Effect And Mechanism Of Dihydromyricetin On Ameliorating Diabetic Cardiomyopathy By Regulating Glucose Absorption And Glycolysis Of Cardiomyocytes Through NR4A1

BackgroundThe global prevalence of diabetes was 9.3%（463 million）in 2019 and will rise to10.9%（700 million）by 2045.Diabetes has become a significant chronic disease and a major public health problem affecting human health.Diabetic cardiomyopathy（DCM）is a kind of heart disease characterized by changes in cardiac structure and ventricular diastolic and systolic function caused by diabetes.It is one of the significant complications of diabetes.Cardiomyocyte hypertrophy,myocardial interstitial fibrosis,and coronary artery microangiopathy were the main pathologic findings of diabetic cardiomyopathy after eliminating other heart diseases such as hypertensive heart disease,coronary heart disease,and rheumatic heart disease.The development of DCM may be associated with disorders of glucolipid metabolism,increased oxidative stress,inflammation,mitochondrial dysfunction,and altered insulin signaling,with diseases of glucolipid metabolism thought to be an early event in the deterioration of cardiac function in patients with DCM.Under physiological conditions,glucose metabolism and fatty acid metabolism can be maintained in a relatively dynamic balance in myocardial tissue.In contrast,during the progression of diabetes,myocardial tissue will become more dependent on fatty acid oxidation as insulin secretion decreases sharply or gradually,resulting in increased circulating glucose levels.As glucose metabolism decreases and fatty acid metabolism increases,this can lead to mitochondrial dysfunction and endoplasmic reticulum stress,leading to structural remodeling and altered cardiac function in the myocardium.The causes of impaired glucose utilization in myocardial tissue in DCM patients include reduced glucose uptake,reduced glycolytic activity,and reduced pyruvate oxidation.In addition,abnormal glucose metabolism has been found to exist in DCM patients and is associated with cardiac insufficiency.Therefore,targeting the altered abnormal cardiac metabolism in patients with DCM by improving myocardial tissue metabolic disorders,especially glucose metabolism,may be an effective means to slow down the process of DCM.A proper diet plays an essential role in preventing and treating various chronic diseases,including diabetes and its complications.Many studies have confirmed that phytochemicals play an essential biological role in preventing and treating various chronic diseases.Dihydromyricetin（DHM）,or ampelopsin,is a flavonoid phytochemical riched in rattan tea,a new food raw material.Previous studies in our laboratory have found that dietary supplementation of DHM can improve the level of serum glucose and biochemical indexes related to glucose and lipid metabolism in patients with non-alcoholic fatty liver disease and may protect the cardiac function of mice with insulin resistance by promoting PGC-1α/FNDC5/irisin pathway in myocardial tissue.Previous studies have shown that DHM can improve the cardiac function of DCM mice,including inhibiting oxidative stress in DCM mice,reducing the expression of mi R-34a to restore damaged autophagy,improving mitochondrial function and inhibiting cardiomyocyte apoptosis.However,the specific mechanism is still not precise.Due to the unique metabolic characteristics of myocardial tissue（including glucose metabolism,lipid metabolism,and so forth）and abnormal myocardial metabolism in patients with DCM,it has not been confirmed whether DHM can delay the occurrence and development of the disease by improving the metabolic disorder of myocardial tissue in DCM.Therefore,we aimed to establish an animal model of DCM and a hyperglycemic model of H9C2 cardiomyocytes,to investigate the effects of DHM on myocardial glucose absorption and glycolysis and reveal the related mechanism.The nuclear receptors（NRs）superfamily senses endocrine and metabolic signals in the body and has become a potential target for the treatment of chronic diseases such as type 2diabetes and obesity,among which NR4A1（also known as Nur77）is a transcription factor of the NR4A family and is involved in the metabolic regulation of many diseases,including obesity,liver disease,and diabetes.Studies have shown that targeting NR4A1 can regulate the critical glycolytic enzymes HK2 and PFK in liver and muscle cells,thereby targeting metabolic syndrome prevention and treatment.It has also been reported that Nur77-KO mice exhibit severe myocardial thinning,rupture,and reduced scar density.At the same time,Nur77-KO mice exhibit elevated Ca²⁺in diastolic and systolic cardiomyocytes,leading to increased myocardial hypertrophy and fibrosis.However,studies on whether myocardial metabolism can be regulated by targeting NR4A1 to improve DCM occurrence have yet to be reported.Therefore,we propose to investigate the effects of DHM on myocardial glucose uptake and glycolysis by establishing an animal model of DCM and an H9C2 cardiomyocyte hyperglycemia model and reveal the relevant mechanism of NR4A1’s role in this study aims to provide a new experimental basis for the application of DHM to DCM control.ObjectiveThe animal model of DCM induced by a high-fat diet combined with STZ and the cell model of H9C2 caused by high sugar was used to study the effect of DHM on the occurrence of DCM and its impact on myocardial glucose uptake and glycolysis and to reveal the possible mechanism of NR4A1 in the improvement of myocardial glucose uptake and glycolysis by DHM.This study aims to provide a new experimental basis for the application of DHM in the prevention and treatment of DCM.Methods1.C57BL/6J mice were induced by a high-fat diet combined with STZ to establish the DCM model,and DHM was evenly mixed with a high-fat diet so that the mice could ingest it freely.The effects of DHM on mice’s body mass and food intake were observed.The effect of DHM on cardiac function in mice was evaluated by echocardiography.The effect of DHM on cardiac glucose and lipid metabolism was evaluated by detecting serum biochemical indexes.The effect of DHM on cardiac pathological changes was evaluated by HE and transmission electron microscopy.Masson and Sirius’s red staining further detected the effect of DHM on cardiac fibrosis.The effects of DHM on myocardial glucose absorption and glycolysis-related gene m RNA and protein expression in mice were detected by tissue immunofluorescence and q RT-PCR assay.2.The RNA-Seq transcriptome technology was employed.Principal component（PCA）analysis was used to screen representative samples.Then the critical molecular targets of DHM for myocardial protection were explored by bioinformatics analysis methods such as differential gene screening,subcellular localization,and KEGG pathway analysis.3.The hyperglycemia model of H9C2 cardiomyocytes was established,and the effect of DHM on cardiomyocyte injury was evaluated by the detection of CCK8 and LDH release rate.The effect of DHM on glucose uptake of H9C2 cells induced by HG was analyzed by a2-NBDG absorption test.NR4A1si RNA was used to silence NR4A1 in order to explore the role of NR4A1 in inhibiting the injury of H9C2 cardiomyocytes induced by high glucose and increasing glucose absorption and glycolysis.The expression of m RNA and protein related to glucose absorption and glycolysis in cardiomyocytes was detected by q RT-PCR and Western blot to reveal the mechanism of NR4A1 in improving glucose absorption and glycolysis of cardiomyocytes by DHM.Results1.DHM can effectively improve the occurrence of DCM in mice induced by a high fat-diet combined with STZ.The systolic and diastolic function of the heart of DCM mice decreased significantly,and the myocardial tissue had inflammatory cell infiltration,prominent fibrosis,and a disorder of glucose and lipid metabolism.The levels of glucose absorption and glycolysis in myocardial tissue were significantly decreased.After intervention with DHM,cardiac function,myocardial fibrosis,myocardial injury,and abnormal glucose and lipid metabolism were significantly improved in DCM mice,and the expression of m RNA and protein related to glucose absorption and glycolysis in myocardial tissue was promoted.2.DHM can significantly change the expression of several metabolism-related genes in the myocardium of DCM mice,among which NR4A1 may be the critical target for DHM to improve the occurrence of DCM and regulate myocardial glucose absorption and glycolysis.The results of RNA-seq showed that there were 334 differential genes between the DCM group and CON group,of which 121genes were up-regulated,and 213genes were down-regulated,while between the DCM+DHM group and DCM group,there were 361differential genes,including 283 genes up-regulated and 78 down-regulated.After DHM intervention,the differential genes were mainly enriched in the metabolic pathway,PI3K-Akt signaling pathway,NF-κB anti-inflammatory signal pathway,and Fox O metabolic regulation.The results of RNA-seq suggested that the expression of several metabolic regulations-related genes（such as Hmgcs2,Gadd45g,NR4A1,Abat,and Thbs4）changed significantly after the intervention of DHM,and the differentially expressed gene NR4A1 may be the potential target of DHM.3.DHM may promote the expression of genes related to glucose absorption and glycolysis in cardiomyocytes through NR4A1 and remission cardiomyocyte injury.DHM could significantly inhibit the injury of H9C2 cardiomyocytes induced by high glucose and ameliorate the levels of glucose uptake and glycolysis of cardiomyocytes.After silencing the NR4A1 gene with NR4A1si RNA,the effect of DHM on cardiomyocyte injury induced by H9C2 was significantly inhibited.At the same time,silencing NR4A1 weakened the effect of DHM on glucose uptake and glycolysis of H9C2 cardiomyocytes induced by high glucose.Therefore,DHM may inhibit cardiomyocyte injury induced by high glucose through NR4A1 regulating glucose absorption and glycolysis.ConclusionDHM may increase the level of myocardial glucose absorption and glycolysis,reduce myocardial injury,improve cardiac diastolic and systolic function,and finally prevent the occurrence of diabetic cardiomyopathy by promoting the expression of NR4A1 and its mediated metabolism-related genes.This study provides a new intervention target for the prevention and treatment of diabetic cardiomyopathy and a new experimental basis for the application of DHM in the prevention and treatment of diabetic cardiomyopathy.