group were transfected with Egr-1 sh RNA and Control sh RNA respectively by recombinant adeno-associated virus serotype 9(r AAV9)through tail vein for two weeks,and then CME model was established.CME+Egr-1 sh RNA+3-MA group received intraperitoneal injection of 3-MA(30mg/kg)30 minutes before establishing CME model,and the other interventions were the same with the CME+Egr-1 sh RNA group.Cardiac function was evaluated by echocardiography and serum cardiac troponin I(c Tn I)was examined by ELISA.HE staining and HBFP staining were used to observe and evaluate the myocardial micro-infarction area.TUNEL staining was used to evaluate myocardial apoptotic index(AI).RT-q PCR,western blot and transmission electron microscope(TEM)were used to detect the expression of Egr-1 m RNA,Bim m RNA and Beclin-1 m RNA,autophagy related protein LC3,p62,Egr-1/Bim/Beclin-1 pathway protein and apoptosis related protein Cleaved caspase-3,and autophagic vacuoles respectively.Results: 1.Compared with the sham group,the cardiac function of the rats in the CME group was significantly decreased.Compared with the CME group,the cardiac function of the rats transfected with Egr-1 sh RNA by r AAV9 was significantly improved,but not in the CME+Control sh RNA group.The specific performances were increase of LVEF,LVFS,CO and decrease of LVIDd and LVIDs.Compared with the CME+Egr-1 sh RNA group,the cardiac function of 3-MA pretreatment group was significantly decreased.2.Compared with the sham group,the level of c Tn I in the CME group was increased significantly.The level of c Tn I was decreased significantly in the CME+Egr-1 sh RNA group compared with the CME group.The level of c Tn I in the 3-MA pretreatment group was increased significantly compared with the CME+Egr-1 sh RNA group.3.Except for the sham group,HE staining of myocardial tissue in the other groups showed that myocardial nuclear dissolved or disappeared around microembolism ball and myocardial tissue was edema degeneration accompanied by a large number of inflammatory cell infiltration.HBFP staining showed that myocardial micro-infarct zone was red and the normal part was yellow.The nucleus was stained blue and many micro-infarction focuses were focally distributed.The micro-infarction size of CME group,CME+Egr-1 sh RNA group,CME+Control sh RNA group,CME+Egr-1 sh RNA+3-MA group were(16.28±2.43)%,(6.52±1.91)%,(15.33±2.02)% and(11.54±1.85)% respectively.Compared with the CME group,the myocardial micro-infarction size in the CME+Egr-1 sh RNA group was decreased significantly(P < 0.05).Compared with the CME+Egr-1 sh RNA group,the myocardial micro-infarction size in the CME+Control sh RNA group and CME+Egr-1 sh RNA+3-MA group were increased significantly(P < 0.05).4.The AI of Sham group,CME group,CME+Egr-1 sh RNA group,CME+Control sh RNA group,CME+Egr-1 sh RNA+3-MA group were(3.9±1.4)%,(29.6±3.8)%,(14.1±2.7)%,(28.3±3.5)% and(23.5±3.1)% respectively.Compared with the Sham group,the myocardial AI in the CME group was increased significantly(P < 0.05).Compared with the CME group,the myocardial AI in the CME+Egr-1 sh RNA group was decreased significantly(P < 0.05).Compared with the CME+Egr-1 sh RNA group,the myocardial AI in the CME+Control sh RNA group and CME+Egr-1 sh RNA+3-MA group were increased significantly(P < 0.05).5.Compared with the Sham group,the expression of Egr-1 m RNA and Bim m RNA in the CME group was increased significantly,while the expression of Beclin-1 m RNA was decreased significantly(P < 0.05).Compared with the CME group,the expression of Egr-1 m RNA and Bim m RNA in the CME+Egr-1 sh RNA group was decreased significantly,while the expression of Beclin-1 m RNA was increased significantly(P < 0.05).Compared with the CME+Egr-1 sh RNA group,the expression of Beclin-1 m RNA in the CME+Egr-1 sh RNA+3-MA group was decreased significantly(P < 0.05).6.Compared with the sham group,the expression of LC3Ⅱ and Beclin-1 protein in the CME group was significantly decreased,while the expression of p62,Egr-1,Bim and C-caspase 3 protein was significantly increased.Compared with the CME group,the expression of LC3Ⅱ and Beclin-1 protein in the CME+sh RNA group was significantly increased,while the expression of p62,Egr-1,Bim and C-caspase 3 protein was significantly decreased.Compared with the CME+sh RNA group,the expression of LC3Ⅱ and Beclin-1 protein in the CME+Control group and CME+sh RNA+3-MA group was significantly decreased,while the expression of p62,C-caspase 3 protein was significantly increased.The expression of Egr-1 and Bim protein in the CME+Control group was significantly increased compared with the CME+sh RNA group,but not in the CME+sh RNA+3-MA group.7.TEM observation indicated that myofibrillar structure was normal and mitochondrial membrane was integral in the sham group,while myofibril fragmentation and mitochondrial swelling were seen in the other groups.Compared with the CME group,the number of autophagic vacuoles was increased in the CME+sh RNA group.The number of autophagic vacuoles was decreased in the CME+sh RNA+3-MA group compared with the CME+sh RNA group.Conclusion: 1.The Egr-1/Bim/Beclin-1 signaling pathway is activated after CME.2.The Egr-1/Bim/Beclin-1 signaling pathway participates in coronary microembolization induced myocardial injury by mediating myocardial autophagy and apoptosis. |
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