| BackgroundCoronary heart disease is one of the diseases that threatening human life and health.According to data statistics,reperfusion is often induced due to myocardial ischemia,necrosis and recanalization of blood vessels.Injury.which in turn leads to heart failure,leads to a high mortality rate in myocardial infarction.Based on the present situation of myocardial ischemia-reperfusion injury,great progress has been made in the treatment of myocardial ischermia-reperfusion injury through clinical practice and research.However,there is a clear sense in predicting the mechanism of myocardial ischemia-reperfusion injury and effectively reducing the effect of ischemia-induced injury in the treatment of myocardial ischemia-reperfusion injury.Insufficient,can not effectively prevent the occurrence of cardiac sudden cardiac events.In view of those above factors,it is urgent to control coronary heart disease and reduce the mortality and complication rate of acute myocardial infarction by accurate detection and effective intervention.At present,most scholars believe that myocardial ischemia-reperfusion injury is a series of cardiac function,metabolism and structural damage caused by lack of adequate blood supply in the process of myocardial open circulation reperfusion,and these injuries often occur and block the coronal Arteries after a certain time.In the past,researchers thought that the only way of myocardial necrosis is cardiomyocyte death caused by I/R injury.However,some scholars have detected apoptosis in heart tissue of I/R rabbit model,and more and more Many studies have also confirmed the presence of apoptosis in the marginal area of myocardial infarction after I/R.cleaved-caspase-3,an asparaginase-cysteine protease,is recognized as a key protease that activates during the early stages of apoptosis and plays a key role in apoptosis and is the last known execution of apoptosis factor.Ischemic preconditioning has been shown to reduce cleaved-caspase-3 activation in many hearts in vivo or in vitro,thereby reducing ischemia-reperfusion-induced cardiomyocyte apoptosis.Myocardial cell necrosis is also one of the main causes of impaired cardiac function after I/R injury.There are a large number of enzymes in myocardial cells,myocardial damage caused by myocardial necrosis,then the enzymes in cardiomyocytes released from the cells into the blood,so that serum levels of myocardial enzymes.Lactate dehydrogenase(LDH)is a glycolytic enzyme in biological organisms that catalyzes the production of pyruvic acid.LDH activity in cardiomyocytes is several hundredfold higher in serum.When myocardial infarction occurs,the level of LDH in serum rises significantly,so it is considered as a molecular marker of cardiomyocyte necrosis.Through a large number of clinical experiments and practical studies,many researchers have found that miRNA is closely related to myocardial ischemia,and has been paid attention to by the medical research field.In the biological body,miRNAs are involved in the regulation of a number of important physiological processes including development of the individual,virus defense,hematopoiesis,organ formation,cell proliferation and apoptosis,and fat metabolism.Through a large number of experiments,we can find that there are differences in the expression of RNA in the body organs with the help of ischemia-reperfusion model.,suggesting that miRNAs may replace the drug directly on the molecular level for the current protection of ischemia-reperfusion injury.However,most studies on the relationship between miRNA and ischemia-reperfusion injury and its mechanism of action are focused on the liver or kidney,and its research in the cardiovascular system is still in its infancy.MiR-139-5p is a member of the miRNA family,which has an important regulating role in RNA transcription and the level of negative regulation of gene expression,and it's of great significance in physiological reaction of inhibiting tumor's occurrence,development,metastasis,invasion and myocardial cell apoptosis,immune autophagy,etc.It is reported that micro RNA-139-5p(miR-139-5p)has high expression in tumor tissues such as colorectal cancer.For example,in the peripheral blood of patients with prostate cancer,the MIR-139-5P and normal values will be significantly different,much higher than that.In addition,the miR-139-5p showed abnormal high expression status in patients with gastric cancer,ovarian cancer,breast cancer and osteosarcoma.But miR-139-5p in ischemic myocardial perfusion damage cells after transcription level of negative regulation of gene expression status has not been reported.miR-139-5p specific regulation which protein pathways has not been set.this paper embarks from the gene miR-139-5p,explore the mechanism of regulation of protein pathways,and it is inflammation and oxidative stress,cell mechanisms such as synergy.In addition,related studies also found that if rats were injured by I/R,the expression level of miR-139-5p would be significantly decreased,so it can be inferred that in the aspect of heart reperfusion injury in organism.MiR-139-5p plays a vital role.However,the role and mechanism of miR-139-5p in myocardial I/R injury are not clear.Therefore,in this study,the I/R rat model was used as the research object to investigate the effect of miR-139-5p on the development of I/R from the perspective of in vivo experiments,and to detect the expression of related proteins of apoptosis and necrosis.To investigate the role of mil39-5P in myocardial ischemia-reperfusion injury and its possible mechanism.Objective1.To investigate the impact of miR-139-5p expression on cardiac dysfunction caused by ischemia-reperfusion injury;2.To investigate the effects of miR-139-5p on on myocardial cell death and cardiomyocyte apoptosis in rat model of ischemia-reperfusion caused by ischemia-reperfusion injury.Methods1.I/R animal model establishmentWista Rats were anesthetized with 1.55%sodium pentobarbital(50 mg/kg)and fixed on the operating table.The skin was incised along the intercostal space 3 to 4 of the sternum and the heart was exposed.Away from the root of the left anterior branch of coronary artery about 2?3 mm at a Department of mixing,a sterile plastic tube inserted and tightened,and then quickly close the chest.Electrocardiographic changes in rats were observed,ST-segment elevation or decrease showed that the coronary artery ligation success,but also to observe the myocardium in the ligature below the color of relatively long tissue dull.After 45 minutes of ligation of the left anterior coronary artery of the rat heart,the plastic tube was pulled out and the coronary perfusion was resumed.The rat was sacrificed 2 hours after the perfusion was resumed,and the heart of the rat was collected.2.The basic indicators of rats in each group test2.1 experimental groupingThe rats were randomly divided into four groups,The number of each group was equal,all of them were 10.Sham group,I/R group,I/R+anta-miR-139-5p group.I/R+anta-Con group.2.2 Myocardial dysfunction detectionThe ejection fraction(EF),fractional shortening(FS)and stroke volume(SV)2.3 Serum lactate dehydrogenase(lactate dehydrogenase.LDH)detectionSerum LDH level in each group was detected by ELISA.2.4 Myocardial histopathological changes were detectedThe myocardial tissue of each group was collected to make paraffin sections.and the pathological changes of myocardial tissue were observed by HE staining.2.5 miR-139-5p expression was detectedRats were taken as subjects to complete the collection of myocardial tissue.Meanwhile,real-polymerase chain reaction was used to complete the detection.The object of detection was MIR-139-5P.2.6 Myocardial infarction area testingTTC staining was used to detect myocardial infarction area in each group.2.7 Cardiomyocyte apoptosis detectionMyocardial apoptosis was detected by TUNEL.2.8 Detection of cleaved-caspase-3 protein expressionWestern blot was used to detect the expression of cleaved-caspase-3 protein in myocardium of each group.3 Statistical analysisThe data obtained from all experiments in this study were entered into theEXCEL table for recording and preservation.The data were then analyzed statistically using SPSS 19.0 software.All statistical analysis results were mean±standard deviation(x ± s)that the average between the two samples using independent samples t test,multiple groups were compared using one-way ANOVA,when P<0.05 indicates that there are statistics Significance of learning.Results1.Myocardial dysfunction detectionCompared with Sham group.EF.FS and SV of I/R group,I/R+anta-Con group and I/R+anta-miR-139-5p group decreased to different extents;Compared with I/R+anta-Con group,there were no significant changes in EF,FS and SV in I/R+anta-Con group EF,FS and SV in 5p group were significantly increased(P<0.05).2.LDH content detectionCompared with the sham-operated group,the expression of LDH had no significant difference in the third sgroup(P<0.05),but also in the I/R group,I/R+anta-con group and I/R+anta-mir-139-5p group(P<0.05).The level showed a downward trend(P<0.05).To sum up,for MIR-139-5P,if it is down-regulated,it will have a significant inhibitory effect on myocardial cells.3.MiR-139-5p expression assayCompared with Sham group,the expression of miR-139-5p in myocardium of I/R group and I/R+anta-Con group had no significant change(P>0.05);Compared with I/R+anta-Con group and the I/R+anta-miR-139-5p group,the expression of miR-139-5p in myocardium decreased significantly(P<0.05)The above results show that miR-139-5p antagomir can significantly inhibit the expression of miR-139-5p in rat myocardial tissue.4.myocardial infarction area detectionCompared with I/R group,there was no significant change in myocardial infarct size in I/R+anta-Con group(P>0.05).Compared with I/R+anta-Con group Compared with I/R+anta-miR-139-5p group,myocardial infarct size was significantly reduced(P<0.05).The above results show that miR-139-5Pexpression can significantly reduce myocardial infarction area after myocardial ischemia-reperfusion in rats.5.myocardial tissue pathological changes detectionThe myocardial cells in Sham group were observed no infarction occurred.There was obvious infarction between I/R group and I/R anta-Con group,but there was no significant difference in infarct area(P>0.05).but in I/R anta-miR-139-5p group,there was no significant difference in infarction area between the two groups(P>0.05).The infarct area decreased significantly(P<0.05).Through these studies,we can conclude that the inhibition of miR-139-5P expression can reduce the infarct size of ischemia-reperfusion myocardium.6.cardiomyocyte apoptosis detectionThere was a siqnificant increase in cardiomyocyte apoptosis in rats(P<0.05).but it was compared with that in I/R group,which was significantly higher than that in control group(P<0.05).In terms of apoptosis,the changes were not obvious(P>0.05).For comparison,To compare Compared with the control group,apoptosis decreased significantly(P<0.05).From the above-mentioned studies,we can draw a conclusion that suppression expression can reduce ischemia-reperfusion myocardial cells.7.cleaved-caspase-3 protein expression detectionCompared with the Sham group,the expression of apoptosis protein in the other three groups was significantly increased(P<0.05).Compared with the ischemia-reper-fusion group,the expression of cleaved-caspase-3 apoptosis protein in the control group had no statistical significance(P>0.05).The expression level of cleaved-caspase-3 in the miR-139-5p down-regulation group was significantly lower than that in the control group(P<0.05).So it can be concluded that the down-regulation of the expression of miR-139-5p in the myocardium of rats after I/R injury may be beneficial toApoptotic protein Will have a significant inhibitory effect.Conclusion1.Rat model of myocardial ischemia-reperfusion with miR-139-5p expression inhibition was successfully established;2.Inhibition of miR-139-5p can effectively alleviate the cardiac dysfunction caused by ischemia-reperfusion injury;3.Inhibition of miR-139-5p can effectively reduce the cardiomyocyte apoptosis and death caused by ischemia-reperfusion injury.Background Ischemia is a common clinical disease that can occur in many tissues and organs.The reason for this disease is the decrease of blood perfusion,which leads to the decrease of oxygen supply to the heart and the abnormal myocardial energymetabolism.Ischemic reperfusion can significantly improve myocardial blood supply,but also aggravate myocardial injury caused by myocardial ischemia,clinical manifestations of arrhythmia,infarct size expansion,but also cause persistent ventricular systolic dysfunction and other symptoms,pathological Manifested as oxidative stress,inflammatoiy response and apoptosis,which are called myocardial ischemia-reperfusion(I/R)injury.With the development of modern medicine and the continuous progress of molecular biology,many scholars have conducted in-depth exploration and research on the factors of ischemia-reperfusion injuty.the pathophysiology and the clinical manifestations of patients.However,The specific mechanism of perfusion injury has not yet been fUlly proved,and still lack of effective prevention and treatment.Although breakthroughs have been made in the research of myocardial I/R injuiy treatment strategies,their curative effect has been greatly hindered because of the complicated pathogenesis of I/R injuiy.Therefore,to further understand the mechanism of myocardial I/R injuiy,looking for an effective therapeutic target is a priority.MiRNAs are involved in the pathogenesis of cardiovascular diseases,pathophysiology and other processes,and can be used as important molecular markers for clinical prediction of related diseases,evaluation of therapeutic effects and prognosis of patients.MiR-139-5p is a member of the miRNA family,which has an important regulating role in RNA transcription and the level of negative regulation of gene expression,and it's of great significance in physiological reaction of inhibiting tumor's occurrence,development,metastasis,invasion and myocardial cell apoptosis,immune autophagy,etc.It is reported that micro RNA-139-5p(miR-139-5p)has high expression in tumor tissues such as colorectal cancer.To taking the peripheral blood in patients with prostate cancer,In terms of relative expression,there was no significant difference between benign hypeiplasia group and healthy control group in the expression of miR-139-5p.In addition,the miR-139-5p showed abnormal high expression status in patients with gastric cancer,ovarian cancer,breast cancer and osteosarcoma.But miR-139-5p in ischemic myocardial perfusion damage cells after transcription level of negative regulation of gene expression status has not been reported,miR-139-5p specific regulation which protein pathways has not been set.this paper embarks from the gene miR-139-5p,explore the mechanism of regulation of protein pathways,and it is inflammation and oxidative stress,cell mechanisms such as synergy.It has been found that there is a close relationship between the abnormal expression of miR-139-5p and acute left ventricular ischemia in humans.Some scholars also found that the expression of miR-139-5p was significantly increased in rat cardiac myocytes undergoing I/R,suggesting that miR-139-5p may play a key regulatory role in the provided cardioprotection.However,the role and mechanism of miR-139-5p in myocardial I/R injury is not clear.Autophagy is the adaptive response that cells maintain their own energy supply under starvation stimulation and plays an important role in maintaining the removal of damaged organelles and proteins.The current research has confirmed that autophagy plays an important role in maintaining the normal morphology and function of cardiomyocytes.Most researchers believe that during myocardial ischemia-reperfusion,autophagy has the "double-edged sword" features.Under normal physiological conditions,there is a low level of autophagy in cai'diomyocytes.When myocardial ischemia-reperfusion occurs,a large amount of oxygen free radicals are produced in the body.Simultaneously,calcium overload and other conditions eventually lead to myocardial cell injuiy and even Necrosis.Autophagy is regulated by autophagy related proteins,which are encoded by autophagy related genes(ATG).Autophagy is mainly mediated by hormones and is a highly coordinated process involving multiple proteins and interrelated signaling pathways.The ULK1-Atgl3-RB1CC1-Atgl0 complex formed by Atgl3 and RB1CC1/Atgl7 interacts with mTORCl and participates in the initial phase of autophagy.In the absence of nutrients and energy,cells display inhibited mTOR activity,while ULK1 is activated,and then control Atgl3 and RB1 CC.autophagy membrane began to fomi.Starvation also activates AMP-activated protein kinase(AMPK).which phosphoiylates and inliibits mammalian TOR(mTOR).Regulatory autophagy pathways include mTOR.PI3K-Akt.p53.CA-AMPK and endoplasmic reticulum pressure.Autophagy-related 4D(ATG4D)is a member of the ATG4 D family of endonucleases and is a key regulator of the autophagic biosynthesis pathvvay^but at present its regulation mechanism and the signal pathway involved are not clear.The AMPK/mTOR/ULK1 signaling pathway has been shown to be involved in the regulation of autophagy-related processes.Adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)has been shown to be activated during myocardial ischemia and mediate autophagy.Activated AMPK regulates ULK1 phosphorylation directly or indirectly and induces autophagy.Studies have shown that AMPK phosphoiylates Raptor site inhibition of mTOR activation,thereby activating ULK1-induced autophagy.It is not clear whether miR-139-5p protects myocardial ischemia reperfusion injuiy by targeting ATG4 D and AMPK/mTOR/ULK1 pathways.Therefore,in this study,the effects of miR-139-5p on cardiomyocyte apoptosis and autophagy after H/R treatment were studied in vitro and the related mechanisms were discussed in order to lay a theoretical foundation for its application in related fields And experimental basis.Objective1.To clarify the role of miR-139-5p in targeting ATG4 D and its effect on cardiomyocyte apoptosis and autophagy after hypoxia/reoxygenation(H/R);2.To clarify the relationship between miR-139-5p and AMPK/mTOR/ULK1 in cardiomyocytes after H/R and its effect on cardiomyocyte apoptosis and autophagy.3.Methods1.H/R induced H9C2 cell injuiy2.Establishment of H9C2 cell H/R model.The effect of H/R on the expression of caspase-3.Bcl-2.Beclin-1 and LC3-II/LC3-I in H9C2 cells was detected by flow cytometry.3.Effect of miR-139-5p on apoptosis and autophagy in H9C2 cells4.The expression of miR-139-5p in H9C2 cells was up-regulated or down-regulated,and the changes of apoptosis after H/R treatment were detected.The expressions of caspase-3.Bcl-2.Beclin-II/ LC3-I protein expression.5.miR-139-5p target gene prediction and validation6.The target gene of miR-139-5p was predicted by biological software and verified by luciferase reporter system to detect the effect of miR-139-5p expression on the expression of ATG4 D protein,and to detect the expression of ATG4 D protein in H/R-treated cells ATG4 D protein changes.7.The role and mechanism of miR-139-5p in regulating apoptosis and autophagy8.The up-regulation/down-regulation of the expression of miR-139-5p and/or ATG4 D in H9C2 cells detected the apoptosis of the cells after H/R treatment,and detected the expressions of caspase-3.Bel-II/LC3-I protein expression levels.The expression of miR-139-5p in H9C2 cells was upregulated/dowm*egulated? and the expression of p-AMPK,AMPK,p-Raptor? Raptor.p-mTOR.mTOR and ULK1 proteins were detected after H/R treatment.9.Statistical analysis The data obtained from all the experiments in the study were entered into the EXCEL table for recording and preservation.The data were then analyzed statistically using SPSS 19.0 software.We use the mean(+standard deviation)method to calculate the results of the analysis(x ± s)that the average between the two samples using independent samples t test,multiple groups were compared using one-way ANOVA? when P<0.05 indicates that there are statistics Significance of learning.Results1.H/R induced H9C2 cell injury Compared with the control group,the proliferation activity of H/R group was significantly decreased(P<0.05).the apoptosis rate was significantly increased(P<0.01),and cleaved-caspase-3.Beclin-1 and LC3-II/LC3(P<0.05),while the expression of Bcl-2 was significantly decreased(P<0.05).so the expression of miR-139-5p(P<0.01).2.Effect of miR-139-5p on apoptosis and autophagy in H9C2 cells Compared with the mimic NC group,the miR-139-5p mimic group cells miR-139-5p expression and apoptosis rate was significantly increased(P<0.05),miR-139-5p inhibitor group cells miR-The expression of 139-5p and the apoptosis rate were significantly decreased(P<0.05).The expression of caspase-3 protein in H/R+miR-139-5p mimic group was significantly higher than that in H/R+mimic NC group,while the expression of Bcl-2,Beclin-1,LC3-II/LC3-(P < 0.05).From the expression level,when compared with H/R+inhibitor NC.the difference is obvious,which can be regarded as significant in statistics caspase-3 protein in H/R+miR-139-5p inhibitor group decreased significantly,while the expression of Bel-2.Beclin-1.LC3-II/LC3-I protein expression levels were significantly increased(P<0.05).3.miR-139-5p target gene prediction and validation ATG4 D is the target gene of miR-139-5p.The expression of ATG4 D protein in miR-139-5p mimic group was significantly lower than that in mimic NC group(P<0.01).Comparing the difference between NC group and NC group,ATG4 D protein in miR-139-5p inhibitor group Was significantly higher(P<0.01).Compared with control group,ATG4 D protein expression in H/R group was significantly increased(P<0.01).Compared with pcDNA3.1 group,the expression of ATG4 D protein in pcDNA-ATG4 D group was significantly higher than that in pcDNAS.l group(P<0.01).Comparing the difference between them and si-NC group.In expression ATG4 D protein in si-ATG4 D group was significantly decreased<0.01).4.The role and mechanism of miR-139-5p in regulating apoptosis and autophagy Compared with H/R+mimic NC+pcDNA-3.1 group,the apoptosis rate of H/R+miR-139-5p mimic group was significantly increased(P<0.05)The apoptosis rate of H/R+miR-139-5p mimic+pcDNA-ATG4 D group was significantly lower than that of the control group(P<0.05).The apoptosis rate of H/R+miR-139-5p inhibitor group was significantly lower than that of H/R+miR-139-5p inhibitor group(P<0.05).compared with H/R+miR-139-5p inhibitor Compared with the control group,the apoptosis rate of H/R+miR-139-5p inhibitor+si-ATG4 D group was significantly increased(P<0.05).Compared with H/R+mimic NC+pcDNA-3.1group,the expression of caspase-3 protein in H/R+miR-139-5p mimic group was significantly increased(P<0.05)(P<0.05).Compared with H/R+miR-139-5p mimic group,the expression level of H/R+miR-139-5p mimic+pcDNA-From the expression point of view,the increase of Caspase-3 in ATG4 D group is very obvious(P<0.05).while the expression of Bcl-2,Beclin-U LC3-II/LC3-I protein was significantlyincreased(P<0.05).The expression ofcaspase-3protein in H/R+miR-139-5p inhibitor group was significantly lower than that in H/R+inhibitor NC+si-NCgroup(P<0.05).Comparing thedifference between H/R+microRNA-13 9-5p group and H/R+microRNA-139-5p group,H/R+miR-139-5p inhibitor+si-The expression of caspase-3 protein in ATG4 D group was significantly increased,while the expression of Bcl-2? Beclin-1,LC3-II/LC3-I protein was significantly decreased(P<0.05).The expression of p-AMPK/t-AMPK,p-Raptor/t-Raptor and ULK1 in H/R+mir-13 9-5P mimic group was significantly lower than that in H/R+mimic NC group(P<0.05),while the expression of p-mTOR7t-mT OR was significantly increased(P<0.05).Compared with H/R+inhibitor NC group.The expression of AMPK/t-AMPK,p-Raptor/t-Raptor and ULK1 were significantly increased(P<0.05),while the expression of p-mTOR/t-mTOR was significantly decreased(P<0.05).Conclusion1.Preliminary confirmed that miR-139-5p can inhibit the expression of ATG4 D after H/R cardiomyocytes apoptosis,inhibition of autophagy;2.MiR-139-5p regulatesapoptosis andautophagyby inhibiting AMPK/mTOR/lJLKl signaling pathway. |
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