| Acute myocardial infarction is a common disease that severely threatens human health.Although emergency intervention and thrombolysis can alleviate the initial myocardial damage to some extent,it is unable to reverse massive necrosis and loss of cardiomyocytes after myocardial infarction.Myocardial cell necrosis after myocardial infarction is the main cause of ventricular remodeling and heart failure,which further affects the prognosis of patients,therefore,novel therapeutic strategies to reduce the myocardial cell death and/or stimulate heart regeneration are highly desirable for the future.Many animal model studies have suggested that stem cell therapy represented by mesenchymal stem cells show a certain role in the treatment of myocardial infarction,because MSCs have paracrine function and differentiation potential.Although studies have proved that MSCs can reduce myocardial infarction area and improve cardiac function in the treatment of myocardial infarction,some scholars have proposed that the clinical application of MSCs is still immature.The main manifestations are as follows: differentiation of MSCs into cardiac myocytes requires special microenvironment and induction of cytokines in vitro and in vivo,and there is no good methods to effectively differentiate MSCs into cardiac myocytes;low survival rate,low differentiation rate and less homing of stem cell transplantation;safety is still not clear,there are tumorigenic risk;allografts is still far away;drug thrombolysis and emergency coronary intervention in acute myocardial infarction are the main treatment methods,transplanting MSCS into the heart is also impractical,the effect of MSCs on acute stage treatment is still unclear.Therefore,new treatment methods are urgently needed to solve these problems.MSCs have paracrine function,and their beneficial effects are largely mediated by paracrine.Exosomes,considered as one of the most important paracrine factors in stem cell therapy,are membranous vesicular bodies secreted from cells to extracellular by exocytosis,and rich in cytokines,abundant proteins,phospholipids and RNA.As the carrier of signal transmission between cells,exosomes are widely involved in the pathologic and physiological regulation of cardiovascular diseases.Studies have shown that exosomes and their microRNA play a very important role in the transmission of intercellular substances and information,especially in the transmission process from stem cells to cardiovascular cells.Exosomes and their miRNA are also considered to be important regulators of cardiomyocytes,endothelial cells,vascular smooth muscle cells,platelets and inflammatory cells,and play an important role in the occurrence and development of atherosclerosis.In addition,the exosomes also has pro-inflammatory,anti-apoptosis,anti-immune hyperplasia,anti-cardiac fibrosis and angiogenesis effects and so on.The above characteristics and functions of exocrine secreted by stem cells provide a new method for treating myocardial infarction.A large number of studies at home and abroad have proved that exosomes derived from stem cells can promote myocardial cell protection and improve cardiac function after myocardial infarction,and at the same time promote angiogenesis.The miRNA contained in exosomes plays an important role in these effects,but the exact mechanism is not very clear.Acute myocardial infarction results in the death of a large number of myocardial cell,and myocardial cell necrosis plays a very important role in the occurrence and development of ventricular remodeling and cardiac dysfunction after myocardial infarction,myocardial cell survival is important for the prognosis of patients with acute myocardial infarction.The process of cell death after myocardial infarction includes apoptosis,necrosis and autophagy.Autophagy is a highly conserved self-digestion in cells,which is a widespread normal physiological process and defense response.It is very important to maintain the homeostasis of myocardial cells.Abnormal autophagy can lead to the development of heart diseases.Autophagy is associated with many heart disease states,such as myocardial infarction,cardiac hypertrophy,cardiomyopathy,cardiac fibrosis,and heart failure.miRNA is an important regulator of autophagy,many kinds of microRNA play an important role in myocardial infarction and myocardial ischemiareperfusion injury by regulating the autophagy level of certain autophagy related genes(ATG)and its regulatory factors.MiRNAs inhibit myocardial cell death by related target molecules,and play an important role in cell fibrosis,ischemic arrhythmia,vascular remodeling and other pathological processes after myocardial infarction.In myocardial ischemia/reperfusion(I/R)injury,miRNA inhibited apoptosis and decreased myocardial infarction area by promoting or inhibiting target genes.MiRNA-301 belongs to the miR-130/301 family,most of the literature reports of miRNA-301 are related to tumors.MiRNA-301 were overexpressed in a variety of oncogenic diseases,such as liver cancer,pancreatic cancer,lung cancer and colon cancer,etc,which affects tumorigenesis and prognosis.Recently,it has been found that miRNA-301 is also expressed in cardiac myocytes,and is involved in the regulation of a variety of biological and pathological processes,such as cell differentiation,inflammatory response and apoptosis.Studies have shown that miRNA-301 expression is down-regulated in surrounding tissues and myocardial cells after acute myocardial infarction,but its role in myocardial infarction and the protective effect of exogenous transfection of miRNA-301 on myocardial infarction are not very clear.The purpose of this study was to investigate the protective effect of miRNA-301 on myocardial infarction and its possible mechanism.Part one Expression of miRNA-301 in exosomes secreted by bone marrow mesenchymal stem cells(BMSCs)Objective:To isolate exosomes from rat bone marrow mesenchymal stem cells and transfect miRNA-301 into exosomes,then observe the expression of miRNA301 in exosomes secreted by bone marrow mesenchymal stem cells,providing data for subsequent studies.Methods:Rat bone marrow mesenchymal stem cells were extracted and cultured,they were identified by flow cytometry.The exosomes were isolated,the morphology of exosomes was observed by transmission electron microscopy and the surface protein markers of exosomes were detected by Western blot.Bone marrow mesenchymal stem cell culture was transferred to the third generation,cells with a fusion degree of about 50% were obtained and divided into four groups: control group,miRNA-301 mimics group(transfected with miRNA-301 mimics),NM group(transfected with irrelevant sequence mimics)and miRNA-301 inhibitor group(transfected with miRNA-301 inhibitor).The expression of miRNA-301 was detected by real-time quantitative PCR(RT-PCR).Results:Bone marrow mesenchymal stem cells were suspended in culture medium in a circular shape with different sizes after the first inoculation.Some cells were adhered to the wall after 24 h,and the cells were seen to gather in sheet shape after 72 h with spindle shape and polygon shape.Large clonal mass was formed on the 7th day.After the 1:2 passage,the cells grew rapidly,and the P3 generation was dominated by spindle cells,showing polar growth,and the cell morphology tended to be stable.The positive rate of CD90,CD44 and CD45 on cell surface was 99.8%,99.3% and 0.4%,which were completely consistent with the basic characteristic of BMSCs.BMSCs cultured in the original generation had the characteristics of high purity and strong homogeneity.The exosomes of BMMSCs observed under transmission electron microscopy were round or oval in diameter of about 60-160 nm,had intact cellular membrane structure,containing low-density substances.The results of Western blot showed that the exosomes of BMSCs expressed significantly exosome surface proteins CD9,CD63 and CD81.There was no significant difference in the level of miRNA-301 expression between the control group and the NM group(P>0.05).Compared with the control group and NM group,the level of miR-301 expression in the miR-301 mimics group was significantly higher(P<0.05).The level of miR-301 expression in the miR-301 inhibitor group was lower than that in the control group,significantly lower than the NM group and the miR-301 mimics group(P<0.05).Conclusion:miRNA-301 is highly expressed in exosomes secreted by bone marrow mesenchymal stem cells after transfection and provides a good basic platform for the follow-up research.Part two Effect of miRNA-301 on rat myocardial infarction area and cardiac functionObjective:Rat model of myocardial infarction was established,and the expression of miRNA-301 was detected by real-time quantitative PCR 4 weeks after local injection of mesenchymal stem cell exosomes or exosomes transfected with miR-301 and miR-301 inhibitor.Based on the myocardial infarction model,masson's trichroml staining was used to evaluate the myocardial infarction area.The left ventricular end-systolic diameter and end-diastolic left ventricular end-diastolic diameter were measured by ultrasound,the left ventricular ejection fraction and the short-axis shortening rate were calculated.The effects of miRNA-301 on myocardial infarction area and cardiac function were investigated.Methods:Rat myocardial infarction model was constructed and phosphate buffer saline,exosomes secreted by BMSCs,exosomes transfected with miR-301 or miR-301 inhibitors were respectively injected in the region around infarction(the boundary of pale and bright red myocardium).All were randomly divided into five groups: sham group(n=6),model group(injected with PBS,n=6),BMSC-Exos group(injected with exosomes,n=6),BMSCmiR-301-Exos group(injected with exosomes transfected miR-301,n=6)and miR-301 inhibitor group(injected with exosomes transfected miR-301 inhibitor,n=6).After 4 weeks,the expression level of miRNA-301 was detected by real-time quantitative PCR.Four weeks after myocardial infarction,the rats were anesthetized by intraperitoneal injection of 10g/L sodium pentobarbital(50mg/kg)and placed in supine position.Taking a left ventricular papillary muscle level two-dimensional plane left ventricular short axis,left ventricular end-systolic dimension(LVESD)and left ventricular end-diastolic dimension(LVEDD)were recorded by high resolution imaging system ultrasound instrument,and the probe frequency is RMV704(40 MHz).The left ventricular ejection fraction(LVEF)and left ventricular short axis shortening rate(LVFS)were calculated automatically by echocardi-ography computer.The mean value of 3 consecutive cardiac cycles is taken.The myocardial tissue sections for 4 weeks after myocardial infarction were taken for Masson's trichroml staining to evaluate the myocardial infarction area.The infarct area of each heart section and the entire left ventricle area were analyzed by image-pro Plus software.Results: The expression levels of miRNA-301 in BMSC-Exos group and BMSC-miR-301-Exos group were both higher than those in model group after 4 weeks of myocardial infarction,with statistical difference among three groups(P<0.05).The levels of miR-301 expression in miR-301 inhibitor group was significantly lower than those in BMSC-Exos group and BMSC-miR-301-Exos group,and also lower than those in the model group(P<0.05).The results of echocardiography showed that comparing with the Model group,both the BMSCs-Exos group and the BMSC-miR-301-Exos group had significantly higher LVEF and LVFS,and significantly lower LVESD and LVEDD(P<0.05).The BMSC-miR-301-Exos group showed the most significant changes,with statistically significant differences when compared with the other two groups.Compared with BMSCs-Exos group and BMSC-miRNA301-Exos group,LVEF and LVFS in miR-301 inhibitor group were significantly decreased,while LVESD and LVEDD were significantly increased(P<0.05).Masson's trichroml staining results showed that compared with model group,the myocardial infarction area of both the BMSC-Exos group and the BMSC-miRNA301-Exos group decreased,.The change in BMSC-miR-301-Exos group was the most significant,and the scar tissue in the myocardial infarction area was significantly thickened and the local swelling was reduced(P<0.05).The myocardial infarction area of the miR-301 inhibitor group was significantly higher than that in the BMSC-Exos group and the BMSC-miR-301-Exos group,and the scar tissue in the myocardial infarction area was also significantly reduced(P<0.05).Conclusion:MiRNA-301 in exosomes secreted by BMSCs can improve rat postmyocardial function and reduce myocardial infarction area.The exosomes secreted by BMSCs after exogenous transfection of miR-301 have more significant effects on the cardiac function and myocardial infarction area after myocardial infarction,which may provide a new way and method for the treatment of myocardial infarction by transfection of miR-301 in vitro.Part three miRNA-301 in exosomes secreted by BMSCs protected myocardial infarction by inhibiting myocardial autophagyObjective: To explore the mechanism of miRNA-301 protecting myocardial infarction by the changes of myocardial autophagy in rats of each group observed under transmission electron microscopy and the levels of autophagy marker protein detected by Western blot.Methods:The myocardial tissue surrounding myocardial infarction was taken,myocardial tissue sections were made,proteins were extracted,and then the LC3-II/LC3-I ratio and P62 protein levels of the autophagy marker protein were detected by Western blot in each group.The changes of myocardial autophagy in rat models were observed by transmission electron microscopy.Results:Compared with the Model group,the LC3-II/LC3-I ratio in the BMSCs-Exos group was significantly reduced(P<0.05),and the P62 level was increased(P > 0.05).Compared with the BMSCs-Exos group,the ratio of LC3-II/LC3-I in the BMSCs-miR301-Exos group significantly decreased(P<0.05)and the level of P62 significantly increased(P<0.05).The difference was statistically significant(P<0.05).The level of LC3-II/LC3-I in the BMSC-miR-301 inhibitor group was higher than that in model group and BMSC-miR-301-Exos group,while the level of P62 was significantly lower than that in two groups,the differences between three groups were statistically significant.Compared with the Model group,the number of autophagosomes in the peripheral myocardial tissue of the rats in the BMSCs-Exos group was significantly reduced,and the number of autophagosomes in the peripheral myocardial tissue of the rats in the BMSCs-miR-301-Exos group was significantly reduced compared with the BMSCs-Exos group.The number of autophagosomes was significantly increased in the BMSC-miR-301 inhibitor group(P<0.05).Conclusion:miRNA-301 in exosomes secreted by BMSCs may play a protective role in myocardial infarction by inhibiting myocardial autophagy. 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