Study Of Sappanone A Attenuating High Fat Diet-induced Myocardial Lipotoxicity Via Nox4 Inhibition Of Ferroptosis

Objective:The proportion of high-calorie food rich in fat in people’s diets is increasing;consequently,long-term high-fat diet-induced metabolic disorders,such as diabetes and obesity,have become a major problem endangering human health.Fatty acid oxidation is an important method of supplying energy to myocardial tissue.In the peripheral blood of patients with obesity and diabetes,the content of free fatty acid（FFA）is increased significantly,and the associated metabolic disorder causes the accumulation of a large number of lipids and their oxidative intermediates in cardiomyocytes,thus leading to cardiomyocyte steatosis.Significant cardiomyocyte steatosis can further cause cardiac dysfunction,myocardial hypertrophy,myocardial fibrosis,and myocardial remodeling,ultimately leading to heart failure.This pathophysiological process is known as myocardial lipotoxicity.The prevention and treatment of myocardial lipotoxicity caused by a high-fat diet has attracted the attention of many scholars.At present,only a few drugs on the market have been proven to have anti-myocardial lipotoxicity activity in animal models.However,many natural compounds have shown promising therapeutic effects in in vitro and in vivo models of lipotoxicity,thus providing us with a good research entry point.Sappanone A（SA）is a new type of isoflavone compound extracted from dried Caesalpinia sappan,which has anti-oxidation and anti-inflammatory activities.Recent studies have found that SA has a good therapeutic effect on myocardial ischemia-reperfusion injury;therefore,we hypothesized that SA might also have a protective effect against myocardial lipotoxicity caused by a high-fat diet.Studies have confirmed that ferroptosis is closely related to the occurrence of cardiovascular diseases,and ferroptosis changes the structure and function of the myocardium,which might be the target for intervention against myocardial lipotoxicity.Therefore,it is essential to explore the role and mechanism of SA in reversing ferroptosis in the myocardium to provide a new treatment method for patients with myocardial lipotoxicity.Methods:1.Establishment of a myocardial lipotoxicity model induced by a high-fat diet in rats:Healthy male Wistar rats were randomly divided into a normal chow diet（NCD）group and a high fat diet（HFD）group.After 20 weeks of normal/high fat diet feeding,the related indexes were detected.（1）The changes in body weight,blood glucose,blood lipid,and insulin were detected.（2）The changes to the cardiac structure and function were detected using echocardiography.(3 Hematoxylin-eosin（HE）staining was used to detect the cross-sectional area of the myocardium.（4）Myocardial fibrosis was detected using Masson staining.（5）A Terminal deoxynucleotidyltransferase-mediated d UTP-biotin nick end labeling（TUNEL）staining assay was used to detect myocardial cell apoptosis.2.Protective effects of SA intervention on the myocardium of high-fat diet-fed rats:Rats were divided into five groups:A high-fat group:Rat were fed with an HFD for20 weeks.The normal saline group:After feeding with an HFD for 12 weeks,from the13th week,1 ml of normal saline was instilled once a day for 8 weeks.The low-,medium-,and high-SA treatment groups:After feeding with an HFD for 12 weeks,from the 13th week,gastric perfusion was performed with low（10 mg/kg）,medium（20mg/kg）,and high（40 mg/kg）doses of SA in 1 m L of saline once a day for 8 weeks.Body weight,blood glucose,blood lipid,insulin,echocardiography,HE staining,Masson staining,Sirius red staining,and TUNEL staining were used to evaluate the effects of SA intervention on abnormal glucose and lipid metabolism,cardiac systolic dysfunction,myocardial hypertrophy,myocardial fibrosis,and myocardial cell apoptosis induced by the HFD in rats.3.Transcriptome sequencing:Transcriptome sequencing was performed on the heart tissue of the constructed animal model to compare and analyze the differential gene expression in the myocardium of the SA treatment group and the HFD group,and the signaling pathways of SA were analyzed using gene ontology（GO）and Kyoto Encyclopedia of Genes and genomes（KEGG）.4.Palmitic acid induction of ferroptosis in cardiomyocytes:Rat H9c2 cardiomyocytes were stimulated with 400μM palmitic acid（PA）for 24 h to establish a myocardial lipotoxicity cell model.The cells were divided into two groups:The control group and the PA group.（1）Lipid deposition in cells was detected using oil red O staining;（2）A Ferro Orange probe was used to detect the content of Fe²⁺;（3）A2′,7′-Dichlorofluorescein diacetate（DCFH-DA）probe was used to detect the reactive oxygen species（ROS）content;（4）The contents of malondialdehyde（MDA）,superoxide dismutase（SOD）,and glutathione peroxidase（GSH）were determined using colorimetry.（5）Western blotting was used to detect ferroptosis marker proteins such as long-chain acyl-Co A synthetases（Acsl4）,glutathione peroxidase 4（Gpx4）,and Ferritin H1（Fth1）.5.SA inhibits ferroptosis in cardiomyocytes:（1）Exploration of the therapeutic concentration of SA:A Cell Counting Kit-8（CCK-8）assay was used to detect the viability of cardiomyocytes.The contents of creatine kinase-MB（CK-MB）and lactate dehydrogenase（LDH）were detected using colorimetry.（2）Effect of SA on PA-induced ferroptosisCardiomyocytes were divided into three groups:A control group,a PA group,and a PA+SA group.（1）The Ferro Orange probe was used to detect the Fe²⁺content;（2）The ROS content was detected using the DCFH-DA probe;（3）The contents of MDA,SOD,and GSH were determined using colorimetry;（4）Western blotting was used to detect the levels of ferroptosis marker proteins Acsl4,Gpx4,and Fth1.（3）The effect of SA on erastin-induced ferroptosisCardiomyocytes were divided into three groups:A control group,an erastin group and an erastin+SA group.（1）CCK-8 assays were was used to detect the viability of cardiomyocytes;（2）The ROS content was detected using the DCFH-DA probe;（3）The contents of MDA,SOD,and GSH were determined using colorimetry;（4）Western blotting was used to detect the levels of ferroptosis marker proteins Acsl4,Gpx4,and Fth1.（4）Erastin reversed the protective effect of SA on PA-induced cardiomyocyte hypertrophy and fibrosisCardiomyocytes were divided into a control group,a PA group,a PA+SA group,and a PA+SA+Erastin group;the related indicators were then detected.（1）Cell hypertrophy was detected using phalloidin staining;（2）Quantitative real-time reverse transcription PCR（q RT-PCR）was used to detect the expression of m RNA encoding Collagen I and Collagen III to evaluate the degree of myocardial fibrosis.6.Bioinformatic prediction of SA binding proteins:The proteins that might bind to SA were predicted using the SEA online database.Gene enrichment analysis was used to determine the relationship between NADPH oxidase 4（Nox4）and cardiomyocyte ferroptosis.The candidate protein Nox4 and SA were subjected to molecular docking using MOE software.7.Effect of SA on ferroptosis through Nox4:The effect of SA on Nox4 activity was determined using colorimetry.Cells were transfected with a Nox4 overexpression plasmid to observe whether Nox4overexpression could reverse the inhibitory effect of SA on PA-induced ferroptosis.Results:1.Compared with those in the NCD group,the 2 h postprandial blood glucose,triglyceride,total cholesterol,and insulin levels of the HFD group were increased significantly;however,the fasting blood glucose level was not significantly increased.The results of echocardiography showed that compared with those in the NCD group,the left ventricular wall thickness（WT）of the HFD group was increased significantly,and the fractional shortening（FS）was decreased significantly.However,there was no significant difference in the left ventricular end-systolic dimension（LVESD）and the left ventricular end-diastolic dimension（LVEDD）.HE staining showed that the cross-sectional area of myocardium in the HFD group was significantly larger than that in the NCD group.Masson staining showed that the percentage of myocardial fibrosis in the HFD group was significantly higher than that in the NCD group.Sirius red staining showed that the percentage of myocardial collagen fibers in the HFD group was significantly higher than that in the NCD group.TUNEL staining results showed that the apoptosis rate of myocardial cells in the HFD group was significantly higher than that in the NCD group.2.Compared with the HFD group,there was no significant difference in blood glucose,blood lipid and insulin levels after 8 weeks of oral SA treatment.The results of echocardiography showed that the WT in the low,medium,and high dose SA treatment groups decreased to varying degrees compared with that in the HFD group.The FS in the middle and high dose SA treatment groups increased significantly compared with that in the HFD group;however,there was no significant difference in the LVESD and LVEDD.In addition,the myocardial cross-sectional area,the percentage of myocardial fibrosis,the percentage of myocardial collagen fibers,and the apoptosis rate of myocardial cells in the low,middle,and high dose SA treatment groups were lower than those in the HFD group.The changes of the above indexes in the middle dose SA treatment group were more significant than those in the low dose SA treatment group,and there was no significant difference between the high dose SA treatment group and the middle dose SA group.3.Compared with that in the HFD group,55 genes were significantly upregulated and 53 genes were significantly downregulated in the SA group according to transcriptome sequencing.GO and KEGG analysis showed that the ferroptosis signaling pathway was the most enriched pathway.Western blotting results showed that the level of Acsl4 was decreased significantly,and the levels of Gpx4 and Fth1 were increased significantly in the myocardium of the SA treatment group.4.Oil red O staining showed that compared with that in the control group,a large number of orange lipid droplets were deposited in the cytoplasm of H9c2 cardiomyocytes after 400μM PA stimulation for 24 h,suggesting that PA successfully established a myocardial lipotoxicity model in vitro.5.Compared with those in the control group,the contents of intracellular Fe²⁺,ROS,and MDA,and the level of Acsl4,were increased significantly,while the contents of SOD and GSH and the levels of Gpx4 and Fth1 were decreased significantly,suggesting that PA induces ferroptosis in cardiomyocytes.6.Compared with those in the control group,SA dose-dependently attenuated the PA-induced decrease in cardiomyocyte viability and reduced the release of Creatine CK-MB and LDH.SA at 25μM had the most significant effect.7.SA could significantly inhibit the PA-induced increase in Fe²⁺,ROS,and MDA content and Acsl4 levels in cardiomyocytes,and induced a relative increase in SOD and GSH content and Gpx4 and Fth1 protein expression in PA-induced cardiomyocytes,suggesting that SA can reverse PA-induced ferroptosis in cardiomyocytes.8.SA could significantly prevent the decrease in cell viability caused by erastin-induced ferroptosis,inhibit the production of ROS and MDA,and increase the activities of SOD and GSH in myocardial cells,suggesting that SA can reverse the occurrence of ferroptosis induced by erastin in myocardial cells.Moreover,erastin reversed the effects of SA on PA-induced cardiomyocyte hypertrophy and fibrosis.9.Bioinformatic analysis revealed a potential binding between SA and Nox4,which regulates ROS production.PA could significantly increase the activity of Nox4 in cardiomyocytes.Compared with the PA group,SA treatment reversed the PSA-induced promotion of Nox4 activity.10.Myocardial cells transfected with the Nox4 overexpression plasmid were treated with SA+PA,and the phenotype of ferroptosis was restored compared with untransfected cells treated with SA+PA alone.Conclusion:1.SA has a protective effect against myocardial lipotoxicity induced by a high-fat diet.2.SA exerts its anti-myocardial lipotoxicity effect by inhibiting myocardial ferroptosis.3.SA reverses ferroptosis by downregulating Nox4 expression to exert its protective effect against myocardial lipotoxicity.