| Objective:Alcoholiccardiomyopathy?ACM?istheischemicanddilated cardiomyopathy caused by long-term ethanol intakeis.ACM is Characteristic of cardiomegaly,arrhythmia,and congestive heart failure.Ethanol intake can lead to enlargement of the heart,structural disorders of myocardial fibers,and decreased myocardial systolic function.Ethanol intake can lead to enlargement of the heart,structural disorders of myocardial fibers,and decreased myocardial systolic function.A study reported that drinking more than 80 grams of ethanol per day for more than10 years,20 to 36 percent of the population could suffer from ACMand if continue drinking,the mortality rate was close to 50 percent within four years.In recent years,the increasing incidence of ACM has seriously affected the quality of people's lives and even threatened human health.Therefore,it is the focus of attention to explore the pathogenesis of ACM and find the early diagnosis and treatment of ACM.Ethanol metabolic processes in the body is first generated acetaldehyde by ethanol dehydrogenase,then acetic oxide in mitochondria by the role of acetaldehyde dehydrogenase generate,and finallycarbon dioxide and water through the tricarboxylic acid cycle.The human body generally has the same amount of ethanol dehydrogenase,while the content of acetaldehyde dehydrogenase has a large individual difference.The vast majority of the content of acetaldehyde dehydrogenase in the human body is low,and it can't timely metabolize effectively after consuming ethanol,but accumulate in the body in the form of acetaldehyde,causing tissue and cell toxicity.The pathogenesis of ACM has not been fully understood.Currently,studies have shown that the pathogenesis of ACM includes the cardiac toxicity of the main metabolites of ethanol.Effects of ethanol on mitochondrial function of myocardial cells;Cardiomyocyte apoptosis;Abnormal energy metabolism of myocardial cells;Heart remodeling;Disorders of the neurohumoral system;Nutrient imbalance;The effect of ethanol on cell signal transduction;Immune and inflammatory responses.The mitochondrial function of myocardial cells and apoptosis play an important role in the pathogenesis of ACM.Mitochondria are the site of ethanol induced reactive oxygen species,and also the target of ethanol induced damage of reactive oxygen species at the same time.Studies have shown that the abnormality of mitochondrial function is the pathological basis of many diseases,and the myocardium,as a high-energy metabolic tissue,is more sensitive to changes in mitochondrial function.Mitochondrial dysfunction in cells was found in cardiovascular disease,ischemic cardiomyopathy,heart failure and multiple secondary or primary cardiomyopathy.Studies have shown that the size of mitochondria in the endocardial cells of patients with ACM is about 2 to 3 times of mitochondria in the normal myocardial cell,also found edema and structuredeformity in some mitochondria,for example,mitochondrial cristae is disordered or disappeared and mitochondrial membrane is incomplete,missing.These changes on mitochondria of cardiac cells are obvious changes compared with control groups.Mitochondrial fusion protein 1?MFN1?plays a key role in regulating Mitochondrial fusion and maintaining the dynamic balance of Mitochondrial morphology.Mfn1 mainly promotes the fusion between the mitochondria for each other on the early stage.When the expression of MFN1 is insufficient,the efficiency of mitochondria fusion drops,fragmentation of mitochondria increases,the movement ability of the mitochondrialabates,and the shape of mitochondria changes obviously.In the process of the onset of ACM,the apoptosis of myocardial cellsplay an important role.The expression level of cell apoptosis proteins Bax,c-myc,of TNF-?and caspase 3 rises significantly after long-term excessive ethanol intake,which promotes cell apoptosis.Apoptosis regulation factor?X-linked inhibitor of apoptosis protein,XIAP?is a member of the inhibitor oftheapoptosis protein family?IAP?,and it is currently known as the most effective IAP proteins in human histocyte.XIAP can inhibit cell apoptosis induced by viral infection or overexpression of the caspase family protein.MicroRNA is single,small and a non-coding RNA molecules with the length of the19-25 nucleotides,which is highly conserved in evolution,and the function is regulating the gene expression on the level of transcriptionby the way of complementary pairing with targeted mRNA.MicroRNA is widely regulated in cells,and a single microRNA can regulate the transcription and translation of hundreds of targeted genes.The expression level of microRNA has obvious differences in different organizations and development stages.The kind of miRNAs expression patterns have differentiation phase and timing,which shows that miRNAs can be involved in regulation of gene expression of these molecules and be of great significance.In recent years the microRNA research more focus on the occurrence,development and prognosis of disease conditions.At the end of2006,scientists had started noticing microRNA played important roles in various pathological process of heart disease and made a series of important research progress on myocardial hypertrophy,arrhythmia,heart failure and other diseases,which made microRNA immediately become a research focus in the field of cardiovascular disease at home and abroad.The study of ethanol-induced cardiomyopathy,found that the expression level of the multiple microRNA in Hypertrophy myocardial tissue was detected anomalies,which hinted that alcoholic myocardiopathy may be associated with abnormal expression of partial microRNA.MicroRNA?suchasmicroRNA-133a,microRNA-125,microRNA-195,microRNA-186-5p,microRNA-488-3p,etc.?have been screened out in our previous work.And the target genes related to microRNA were predicted by bioinformatics software?TargetScan,miRanda et al.?:micra-186-5p/XIAP and micra-488-3p/MFN1.In this study we will deal with AC16 myocardial cells with different concentrations of ethanol,investigate the expression levels of microRNA-488-3p/MFN1 and microRNA-186-5p/XIAP,analysize mitochondrial function and myocardial cell apoptosis,further explore the specific molecular mechanism of ethanol-inducedcardiomypathy damageand lay a good foundation for the diagnosis and treatment of ACM.Methods:1 Cell cultureAC16 myocardial cells were selected,and the culture medium of cultured cells was F12 medium,including 10%fetal bovine serum,100 ug/ml streptomycin and 100U/ml penicillin.Culture conditions for 37?and 5%CO2.2 Cell transfectionTransfection of this experiment:plasmid pCMV6-XIAP,pCMV6-MFN1 and no-load plasmid;Reagent Lipofectamine 3000 and DharmaFECT.3MTT assay measures the proliferation of AC16 myocardial cells after ethanol treatment.The reagents used in this experiment were fetal bovine serum,PB buffer liquid and0.25%trypsin.To vaccinate AC16 myocardial cells in orifice plate?repeat each sample in three holes?.Put the orifice plate in 37?constant temperature box for incubation?37?and 5%CO2?.Use enzyme standard instrument measure absorbance value respectively for five days in 450 nm dispose.The number of days of cell culture was horizontal axis,the absorbance was a vertical axis,and then draw the line chart.4 Flow cytometry was used to detect the level of cell apoptosis,the content of cellreactive oxygen radical and mitochondrial membrane potential.?1?mitochondrial membrane potential:The transfected cells were digested,thenwith the low-speed centrifugation?1200rpm,10min?.Discarded Supernatant.Cultivated the heavy suspension cells with serum free medium,then incubated the cells with CO2 incubator?37??.After the incubation with CCCP,JC-1?200uM?was added to continue incubating with the incubator.Then 4?,1200rpm centrifuged for 10 min,and discarded supernatant.Added PBS,and centrifugated?1200rpm,4?,10min?.Discarded supernatant,added PBS and play well gently the heavy suspension cells.The changes of mitochondrial membrane potential were detected and analyzed by the flow cytometry.?2?Apoptosis:The transfected cells were digested,then with the low-speed centrifugation?1200rpm,10min?and washed with precooled PBS.Added Annexin V FITC,played well gently,and incubated avoiding light.Add PI to the EP tube,played well gently,and incubated avoiding light.After incubation,1 x binding buffer was added and played well gently.Cell apoptosis was detected and analyzed by the flow cytometry.?3?reactive oxygen radicals:The transfected cells were digested,then with the low-speed centrifugation?1200rpm,10min?.Discarded Supernatant.Cultivated the heavy suspension cells with serum free medium,added CELLROX?2.5 mmol/L?dye,and incubated with 37?CO2incubator.Centrifuged for 10 min?1200 rpm,4??after the incubation.Added PBS,and centrifuged?1200 rpm,4?,10 min?.Discarded Supernatant,added PBS,and play well gently the heavy suspension cells.Cell reactive oxygen radical changes were detected and analyzed by the flow cytometry.5 Mitotracker staining fluorescence microscopy observed the changes in mitochondrial morphology of myocardial cells after treatment with ethanol.Digest and collect the cells,andClimbed the slides.hey crawl the cells.Diluted mitotrack?1 mmol/L?to 100 nM?1:10 000?working liquid with serum free medium dilution at room temperature after transfection for 72h.Added mitotrack working liquid to each hole and incubated with the incubator?37?CO2,25 min?.Then added Hoechst to each hole and incubated for 5 min with the CO2 incubator.After the incubation,washed the cells with no serum culture medium at room temperature three times.Collect images with the fluorescence microscop.6 Biluciferase reporter gene was used for fluorescence detection of target genes.MFN1 was the target gene of microRNA-488-3p directly through luciferase report,and XIAP was the target gene of microRNA-186-5p directly.7 The expression levels of MFN1 and XIAP were determined by Western Blot.The antibodies used in this study included XIAP,MFN1,ACTIN and second antibodies which were coupled by horseradish peroxidase?Abcam,USA?.RIPA cell lysate contained protease inhibitors and phosphatase inhibitors.The protein was quantified using BCA protein quantitative kits.8 RNA extraction and RT-PCR were used to measurethe expression levels of microRNA-488-3p,MFN1,microRNA-186-5p and XIAP.The total RNA of sample was extracted using Trizol reagent,and SYBR Green master mix kit was used in the real-time fluorescence quantitative PCR experiment.The instrument used for PCR was 7500 real-time PCR System.The gene of internal reference used beta-actin.MicroRNA of internal reference used U6.The calculation method of gene amplification ratio based on 2-??CT.This experiment was repeated for three times.9 Statistical analysisThe software of statistical analysis was SPSS16.0.T test was used to analyze the obtained data between the treatment group and the control group.P<0.05 indicated a significant difference between groups.Results:Part one1.Ethanol induced mitochondrial dysfunction in cardiomyocytes.When AC16 cardiomyocytes are treated with ethanol,it leads to mitochondrial dysfunction:the levels of mitochondrial membrane potential decrease.the morphology of mitochondria changes:mitochondrial division increases,and fusion decreases.The content of reactive oxygen species increase.It is dependent on concentration and action time of ethanol2.MFN1 is the direct target gene of microRNA-488-3p.The luciferase reporter gene analysizes that 3'UTR region of MFN1iscombined with the site of microRNA-488-3p directly,therefore MFN1 is the direct target gene of microRNA-488-3p.MicroRNA-488-3p displays the negative regulation function,inhibiting expression of MFN1 directly,so as to regulate the mitochondrial dysfunction in AC16 myocardial cell induced by ethanol.3.MicroRNA-488-3p and MFN1 are involved in regulating mitochondrial dysfunction induced by ethanol.After the AC16 myocardial cells are dealed with ethanol,the expression of microRNA-488-3p is up-regulated and the expression of MFN1 is down-regulated.It is dependent on concentration and action time of ethanol.By the transfection technology,when the level of microRNA-488-3p in cells is up-regulatedthe damage of mitochondrial function isdeteriorative.When the level of microRNA-488-3p in cells is down-regulatedand the levelof MFN1 is up-regulated,the damage of mitochondrial function is improved.4.MicroRNA-488-3pregulatesthemitochondrialdysfunctionofalcoholic cardiomyocytes with MFN1 as the target gene.The expression level of MFN1 is detected by transfecting microRNA-488-3p mimic/inhibitor in order to response to the expression of microRNA-488-3p.When the level of microRNA-488-3p is up-regulated,the level of MFN1 is down-regulated.When the level of microRNA-488-3p is down-regulated,the level of MFN1 is up-regulated.Part two1.Ethanol inhibits proliferation of AC16 myocardial cell.MTT assaydetects that the viability of myocardial cells decreases after AC16myocardial cells are treated with ethanol.It is suggested that ethanol inhibits the proliferation of AC16 myocardial cells with concentration and action time-dependence.2.Ethanol induces apoptosis in cardiomyocytes.Flow cytometry detects the level of apoptosis and finds that ethanol treatment can induce the raised level of apoptosis of myocardial cells and the level of apoptosis can further increase with the increase of ethanol concentration and action time3.XIAP is the direct target gene of microRNA-186-5p.The luciferase reporter gene analysizes that3'UTR region of XIAP is combined with the displays the negative regulation function,inhibiting expression of XIAP directly,so as to regulate the process of AC16 myocardial apoptosis induced by ethanol.4.MicroRNA-186-5p and XIAP are involved in regulating the apoptosis of cardiomyocytes induced by ethanol.The expression of microRNA-186-5p was up-regulated in AC16 myocardial cells after ethanol intake,and the expression of XIAP was down-regulated.It is dependent on concentration and action time of ethanol.By the transfection technology,when the expression of microRNA-186-5p in cells is up-regulated,the level of myocardial apoptosis increases.When the expression of XIAP in cells is up-regulated,the level of myocardial apoptosis decreases.5.MicroRNA-186-5p regulates myocardial apoptosis induced by ethanol with XIAP as the target gene.The expression level of XIAP is detected by transfecting microRNA-186mimic/inhibitor in order to response to the expression of microRNA-186-5p.When the expression level of microRNA-186-5p is up-regulated,the level of XIAP is down-regulated.When the level of microRNA-186-5p is down-regulated,the expression of XIAP is up-regulated.Conclusion:1 AC16 myocardial cells dealed with the ethanol have mitochondrial dysfunction and high level of apoptosis.The degree of mitochondrial dysfunction is more serious and the level of apoptosis is more increase with greater concentration and action time of ethanol.2 The express of microRNA-186-5p was up-regulated and XIAP was down-regulated in myocardial cells treated by ethanol and microRNA-186-5p regulated the apoptosis of myocardial cells with the target gene of XIAP.3 The express of microRNA-488-3p was up-regulated and MFN1 was down-regulated in myocardial cells treated by ethanol and microRNA-488-3p regulated Mitochondrial function of cardiac myocytes with the target gene of MFN1. |
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