| With the continuous development of society and economy,significant changes in lifestyle,accelerated aging of the population,and intensified prevalence of high-risk factors for cardiovascular disease,the number of people suffering from cardiovascular disease continues to increase.The country’s medical burden on cardiovascular disease and the economic pressure on the patient’s family are increasing day by day,which has become a major public health problem in my country.A series of pathophysiological processes such as apoptosis and metabolic stress gradually progress in the myocardial infarction area and the peri-infarct area,leading to the occurrence and development of left ventricular remodeling.Research on drugs for delaying left ventricular remodeling is in full swing.At the same time,the search for non-drug strategies to treat cardiovascular disease has also attracted the attention of many scholars,and cardiac rehabilitation has emerged as the times require.Cardiac rehabilitation aims to improve the quality of life of rehabilitation participants,prevent the occurrence and recurrence of cardiovascular events,and help them return to their families and society.Cardiac rehabilitation includes five prescriptions: drug prescription,exercise prescription,diet prescription,psychological prescription,sleep and psychological prescription.Drug prescription is the cornerstone of cardiovascular disease treatment,and exercise prescription is the core of cardiac rehabilitation.Previous studies have confirmed the protective effect of exercise on the heart,but its specific mechanism of action has not been fully elucidated.Skeletal muscle,as the main participant in the sports process,can not only play a role in providing power,but also act on the surrounding tissues or distant organs in the form of autocrine or paracrine by secreting movement-related factors through muscle contraction,serving as a messenger of inter-organ disturbance.BAIBA is a small molecule(103.6 Da)produced by skeletal muscle during exercise.It can extend the beneficial effects of exercise from skeletal muscle to other tissues and organs through the endocrine system to regulate metabolic function.BAIBA can activate fatty acid β oxidation pathway in the liver and convert white adipose tissues into brown adipose.It can improve insulin resistance and myositis through the autocrine/paracrine system,prevent diet-induced obesity,and prevent metabolic dysfunction in type 2 diabetes.Based on the beneficial regulatory effect of BAIBA on metabolism,we explored whether exercise played a myocardial protection role through BAIBA,to provide new ideas and evidence for the rehabilitation treatment of patients with heart failure after myocardial infarction,and to explore the targets of accurate treatment.This study is divided into five parts.In the first part,an animal model of heart failure after myocardial infarction was constructed to verify the protective effect of exercise on the heart,and to explore the relationship between exercise and BAIBA.In the second part,the model of heart failure after myocardial infarction was constructed at the animal level to verify the protective effect of BAIBA on the heart.In the third part,the differentially expressed genes after BAIBA intervention were analyzed by transcriptomic sequencing,and the overexpression and inhibition models were established by cell transfection at the cellular level to verify the protective effect of differential genes on myocardium.In the fourth part,the differential metabolites after BAIBA intervention were analyzed by metabolomic sequencing,combined with transcriptomic analysis to explore the specific action pathways of BAIBA,and to verify the action pathways at the animal and cellular levels.The fifth part explores and validates the protective mechanism of BAIBA in H2O2-induced cardiomyocyte apoptosis and metabolic stress models through pathway inhibitors.Part Ⅰ Protective effect of exercise on cardiac function in rats with heart failure after myocardial infarction Objective:The heart failure(HF)model after myocardial infarction(MI)was established at the animal level,and the rats with heart failure received exercise intervention to verify the effect of exercise on ventricular remodeling after myocardial infarction.Methods:The rat model of HF after MI was constructed by ligation of the anterior descending branch of the coronary artery,and the success of the modeling was determined by echocardiography four weeks after operation.Rats with heart failure were sequenced according to cardiac function.The serpentine grouping was divided into sham operation group(Sham)(n=6),heart failure group(HF)(n=6),heart failure exercise group(HFE)(n=6)and sham operation exercise group(Sham E)(n=6).Rats in the heart failure exercise group and the sham operation exercise group received exercise intervention(50%–60% of maximum intensity,60min/d,5 days/week,and gradient of 0%).After 8 weeks of exercise training,another color Doppler echocardiography was performed.The peripheral blood was collected for mass spectrometric analysis of β-aminobutyric acid(BAIBA).The myocardial tissue was taken out after thoracotomy to extract RNA and protein from the myocardial tissue.The histological morphology was observed by hematoxylin-eosin(HE)staining,MASSON staining and TUNEL staining.The mitochondrial morphology in the myocardial tissue around the infarction was observed by electron microscopy in each group.The ATP level is detected by a spectrophotometer;Western Blot was used to detect the protein expression.Result:1.Exercise intervention can effectively improve the cardiac function of rats with HF after MI.The myocardial tissue fibrosis and apoptosis were reduced,the mitochondrial morphology of myocardial tissue was improved,the metabolic stress X was improved,and the protein expression of myocardial apoptosis markers was improved.2.Exercise can significantly increase the expression of BAIBA in the circulation of rats with or without heart failure.Part Ⅱ Protective effect of exercise-generated BAIBA on cardiac function in rats with heart failure after myocardial infarction Objective:The heart failure model after myocardial infarction was established at the animal level.Rats with heart failure were intervened by BAIBA to explore the effects of BAIBA on cardiac remodeling after myocardial infarction.Methods:The rat model of heart failure after myocardial infarction was constructed by ligation of the anterior descending branch of the coronary artery,and determined to be successful by echocardiography 4 weeks after operation.Rats with heart failure were sequenced according to cardiac function.The serpentine rats were divided into sham operation group(n=6),heart failure group(n=6),and BAIBA group(n=6).Rats in the BAIBA group were intragastrically administered with BAIBA at 75 mg/kg/day,and rats in the heart failure group and the sham operation group were intragastrically administered with an equal volume of vehicle(dd-H2O).After 8 weeks of BAIBA intervention,echocardiography was performed again.The myocardial tissue was taken out after thoracotomy,and the RNA and protein in the myocardial tissue were extracted.The histological morphology was observed by HE staining,MASSON staining and TUNEL staining.The mitochondrial morphology in the myocardial tissue around the infarction was observed by electron microscopy in each group.Application of spectrophotometer to monitor ATP level;The m RNA and protein expressions of apoptotic markers in the ventricular tissues around the infarction were detected by q PCR and western blot.Result:BAIBA intervention effectively improved the cardiac function of rats with heart failure after MI,and the results showed that compared with the heart failure group,the cardiac function of the BAIBA group was significantly improved,the myocardial tissues were arranged neatly,and the degrees of myocardial fibrosis and apoptosis were alleviated.The morphology of myocardial mitochondria was improved,as well as metabolic stress,and the m RNA and protein expressions of myocardial apoptosis markers were decreased.Part Ⅲ Transcriptomic sequencing to explore the gene targets and verification of the protective effect of BAIBA on cardiac function in rats with heart failure after myocardial infarction Objective:To analyze the transcriptomics information of myocardial tissue in rats after BAIBA intervention and explore the gene targets of BAIBA intervention.Methods:RNA sequencing was performed on the tissues around the infarction in the heart failure group and the BAIBA intervention group,and the differentially expressed genes(DEG)up-regulated and down-regulated with the intervention of BAIBA were screened out.In an in vitro experiment,H9C2 cells were subjected to expression verification of candidate molecules with significant changes in sequencing results by using q PCR technology.In the in vivo experiment,the expression of the target gene in the myocardial tissue of rats in the sham operation group,the heart failure group,the heart failure exercise group,and the exercise group of the sham operation group was verified.After verification,H9C2 cells were used as the object again,and the cell model of apoptosis and metabolic stress was constructed by H2O2.On the basis of BAIBA intervention,Lippo 2000 was used to overexpress and inhibit the target gene.XII The expression of apoptosis-related proteins was detected by WB,and the ATP level was detected by spectrophotometer.Result:1.Compared with the heart failure group,the expression of 18 DEGs in the myocardium of BAIBA group tended to be down-regulated,and the expression of 11 DEGs in the myocardium of BAIBA group tended to be up-regulated.2.Both exercise and BAIBA can increase the expression of mi R-208 b,and the expressions of BAIBA and mi R-208 b are in a dose-dependent manner.3.BAIBA exerts the protective effect on myocardial tissue through mi R-208 b,and antagonizes the H2O2-induced apoptosis and metabolic stress in H9C2 cells.Part Ⅳ Metabolomics sequencing and combined analysis to explore the key pathways of BAIBA’s protective effect on cardiac function in rats with heart failure after myocardial infarction Objective:Metabolomics analysis was performed on the tissues in the peri-infarction area of rats in the heart failure group and the BAIBA intervention group to explore the molecular pathways of BAIBA intervention.It was also verified in animal models and H2O2-induced metabolic stress model of H9C2 cells.Methods:Metabolomics sequencing was performed on the tissues around the infarction in the heart failure group and the BAIBA intervention group,and the differentially expressed metabolites that were up-regulated and down-regulated with the intervention of BAIBA were screened out.The differentially expressed metabolites and differentially expressed genes were jointly analyzed to identify the protective molecular pathways of BAIBA on cardiac function in rats with heart failure after MI.In the in vivo experiment,WB was used to detect the expression of pathway-related proteins in the peri-infarction area of myocardial tissue.In the in vitro experiment,H9C2 cells were used to construct a metabolic stress cell model using H2O2,and WB technology was used to verify the effect of different concentrations of BAIBA on the expression of pathway-related proteins.H9C2 cells were used as the object,and a metabolic stress cell model was constructed by H2O2.On the basis of BAIBA intervention,mi R-208 b analog,mi R-208 b inhibitor and mi R-208 b NC were transfected with Lipoprotein 2000.The expression of pathway-related proteins was detected by WB.Result:1.A total of 40 metabolites were differentially expressed in the myocardium of BAIBA group in positive and negative ion modes when compared with the heart failure group.2.Joint analysis of DEGS and differential metabolites showed that the key signals regulated by BAIBA were enriched in the AMPK pathway.3.BAIBA effectively increased the expression of phosphorylated AMPK in vitro and in vivo.4.BAIBA mediates the activation of AMPK through mi R-208 b.5.In addition to AMPK signaling pathways,BAIBA intervention can also regulate HIF-1 signal,Fox O signal,fructose and mannose metabolism,insulin secretion,glucagon signal,insulin resistance and other metabolic-related signaling pathways.Part Ⅵ Validation of AMPK signaling pathway and its downstream proteins in vivo and in vitro Objective:To investigate the effects of BAIBA on the mi R-208b/AMPK signaling pathway protein in in vivo and in vitro models of heart failure after myocardial infarction in rats.Methods:In the in vivo experiment,WB was used to detect the expression of AMPK pathway downstream protein in the myocardial tissue of the peri-infarction area in rats after BAIBA intervention.In the in vitro experiment,H9C2 cells were used as the object,and a metabolic stress cell model was constructed by H2O2.On the basis of BAIBA intervention,compound C(AMPK inhibitor)was used to detect the expressions of apoptosis-related proteins and AMPK downstream proteins by WB,and the ATP level was detected by using a spectrophotometer.The generation of ROS was observed by a fluorescence microscope,and the changes of mitochondrial membrane potential were detected by a confocal microscope.Result:1.BAIBA increases the expression of proteins related to AMPK downstream fatty acid metabolism and mitochondrial function in myocardial tissue.2.BAIBA improves the H2O2-induced fatty acid metabolic disorders in H9C2 cells and the decline in the expression of mitochondrial function-related proteins.3.BAIBA improves the H2O2-induced metabolic stress and apoptosis of H9C2 cells.4.The protective effects of BAIBA on metabolic stress and apoptosis of H9C2 cells were offset by the AMPK inhibitors.Conclusion:In the in vivo model of heart failure after myocardial infarction and the in vitro model of metabolic stress induced by H2O2 in H9C2 cells,exercise-generated BAIBA affects the expression of genes and metabolites related to apoptosis and metabolism,relieves metabolic stress and apoptosis,and delays ventricular remodeling through the MIRI-208B/AMPK signaling pathway. |
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