| In recent years,with the increasing number of obese people,more and more attention has been paid to the potential harm of obesity to human health.Insulin resistance is a key feature of obesity,which is caused by disorders in the regulation of energy metabolism organs caused by excessive accumulation of lipids in adipose tissue.A large number of clinical and epidemiological evidences show that obesity is related to coronary heart disease,heart failure and other cardiovascular diseases,and is an independent risk factor of cardiovascular diseases,which can directly or indirectly increase the morbidity and mortality of cardiovascular diseases.However,the mechanism of obesity-induced myocardial injury is not yet clear.Insulin resistance may cause myocardial injury by inducing glucose metabolism and lipid metabolism disorder,myocardial calcium homeostasis imbalance,etc.At present,the prevention and treatment strategies of myocardial injury induced by insulin resistance are mainly to control body weight and blood sugar,and there is a lack of appropriate intervention drugs.Therefore,the rational application of transcriptomics and other life omics methods is expected to reveal the pathogenesis of insulin-resistant myocardial injury and develop prevention and treatment drugs targeting the pathogenesis.Histone deacetylase inhibitor(HDACi),Sodium butyrate(NaBu),is the sodium salt of short-chain fatty acid butyrate.NaBu not only serves as an energy source for the colon,but also plays a positive role in improving body weight and glucose metabolism.Studies have found that NaBu intervention in obese animal models can significantly reduce diet-induced weight gain,improve insulin sensitivity,and improve glucose tolerance.At the same time,NaBu can also promote the expression of irs-1,thus increasing insulin sensitization.The expression of GLUT4 and AKT in skeletal muscle is promoted to improve insulin signaling.Na Bu can also maintain blood glucose level and regulate the expression of genes related to insulin synthesis and secretion,so as to improve glucose homeostasis.Other studies have shown that Na Bu can enhance the expression of Bcl-2,reduce the level of BAX,and significantly reduce the apoptosis of colon cells in mice.It has also been reported that Na Bu promotes islet cell proliferation and inhibits cell apoptosis in C57BL/6J obese mice,suggesting that NaBu can regulate blood glucose homeostasis by improving islet cell function.It is suggested that sodium butyrate can be used as a research target to reduce the incidence of complications and mortality in obese patients.Insulin resistance leads to impaired insulin action.Insulin is an important hormone to maintain the metabolic homeostasis of the functional body.It can dephosphorylate pPFKFB1 and activate its kinase activity through the PI3K-AKT pathway,thus promoting the glycolysis pathway.How glycolysis pathways change in insulin resistance models is not entirely clear.Nur77 protein is a member of the orphan nuclear receptor superfamily.and its expression is regulated by histone deacetylase(HDAC).Nur77 is involved in the regulation of glucose metabolism and the glycolysis pathway,and the decrease of its expression is not only related to the expression of GLUT4,but also to the decrease of the expression of a series of genes related to glucose and glycogen metabolism.It has been reported in the literature that Nur77 is a new regulator of glucose metabolism in skeletal muscle,and Nur77 can interact with and stabilize PEPCK1 in the cytoplasm to activate gluconeogenesis in HCC cells,thereby neutralizing the glycolysis process.Recent research reports indicate that Nur77 can directly bind to two NBRE on the WFDC21 P promoter to activate its transcription and inhibit the glycolysis process].However,the detailed mechanism by which Nur77 inhibits glycolysis in cardiomyocytes remains to be supplemented.In summary,we speculate that Nur77 may be an important protein inhibiting the glycolytic pathway,and HDAC i sodium butyrate may be involved in obesity-induced insulin resistance by regulating the expression of Nur77.Research objective:In this study,RNA-seq transcriptomics were used to screen differentially expressed genes in the myocardium of insulin resistance model mice,and key genes were verified by in vitro and in vivo experiments.Meanwhile,based on in vitro insulin-resistant cardiomyocyte model,the protective effect of sodium butyrate on myocardial insulin resistance and the effect of Nur77 pathway on myocardial glycolysis pathway were investigated.Research method:In the animal part,male C57BL/6J mice were selected to simulate the obesity-induced insulin resistance model by conducting an 8w high-fat diet.After 8w,the body weight of the mice was measured.Serum myocardial enzyme was detected by ELISA.Serum levels of inflammatory cytokines il-6 and TNF-were determined by ELISA.The morphology of mouse cardiomyocytes was detected by HE staining.Western blot and qPCR were used to detect the expression of potential differentially expressed genes in mouse myocardium,verify the transcriptome results,and screen out the target genes for the main study.In the cell experiment,H9c2 cell line was used to establish the insulin resistance model induced by palmitic acid.MTT method was used to determine the optimal concentration and time of palmitic acid in H9c2 cells.The expression of differential genes was detected by qPCR.The expressions of Nur77 and HK2 were detected by Western blot.The expression of Nur77 and HK2 was detected by Western blot and qPCR.The expression of Nur77 and HK2 was detected by Western blot and qPCR.MTT method was used to determine the optimal concentration of sodium butyrate in H9c2 cells.The contents of TC and TG were detected by ELISA.The expressions of caspase-3,bcl-2,Nur77,HK2,ERK and CREB were detected by Western blot.The expression of Nur7 and HK2 was detected by qPCR.The expression of Nur77 and HK2 was detected by Western blot and qPCR.The expression of Nur77 and HK2 was detected by Western blot.Research results:(1)Mice were continuously given a high-fat diet of 8w to establish an obesity-induced insulin resistance model.Biochemical results showed that the significant increase of body weight in mice was accompanied by significant changes in blood glucose and lipid,suggesting the success of the model establishment.The expression of myocardial enzymes and pro-inflammatory factors increased,and histological examination showed that myocardial hypertrophy resulted in myocardial injury such as fibrosis,which could be used for further transcriptome analysis.(2)Based on transcriptome analysis of IR induced myocardial injury,184 differential genes were screened out.Based on bioinformatics methods,the glycolysis pathway was identified as the key differential pathway,and Nur77,Lpar-2,Pck1,GCK,Lamb3,fgf21,alox15 and ccna1 were selected as the main differential genes.The expression of the differentially expressed genes was detected by qPCR,and the results showed that the main differentially expressed genes showed significant changes in myocardial expression,and HK2,as the first key enzyme in the glycolytic pathway,significantly decreased in the glycolytic pathway.Based on pa-induced H9c2 insulin resistance model,qPCR detected differential gene expression and found that the expression trends of Nur77,Lpar-2,Pck1,GCK,lamb3,fgf21,alox15 and ccna1 were consistent with those in vivo.It was also found that palmitic acid could up-regulate the expression of Nur77 and down-regulate the expression of HK2,a key enzyme in the glycolytic pathway.Through Nur77 agonizing agent and Nur77 siRNA,it was proved that palmitic acid up-regulated the expression of Nur77 and down-regulated the expression of HK2,a key enzyme in the glycolytic pathway.(3)MTT results showed that the optimal concentration of sodium butyrate was 1mM.Western blot and qPCR results showed that sodium butyrate could improve the high expression of caspase-3,Nur77,p-ERK and p-CREB proteins caused by palmitic acid.Reduce the low expression of bcl-2 and HK2 caused by palmitic acid.ELISA showed that sodium butyrate could improve the expression of TC and TG caused by palmitate.The results of Western blot and qPCR showed that sodium butyrate could improve the high expression of HK2 caused by palmitic acid by reducing the expression of Nur77.Research conclusion:(1)Insulin resistance in mice induced by high-fat diet model is established,based on the IR induced myocardial injury transcriptome analysis,select 184 different genes,clear glycolytic pathway is the key difference between pathway,screening and verify the Nur77,wouldn't-2,pck1 and houndreds types product as like GCK key base in myocardium and significant changes in insulin resistance model of myocardial cells,and Nur77 and its regulation of glycolytic pathway genes is the key to the differentially expressed genes.(2)In the H9c2 insulin resistance model induced by palmitic acid,palmitic acid may mediate the inhibition of glycolytic pathway by regulating the Nur77 pathway to inhibit the expression of HK2,a key enzyme in the glycolytic pathway.(3)By inhibiting the expression of Nur77 in the H9c2 insulin resistance model,sodium butyrate can affect the glycolysis pathway,so as to improve cell survival rate and reduce cell apoptosis. |
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