Study On The Effect And Mechanism Of MicroRNA-144-3p In Myocardial Lipotoxicity Induced By High Fat

Objective:Obesity has become a serious threat to human health in my country and even in the world.60% to 70% of the heart’s energy supply comes from fatty acids.In severely obese patients,the level of free fatty acids(FFA)in the peripheral blood increases,coupled with FFA utilization barriers,resulting in insufficient lipid oxidation,resulting in a large amount of lipids and their intermediate products Deposition in the heart causes fatty degeneration of myocardial cells,resulting in cardiac dysfunction or myocardial hypertrophy.This pathophysiological process is called myocardial lipotoxicity.However,there is currently no drug treatment for this disease.Micro RNA(micro RNA,miRNA)is a new class of metabolic non-peptide regulators,mainly acting on the post-transcriptional level.It is known that miRNAs play a key role in a variety of basic biological processes.It has been demonstrated that miRNAs inhibited or induced by lipid overload in cell culture have been shown to regulate the process of lipid-induced cell death by interacting with specific RNA targets.However,so far,the mechanism of miRNA-mediated lipotoxicity to myocardium is still unclear.Therefore,this study aims to clarify the role of some miRNAs in high-fat-induced myocardial lipotoxicity and related mechanisms.Method:1.Wistar rats were randomly divided into two groups(n=15),fed with normal feed diet(NCD)or high-fat feed diet(HFD)for 20 weeks.Body weight test is used to assess growth and development,blood biochemical test to assess blood sugar and blood lipids,echocardiography to assess cardiac function,HE staining to assess myocardial hypertrophy,MASSON staining and Sirius Red staining to assess myocardial fibrosis,PCR to detect myocardial hypertrophy markers to assess myocardium Hypertrophy.Use miRNA microarray to analyze miRNA expression,and use quantitative real-time PCR(q RT-PCR)to verify the microarray data.2.H9c2 cardiomyocytes cultured under normal conditions(37°C,5% CO2)were added with palmitic acid working solution to prepare a high-fat-induced H9c2 myocardial lipotoxicity model.The cells in the normal group were not treated with drugs;the cells in the high-fat group were treated with palmitic acid,according to the concentration gradient(100u M,200 u M,400 u M,800 u M)and time gradient(12h,24 h,36h,48h)to be treated with palmitic acid,through CCK-8 Evaluate the viability of cardiomyocytes,evaluate the lipid deposition in cardiomyocytes by oil red O staining,and use quantitative real-time PCR(q RT-PCR)to verify the microarray data.3.Transfect miRNA-144-3p mimics/ miRNA-144-3p inhibitor into H9c2 cardiomyocytes cultured under normal conditions(37℃,5% CO2),overexpression/inhibit miRNA-144-3p,and then add palmitic acid The working fluid was used to prepare the myocardial high-fat model.The transfection efficiency was evaluated by fluorescence coloring under the microscope and the expression of mi R-144-3p by PCR,the viability of myocardial cells was evaluated by CCK-8,the damage of myocardial cells was evaluated by the content of lactate dehydrogenase,and the damage of myocardial cells was evaluated by Hoechst staining and flow cytometry.For cardiomyocyte apoptosis,the expression of apoptotic protein Cleaved caspase3 was detected by WB.4.Wistar rats were fed with normal feed diet(NCD)or high-fat diet(HFD)for 20 weeks after random tail vein injection of heart-specific adeno-associated virus overexpression/inhibition of miRNA-144-3p,and fluorescence was observed under a microscope Chromogenic and q RT-PCR detection of mi R-144-3p expression to evaluate transfection efficiency,weight measurement to evaluate growth and development,blood biochemical detection to evaluate blood glucose,blood lipids and other related test indicators,and heart weight/weight ratio and heart weight/tibia The ratio of length was used to evaluate myocardial hypertrophy,HE staining was used to evaluate myocardial hypertrophy,and MASSON staining and Sirius Red staining were used to assess the degree of myocardial fibrosis and coronary atherosclerosis.5.Predict the mi R-144-3p target gene by Pic Tar,starbase and Targetscan,and verify the target gene binding by luciferase,PCR,WB and immunofluorescence.Transfect miRNA-144-3p mimics/ miRNA-144-3p inhibitor into H9c2 cardiomyocytes cultured under normal conditions(37°C,5% CO2),overexpression/inhibit miRNA-144-3p,and add it through CCK-8 evaluation for cell survival after ferroptosis inducer/inhibitor,iron content is detected by iron probe and tissue iron concentration measurement;intracellular reactive oxygen levels are detected by ROS kit,and lipid peroxidation of cells is assessed by MDA kit.JC-1 detects changes in mitochondrial membrane potential;transfects miRNA-144-3p inhibitor and si Acsl4+miRNA-144-3p inhibitor into H9c2 cardiomyocytes cultured under normal conditions(37°C,5% CO2),and passes CCK-8To detect cell viability,the ROS kit detects the level of reactive oxygen species in the cell,and the iron probe detects the iron content.Result:1.The body weight and blood sugar of obese rats induced by high-fat diet(HFD)are higher than those of conventional diet rats,the inner diameter of the left ventricular end contraction and the thickness of the ventricular wall are increased,the short axis shortening rate is reduced,and the cardiomyocytes show hypertrophy,fibrosis and decline.Microarray analysis and PCR detection showed that miRNA-141-3p and miRNA-144-3p were up-regulated in the heart of obese rats induced by high fat.2.According to the concentration gradient(100u M,200 u M,400 u M,800 u M)and time gradient(12h,24 h,36h,48h),the activity of cardiomyocytes treated with palmitic acid decreased,the lipid deposition in the cardiomyocytes gradually increased,and the cardiomyocyte miRNA-144-3p gradually increased.3.Overexpression of miRNA-144-3p can improve the viability,toxicity and apoptosis of H9c2 cardiomyocytes induced by high fat.Inhibition of miRNA-144-3p can aggravate the damage,toxicity and apoptosis of H9c2 cardiomyocytes induced by high fat.4.Overexpression of miRNA-144-3p can improve myocardial hypertrophy,fibrosis,and coronary atherosclerosis in HFD rats,and inhibiting miRNA-144-3p can aggravate myocardial hypertrophy,fibrosis,and coronary atherosclerosis in HFD rats.5.miRNA-144-3p targets Acsl4.Overexpression of miRNA-144-3p can alleviate the ferroptosis of H9c2 cardiomyocytes,reduce the iron deposition,reactive oxygen accumulation,lipid peroxidation and decrease of mitochondrial membrane potential induced by high fat.Inhibition of miRNA-144-3p can aggravate the ferroptosis of H9c2 cardiomyocytes,and exacerbate high-fat-induced iron deposition,reactive oxygen species accumulation,lipid peroxidation,apoptosis,and decrease in mitochondrial membrane potential.Silencing Acsl4 alleviates the inhibition of miRNA-144-3p High fat induces cardiomyocyte damage.Conclusion:1.The expression of miRNA-141-3p and miRNA-144-3p in rat myocardium induced by high fat was significantly up-regulated;2.Overexpression of miRNA-144-3p improves the myocardial lipotoxic injury induced by high-fat in rats,and inhibiting miRNA-144-3p aggravates the high-fat-induced myocardial lipotoxic injury in rats;3.miRNA-144-3p inhibits ferroptosis by targeting Acsl4 to improve the lipotoxic injury of cardiomyocytes induced by high fat.