Metformin Protects The Heart By Decreasing The Level Of MiR-34a In Myocardial Reperfuision And The Relevant Mechanism Research

Myocardial ischemia-reperfusion injury is an important cause of myocardial injury after thrombolysis or percutaneous coronary intervention(PCI)in patients with acute myocardial infarction and is a myocardial secondary injury caused by reopening of blood vessels.Although the ischemic postconditioning method is considered to have a certain mitigation effect,but the operation is complex,controllability is poor,and still cannot completely avoid reperfusion injury.Therefore,the study of effective methods to relieve myocardial ischemia and reperfusion injury has important clinical significance.Myocardial ischemia reperfusion injury involves cardiomyocyte apoptosis,autophagy,oxidative stress,inflammatory response,mitochondrial permeability transition pore,etc.,but its specific molecular mechanism has not been studied clearly.microRNA as a widely expressed in all stages of development of all tissues and individuals regulates various physiological and pathological processes of small molecules;also involves in cardiovascular diseases such as cardiac hypertrophy,heart failure,myocardial infarction and other diseases regulatory process.Apoptosis is a common regulatory process for microRNAs.Researches have shown that pro-apoptotic p53 protein up-regulates the expression of miR-34 a directly at the transcriptional level;Bcl-2 which is an inhibitor of apoptosis is a target molecule of miR-34 a.In tumor cells,p53 can increase the expression of miR-34 a to reduce the expression of Bcl-2,and thus increase the apoptosis of tumor cells,which plays an anti-tumor effect.However,our previous study found that miR-34 a was significantly increased in the rat cardiac H9C2 cell after oxygen glucose deprivation and reperfusion which is used to simulate ischemia reperfusion injury.Is the increased miR-34 a associated with apoptosis that causes myocardial ischemia-reperfusion injury? There is no report till now.Metformin is recommended by the International Medication Guide for high safety,less side effects first-line oral medications for treatment of type 2 diabetes.It reduces the level of blood glucose by inhibiting liver gluconeogenesis and glucose release;it is also possible to(1)inhibit tumorigenesis by modulating the expression of microRNAs.For example,metformin increases Let-7 expression to inhibit breast cancer;increases mi R-26 a to inhibit pancreatic cancer;(2)have a protective effect on cardiovascular,such as metformin can reduce all-cause mortality and the heart infarction rate of type 2 diabetes patients;reduce patients with myocardial infarction and experimental animal myocardial infarction area and serum myocardial enzyme levels;promote myocardial remodeling after heart infarction.Does metformin also protect myocardium from ischemia reperfusion injury? If the protection exists,then what is its protection mechanism? Is it contrary to the mechanism in tumor by reduction of miR-34 a to inhibit apoptosis in heart? There is no related report recently.In order to investigate the above problems,the following three parts were studied in experimental animals,cardiomyocytes and clinical samples:(1)the protective effect of metformin on myocardial ischemia reperfusion injury in rats;(2)metformin protects the oxygen glucose deprivation injury on rat H9C2 cell and its mechanism(3)The protective effect of metformin on myocardium in patients with diabetes and acute myocardial infarction after percutaneous coronary intervention.Part I the protective effect of metformin on myocardial ischemia reperfusion injury in ratsObjective:(1)to explore the role of mi R-34 a in myocardial ischemia-reperfusion injury;(2)whether metformin is protective of ischemia-reperfusion injury and whether it is associated with miR-34aMethods:1 Ligate the left anterior descending artery(LAD)to establish a model of myocardial ischemia-reperfusion injury in rats.2 Use rats myocardial TTC and Evans’ blue staining,mouse myocardial TTC staining,rats electrocardiogram,rat hematoxylin-eosin staining(HE)and rats / mouse serum related biochemical marker to observe the injury thus to confirm the model was established successfully.3 The percentage of apoptotic cells was detected by TUNEL staining in rat myocardium;RT-qPCR was used to detect the mRNA expression of Bcl-2 and p53 in the myocardium.The protein expression level of Bcl-2,p53,caspase 3 and cleaved caspase 3 were detected by western blot,which reflect cardiomyocyte apoptosis.4 The correlation analysis of the above results shows that myocardial ischemia-reperfusion injury is associated with apoptosis;miR-34 a is associated with cardiomyocyte apoptosis.5 rats were treated with metformin(3 mg/kg)in the jugular vein after 2 h of myocardial ischemia.Using the above methods to observe the above indicators,analysis of correlation,proved that metformin decreased apoptosis by reducing the level of miR-34 a expression,thereby reducing myocardial damage.Result:1 Rats/mouse myocardial ischemia reperfusion injury model was successfully establishedAfter myocardial ischemia and reperfusion in SD rats and C57BL/6J mice,there is white infarction area in the LAD blood vessel donor area;In ECG of rat,ST segment is elevated,and serum myocardial enzymes increased significantly(P<0.05).The muscle fibers of the rats in the LAD blood supply were irregular;the texture was blurred,and the fibers were dissolved,broken and deep dying compensated sarcoplasm reticulum.The results show that myocardial ischemia reperfusion injury model was established.2 myocardial ischemia reperfusion injury and myocardial cell apoptosis increasedTUNEL staining showed that the percentage of cardiomyocytes after myocardial ischemia reperfusion injury increased by nearly ten times(P<0.01).The expression of Bcl-2(apoptosis inhibitor)and p53(apoptosis promoter)mRNA and protein in cardiomyocytes were decreased and increased(P<0.01).The expression of cleaved caspase 3 which is the executive protein of apoptosis was increased.The results showed that myocardial cell apoptosis increased after ischemia reperfusion injury.There was a significant positive correlation between the level of serum CKMB and the apoptosis rate of TUNEL stained cells(r=0.8657,P<0.01),which indicated that myocardial injury and cardiac function were related to apoptosis.3 The increase of apoptosis after myocardial ischemia reperfusion injury was associated with an increase in miR-34 a expressionThe expression of miRNA-34 a in myocardium increased after myocardial ischemia reperfusion injury(P<0.05).The expression level of miR-34 a was negatively correlated with the mRNA expression level of Bcl-2(miR-34 a downstream target gene)(r=-0.499,P<0.01),and the percentage of apoptotic cells detected by TUNEL(r=0.5024,P<0.01)and p53(miR-34 a upstream regulatory protein)were positively correlated(r=0.7599,P<0.01).The results showed that the expression of miR-34 a was upregulated by the expression of p53 after ischemia-reperfusion injury,and then down-regulated the expression of the downstream target gene Bcl-2.4 metformin can reduce myocardial cell apoptosis and reduce myocardial ischemia reperfusion injuryMetformin can decrease the myocardial infarct size of SD rats with ischemia reperfusion injury(P<0.05);reduce the irregular arrangement of myocardial tissue;reverse the activity of myocardial enzymes related to myocardial injury(P<0.05);reduce the proportion of apoptotic cells P<0.01).With the decrease of miR-34 a expression,metformin was also able to upregulate the apoptosis of Bcl-2,downregulate the expression of p53 mRNA and protein,and decrease the cleaved caspase 3 protein level(P<0.05).The results showed that metformin could protect the ischemia reperfusion myocardium by reducing the apoptosis of the cells,which may be related to the down regulation of miR-34 a.Summary: After myocardial ischemia and reperfusion,heart function is injured by myocardial apoptosis with the increase of miR-34a;metformin can reduce mi R-34 a in order to reduce myocardial cell apoptosis and improve myocardial ischemia reperfusion injury.Part 2 metformin protects the oxygen glucose deprivation injury on rat H9C2 cell and its mechanismObjective: It was proved that metformin attenuated cardiomyocyte apoptosis by downregulating miR-34 a,and then explored the protective mechanism of metformin on ischemia reperfusion myocardiumMethods:1 H9C2 cardiomyocytes were subjected to ischemia reperfusion injury by glucose deprivation and reperfusion similar with in vivo(glucose deprivation 6 h,reperfusion 2 h).2 TUNEL staining and flow cytometry test the apoptosis myocardial cell proportion.The expression of miR-34 a and Bcl-2 mRNA was detected by RT-qPCR.The protein expression of Bcl-2,caspase 3 and its activated form cleaved caspase 3 were detected by western blotting.3 mi R-34 a mimic and inhibitor were transfected into H9C2 cells to overexpress or knock down miR-34 a,and the above indexes were detected by RT-qPCR and western blot at different transfection time or different transfection concentration.4 After oxygen glucose deprivation for 6 h,with the treatment of metformin on the time of reperfusion,MTS detects the cytotoxicity of different concentrations of metformin.The expression of miR-34 a,pre-miRNA34 a and p53 mRNA was detected by RT-qPCR.The protein expression of caspase 3 and its activated form cleaved caspase 3,Bcl-2,p53 and SIRT1 were detected by Western blot.The activity of SIRT1 deacetylase was detected by SIRT1 activity fluorescence assay.Result:1 H9C2 rat cardiomyocytes were subjected to oxygen glucose deprivation and reperfusion imitated myocardial ischemia reperfusion injuryAfter oxygen glucose deprivation and reperfusion,the apoptosis cell proportion is increased(P<0.001).With the increase of miR-34 a,the level of Bcl-2 mRNA and protein decreased,and the protein level of cleaved caspase 3 increased(P<0.05).The results showed that myocardial ischemia reperfusion injury was simulated by oxygen glucose deprivation and reperfusion in H9C2 cells.2 miR-34 a expression is associated with apoptosis induced by oxygen glucose deprivation and reperfusionTUNEL staining shows that miR-34 a mimic can imitate the injury of OGD/R by dose-dependently increasing H9C2 cell apoptosis,while miR-34 a inhibitor can dose-dependently reduce the H9C2 cell apoptosis after OGD/R.After miR-34 a overexpression or knockdown,the expression level of miR-34 a increased significantly or decreased with the increase of stimulation time and concentration(P<0.001),and obtained the optimal stimulation time(6 h)and the optimal stimulating concentration(160 nM for overexpression,240 nM for knockdown).The expression of cleaved caspase 3 was increased or decreased,and the expression of Bcl-2 mRNA and protein was decreased or increased(P<0.01).The addition of the negative control did not cause any change.3 the MTS is used to detect the different concentrations of metformin cytotoxicity;after the recovery of normoxic hyperglycemia culture,it is administrated of metformin treatment.After completing the reperfusion process,western blot analysis the miRNA34 a expression level,caspase 3 and its activated form,Bcl-2 protein expression level.The expression level of pre-miRNA34 a and p53 mRNA were detected by RT-PCR in OGD/R group and OGD/R+Met group.The expression of p53 and SIRT1 protein was detected by Western blot.The activity of SIRT1 deacetylase was detected by SIRT1 activity fluorescence assay.3 Metformin protects H9C2 from oxidative glucose deprivation and reperfusionMTS cell viability results showed decreased cell viability after oxygen glucose deprivation and reperfusion,but metformin did not show significant cytotoxic effects.Metformin can alleviate the apoptosis caused by OGD/R Treatment of metformin when overexpressing mi R-34 a can reduce the expression of the active form of apoptosis related protein Caspase 3,the expression of Bcl-2;knock down miR-34 a can achieve similar treatment of metformin,if given metformin at the same time can cause apoptosis related protein expression decreased more obviously.4 Study on the mechanism of metformin affecting the expression of mi R-34aThe expression of pre-miR-34 a was higher in OGD/R group.After administration of metformin,the expression of pre-miR-34 a was lower than that of OGD/R group.The mRNA level and protein level of p53 increased in OGD/R group.After using metformin,p53 decreased at mRNA and protein levels.The protein expression of SIRT1 was decreased after OGD/R stimulation,but it did not change after the use of metformin.By OGD/R stimulation,SIRT1 activity decreased;after the use of metformin the enzyme activity increased significantly.Ac-p53(Acetyl K381)is increased after OGD/R,then decreased by the treatment of metformin.Summary: Upregulation of miR-34 a expression in oxygen glucose deprivation is one of the causes of apoptosis in H9C2 cardiomyocytes.Metformin increased the expression of p53 on mRNA and protein by up-regulating the activity of deacetylase SIRT1,while p53 was the upstream regulatory protein of miR-34 a.When its acetylation formation expression was down-regulated,the expression of miR-34 a was down-regulated,thereby reducing the apoptosis caused by oxygen glucose deprivation and reperfusion.Part 3 The protection of metformin on diabetic patients with acute myocardial infarction after percutaneous coronary interventionObjective: To investigate the protective effect of metformin on myocardial myocardium in patients with myocardial ischemia reperfusion injury and its effect on the serum miR-34 a levelMethods:1 90 patients with acute ST-segment elevation myocardial infarction were randomly selected.The venous blood was collected before and after PCI.The serum was divided into two parts.One part is for biochemical analysis for serum myocardial enzymes.The other part is used to detect the expression of mi RNA34 a.Preoperative standard 12-lead electrocardiogram was performed when admission,and ECG was detected again 90 min after PCI.Sum the artificial measurement of all leads ST segment elevation relative to the baseline,the value is called the sum ST segment elevation.The leads include V1-V6,I,aVL,II,III,aVF;and V1-V6 lead are seemed as representative leads for anterior myocardial infarction.2 patients were divided into four groups:(1)patients taking metformin only before the onset of the disease,(2)patients taking statins only,(3)patients taking metformin and statins at the same time,(4)patients taking none of them.The expression of miRNA34 a before and after PCI in each group was detected by RT-qPCR,and its correlations with the increase of sum ST segment elevation and the changes of CKMB were analyzed.The level of mi R-34 a expression was compared between the patients with the same degree of infarct.Result:1 In patients with PCI undergoing acute myocardial infarction,serum mi R-34 a was associated with myocardial injuryThe results showed that the expression level of miR-34 a before and after operation was significantly correlated with the increase of ST segment and the level of serum CKMB.The difference of sum ST segment elevation value between preoperative and postoperative has negative correlation with the changes of miR-34 a expression.The difference of CKMB between the preoperation and postoperation was weakly correlated with the increase of mi R-34 a.2 taking metformin before the presence of acute myocardial infarction has protective impact on myocardial ischemia reperfusion injuryThe expression levels of serum miR-34 a were measured and compared between the two groups before and after PCI.The results showed that mi R-34 a Expression levels were significantly increased after PCI.The levels of serum miR-34 a in patients with the same agree of infarction were compared between the four groups and found that the level of miR-34 a was lower than that of the single statins given;there was no statistically significant difference between the two drugs used with taking metformin only;it is lower compared with taking other drugs;compared with other diabetes treatment,it is lower;the miR-34 a expression levels on patients taking only statins and patients taking both drugs have no statistically significant difference.Summary: The level of serum miR-34 a in patients with myocardial infarction before and after PCI was associated with myocardial injury.Metformin protects against myocardial ischemia-reperfusion injury by affecting the expression of miR-34 a.Conclusion:1 myocardial can occur after injury myocardial ischemia reperfusion.This process has relation with myocardial cell apoptosis increase,and increased apoptosis is associated with the miR-34 a level.Metformin can decrease the expression of mi RNA34 a by down-regulating the expression of p53 protein and the expression of Bcl-2 mRNA and protein,and finally decrease the expression of Caspase 3 and its activation form.The myocardial apoptosis will trim.2 metformin increased the expression of p53 on mRNA and protein by up-regulating the activity of deacetylase SIRT1.Because p53 was the upstream regulatory protein of miR-34 a,so when its expression was down regulated,the expression of miR-34 a was down regulated.Thereby the apoptosis caused by the ischemia-reperfusion injury was reduced.3 The level of serum miR-34 a in patients with myocardial infarction before and after PCI was associated with myocardial injury.Metformin protects against myocardial ischemia reperfusion injury by affecting the expression of miR-34 a.