| Myocardial ischemia is accompanied by the imbalance of cardiac energy production and utilization.With the development of metabonomics detection technology,the current understanding of the changes of myocardial energy metabolism is enriched.These metabolic changes are mainly due to the reduction of oxygen supply,resulting in the blocking of metabolic pathways such as oxidative phosphorylation and tricarboxylic acid cycle,the activation of anaerobic glycolysis production pathway,and the accumulation of glycolysis product lactic acid,which will cause intracellular acidosis and induce further myocardial dysfunction.Therefore,the regulation of tricarboxylic acid cycle and glycolysis may be a new strategy for intervention of hypoxia induced myocardial injury.Phosphoenolpyruvate carboxykinase 2(PCK2)is a key enzyme linking tricarboxylic acid cycle with glycolysis and gluconeogenesis.It has been reported that phosphoenolpyruvate carboxykinase subtypes are involved in physiological processes and the progress of many diseases.Overexpression of PCK2 may lead to abnormal energy metabolism in tumor cells.Inhibition of PCK2 expression can alleviate myocardial pathological hypertrophy,suggesting that PCK2 is involved in regulating the metabolic process of injured myocardium.However,the role of PCK2 in the metabolism of ischemic myocardium has not been reported.Previous studies of our group showed that trichostatin A(TSA)can significantly reduce myocardial ischemia injury in rats and participate in the repair of myocardial ischemia injury.However,the regulatory effect of TSA on myocardial metabolism has not been elucidated.Previous studies have shown that TSA can interfere with cell proliferation and function by regulating the metabolic reprogramming of tumor cells,including tricarboxylic acid cycle and glucose metabolism.Therefore,we speculate that TSA may affect myocardial ischemic injury by regulating myocardial metabolism.Objetives:In this study,we intend to use bioinformatics methods to analyze the pathway of TSA,screen the signaling pathways and key genes involved in the regulation of ischemic myocardial metabolism,and verify the role of key differential genes in ischemic myocardium through in vivo and in vitro experiments;To further explore the mechanism of TSA intervention in myocardial ischemia,based on the key differential genes regulating myocardial ischemia metabolism.Methods:In this study,myocardial ischemia model was established by ligating left anterior descending coronary artery;TTC staining was used to detect the effect of TSA on infarct size;MTT method was used to detect the effect of TSA on the survival rate of H9c2 cells under oxygen glucose deprivation;The target of TSA and regulatory genes of myocardial ischemic metabolites were enriched and analyzed by David network analysis platform;RT-q PCR was used to verify the differentially expressed genes regulating myocardial metabolism and the effect of TSA on their expression;The expression of key differential genes in H9c2 cells was inhibited by transient transfection of si RNA;Western blot and immunofluorescence were used to detect the expression of target protein in myocardial ischemia tissues and H9c2 cells under oxygen glucose deprivation;JASPAR database was used to analyze the interaction sites between transcription factors and key differential genes;The activity of succinate dehydrogenase,a key enzyme of tricarboxylic acid cycle,in rat myocardial tissue and H9c2 cells,and the contents of pyruvate and lactic acid in rat serum and H9c2 cell supernatant were detected by colorimetry to evaluate myocardial metabolism.Results:1.The protective effect of TSA on ischemic myocardiumTTC staining results showed that TSA could significantly reduce the infarct size of myocardial ischemia rats(P < 0.05);MTT detection results showed that TSA could significantly increase the survival rate of H9c2 cells in oxygen glucose deprivation rats(P < 0.01).It is suggested that TSA can improve myocardial injury induced by ischemia.2.Bioinformatics analysis of TSA target genes and potential regulatory genes of ischemic myocardium metabolismBased on Pub Chem and CTD database,we searched the target genes of TSA and analyzed the pathway enrichment,and found 864 pathways involved in the regulation of TSA,of which metabolism was the second.Through GO terms enrichment analysis and KEGG pathway analysis of TSA target genes involved in the regulation of metabolic pathways,165 metabolic pathways were obtained,among which TSA plays an important role in the regulation of phosphatidylinositol signaling pathway and glucose metabolism pathway.The metabolic biomarkers of myocardial ischemia were retrieved based on Pub Med database.The metabolic marker genes were obtained by constructing metabolite gene network analysis,and 651 metabolic regulatory genes that have not been proved to be related to myocardial ischemia were screened.PPI analysis and other methods were used to identify 183 core genes,and Go terms enrichment analysis and KEGG pathway analysis were carried out to confirm that tricarboxylic acid cycle plays an important role in the metabolic pathway enriched.3.TSA affects the expression of PCK2,a potential regulatory gene in ischemic myocardiumBased on the bioinformatics analysis of TSA target genes,Western blot results showed that TSA could activate Akt m TOR signaling pathway in ischemic myocardium(P < 0.05),suggesting that TSA could protect ischemic myocardium and regulate myocardial metabolism by activating Akt / m TOR.RT-q PCR results showed that PCK2 was the most significantly differentially expressed gene related to tricarboxylic acid cycle in ischemic myocardium(P < 0.01),while TSA could significantly reduce the expression of PCK2 m RNA in ischemic myocardium of TSA rats(P < 0.01).Western blot and immunofluorescence results showed that the expression of PCK2 protein in myocardial ischemia tissues and H9c2 cells under OGD condition was significantly down regulated after TSA Administration(P < 0.05).4.Regulatory mechanism of TSA on metabolism of ischemic myocardium based on PCK2Succinate dehydrogenase,a key enzyme of the tricarboxylic acid cycle,was significantly decreased in myocardial ischemia rats(P < 0.01);Succinate dehydrogenase activity in H9c2 cells was significantly decreased under oxygen glucose deprivation(P < 0.01),and increased after transient transfection of si RNA to inhibit pck2(P < 0.05).Pyruvate and lactate,the key products of glycolysis,were significantly accumulated in the serum of myocardial ischemia rats(P < 0.05),and in the supernatant of H9c2 cells under OGD conditions(P < 0.05).Transient transfection of si RNA to inhibit PCK2 reduced the accumulation of pyruvate and lactate(P < 0.05).After TSA administration,the activity of succinate dehydrogenase in H9c2 cells increased significantly(P < 0.01),while the contents of pyruvate and lactic acid in serum and cell supernatant decreased significantly(P < 0.05).It is suggested that TSA can improve the metabolism of ischemic myocardium by inhibiting PCK2.EPD database and Jaspar database search analysis found that Fox O1 and NR4A1 are upstream transcription factors of PCK2.PCK2 has four potential transcriptional binding sites with Fox O1 and five potential transcriptional binding sites with NR4A1.Western blot analysis showed that the expression levels of Fox O1 and NR4A1 in myocardial ischemic tissues and H9c2 cells were significantly increased after TSA Administration(P < 0.01).Conclusions:1.Animal experiments show that PCK2 is a key gene regulating tricarboxylic acid cycle and glycolysis in ischemic myocardium of rats;2.Inhibition of PCK2 expression could enhance the activity of succinate dehydrogenase in H9c2 cells,and reduce the accumulation of lactic acid and pyruvate in the supernatant of H9c2 cells;3.TSA can up regulate the expression of Fox O1 and NR4A1,inhibit the expression of PCK2,regulate myocardial metabolism and improve myocardial ischemia injury. |
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