Exploring The Molecular Mechanism Of Inhibition Of Ferroptosis In Cardiomyocytes Of Coronary Heart Disease Model By Ershen Decoction Based On Nrf2/GPX4 Signaling Pathway

Objective:1 Utilizing bibliometric analysis with Cite Space,the current state and trends of research in"ferroptosis"and"cardiovascular diseases and ferroptosis"are analyzed to identify the knowledge structure and the evolution of hotspots.2 Through a coronary heart disease rat model and an Erastin-induced H9C2 cell ferroptosis model,focusing on the regulation of the Nrf2/GPX4 signaling pathway to inhibit ferroptosis in coronary heart disease myocardial cells,the protective effects and molecular mechanisms of Ershen Decoction on myocardial cells in coronary heart disease are revealed,providing experimental evidence for the treatment of coronary heart disease with Ershen Decoction.Methods:1.Literature study1.1 The WOSCC database was used with the search terms set as the subject,TS=（"ferroptosis"OR"ferroptotic"）,the time span from January 1,2012,to December 31,2023,the language limited to"English,"and the type of literature limited to"Article OR Review."Based on Cite Space software and Excel’s"three-line table,"this study further analyzes the annual trend of publications,distribution of countries and institutions,authors and authors’co-citation analysis,journals and journals’co-citation situations,keyword analysis,literature co-citation analysis,and creates a network mapping.1.2 Using the WOSCC database,set TS as the search term:TS=（"cardiovascular disease"OR"CVD"OR"cardiovascular"OR"heart"OR"circulation"）AND TS=（"ferroptosis"OR"ferroptotic"）.The time span should be from January 1,2012,to December31,2023.Limit the language to"English"and the document type to（"Article"OR"Review"）.Using the CNKI database,search for the theme=（"cardiovascular disease"OR"cardiovascular"OR"heart"）AND theme=（"ferroptotic"）.The time span should be from January 1,2012,to December 31,2023.Limit the language to Chinese,and manually exclude bibliographies,conferences,and other papers.Based on the Cite Space software and Excel’s data"trilinear table",we further analysed the annual trend of publications,the distribution of countries and institutions,the authors and authors’co-citation analyses,the journals and journals’co-citation analyses,keyword analysis,literature co-citation analysis,and network mapping.2.Experimental studies2.1 In vivo experiment:Sixty SPF-grade healthy male SD rats（weight 180-220g）were selected.A coronary heart disease rat model was established by continuous feeding with a high-fat diet combined with vitamin D3（600,000 IU/kg）and pituitrin hormone（30μg/kg）for12 weeks.The rats were randomly divided into six groups:a blank control group（Ctrl）,a model group（Model）,an Ershen Decoction high-dose group（ESH）,an Ershen Decoction medium-dose group（ESM）,an Ershen Decoction low-dose group（ESL）,and a positive drug group（Y）,with 10 rats in each group.According to the equivalent dose ratio of"human-rat"in"Pharmacology of Traditional Chinese Medicine,"the ESH-L groups were administered35.2 g/kg,17.6 g/kg,and 8.8 g/kg respectively by gavage.The Y group was given atorvastatin calcium tablets at 0.9 mg/kg.This was done once daily for 6 consecutive weeks.During the experiment,the general condition of the rats in each group was observed.AⅡ-lead routine electrocardiogram was collected after the last intraperitoneal injection of pituitary posterior lobe hormone.Hematoxylin-eosin staining（HE）was used to observe the pathological morphology of myocardial tissue in each group.Masson staining was used to observe the degree of fibrosis injury in myocardial tissue in each group.Transmission electron microscopy was used to detect ultrastructural changes in myocardial tissue in each group.Enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of total cholesterol（TC）,triglycerides（TG）,high-density lipoprotein cholesterol（HDL-C）,and low-density lipoprotein cholesterol（LDL-C）in plasma of each group.ELISA was also used to detect the levels of lipid peroxides（LPO）,reactive oxygen species（ROS）,superoxide dismutase（SOD）,malondialdehyde（MDA）,4-hydroxynonenal（4-HNE）,glutathione（GSH）,and ferrous ions(Fe²⁺)in myocardial tissue of each group.Reverse transcription polymerase chain reaction（RT-PCR）and Western blotting were used to detect the m RNA and protein expression of FTH1,Keap-1,Nrf2,HO-1,SLC7A11,and GPX4 in myocardial tissue of the rats.2.2 In vitro experiment:Establishing an Erastin-induced ferroptosis model in H9C2 cells,the Cell-Counting-Kit-8（CCK8）method was used to detect cell viability;ELISA was used to detect Fe²⁺levels to confirm the successful establishment of the ferroptosis model.The experiment was divided into the following groups:control group（10%rat serum from the control group）,model group（Erastin+10%rat serum from the control group）,low-dose Ershen Decoction group（Erastin+10%rat serum with low dose of Ershen Decoction）,medium-dose Ershen Decoction group（Erastin+10%rat serum with medium dose of Ershen Decoction）,and high-dose Ershen Decoction group（Erastin+10%rat serum with high dose of Ershen Decoction）.The effects of Ershen Decoction on cell viability and LDH activity were detected using CCK8 and LDH assay kits.The effects of Ershen Decoction on key indicators of lipid peroxidation within H9C2 cells（ROS,SOD,GSH,MDA,4-HNE）and intracellular Fe²⁺were detected using ROS,SOD,GSH,MDA,4-HNE,and Fe²⁺assay kits.RT-PCR and Western blot were used to detect the expression of FTH1,Keap-1,Nrf2,HO-1,SLC7A11,GPX4 m RNA and proteins.To further investigate whether Ershen Decoction regulates ferroptosis-mediated H9C2 cell damage through the Nrf2/GPX4 signaling pathway,the same batch of H9C2 cells was selected and divided into 6 groups:blank control group（Ctrl）,model group（Model）,Erastin+10%high-dose group rat medicated serum group（ES）,Erastin+ferrostatin-1 group（Fer-1）,and Erastin+10%high-dose group rat medicated serum+ML385 group（ES+ML385）.Cell viability and LDH activity in each group were detected using CCK8 and LDH assay kits;the effects of key indicators of lipid peroxidation within cells（ROS,SOD,GSH,MDA,4-HNE）and intracellular Fe²⁺were detected using ROS,SOD,GSH,MDA,4-HNE,and Fe²⁺assay kits.RT-PCR and Western blot were used to detect the effects of Ershen Decoction on the expression of Keap-1,Nrf2,HO-1,SLC7A11,GPX4m RNA,and proteins.Results:1.Literature Study1.1 Based on bibliometric analysis of ferroptosis research,10,188 papers were published by 57,309 authors from 5,475 institutions across 100 countries/regions in 1,374 academic journals.Publications related to ferroptosis are rapidly increasing.Among these,the journal with the most ferroptosis-related papers is"Frontiers in Oncology",while the most cited journal is"Cell".The main publishing and co-cited journals are in the fields of molecular and biology.China is the country with the highest number of publications,followed by the United States.Central South University is the institution with the highest number of publications,while the University of Pittsburgh and Columbia University are the most authoritative institutions.Additionally,Tang Daolin is the author with the most publications on the topic（70 papers）,and the most cited author is Dixon SJ（5,894 citations）.Keyword and co-citation analysis revealed that research hotspots and directions in ferroptosis focus mainly on the mechanisms,particularly reactive oxygen species,oxidative stress,lipid peroxidation,and the Nrf2 signaling pathway.1.2 Based on bibliometric analysis of research on"Cardiovascular Diseases and Ferroptosis",a total of 903 articles were included in the WOSCC and CNKI databases,with645 in English and 258 in Chinese.Contributions to this research were made by 4,437 authors from 859 institutions across 48 countries,with the number of publications showing an upward trend each year.China has the highest number of publications,followed by the United States.The top ten institutions in terms of publication frequency are all from China,with Peking Union Medical College ranking first.Chattipakorn Nipon is the most prolific author,while Dixon SJ from the Department of Biology at Stanford University is the most frequently co-cited author.The journal with the highest number of publications is"Frontiers in Cardiovascular Medicine"（27 publications,4.12%）,and the most cited journal is"Cell"（503citations）.Keyword and co-citation analysis revealed that research hotspots in the field of cardiovascular diseases and ferroptosis are mainly focused on mechanisms and disease processes such as oxidative stress,apoptosis,lipid peroxidation,heart failure,and ischemia-reperfusion injury.Additionally,traditional Chinese medicine may become an emerging research trend in the study of cardiovascular diseases and ferroptosis.2.Experimental Research2.1 In Vivo Experiments:（1）General condition of rats:Compared to the Ctrl group,the Model group rats showed poor fur neatness and glossiness,decreased activity,impaired mobility,poor mental state,and symptoms of lethargy and fatigue.After treatment with Ershen Decoction,the rats showed improvements in fur brightness and neatness,agility,and overall vitality.（2）Electrocardiogram（ECG）:Compared to the Ctrl group,the Model group exhibited a downward shift in the S-T segment.The ESM group,ESH group,and Y group showed a smaller downward shift in the S-T segment,closer to the level of the Ctrl group,indicating that Ershen Decoction improved myocardial ischemia in the coronary heart disease model rats.（3）HE staining:The Ctrl group exhibited good structural integrity of the heart tissue,with no abnormalities in the heart wall and chamber.Myocardial fibers were uniformly stained,with clear cell boundaries,orderly arrangement,distinct cross-striations of myocardial cells,and no significant pathological changes in the stroma and vascular structures.Compared to the Ctrl group,the Model group showed marked vascular necrosis and calcification,proliferation of connective tissue,and mild to moderate infiltration of inflammatory cells,with varying degrees of myocardial cell necrosis.After treatment with Ershen Decoction,the instances of vascular necrosis and unclear structures were reduced,local fibrous tissue proliferation was mild,and only slight infiltration of inflammatory cells was observed（4）Transmission electron microscopy:In the Ctrl group,myofibrils are neatly arranged without obvious fractures.The sarcomeres are intact and uniformly wide.The nuclei（N）are irregular in shape.The mitochondrial（M）matrix is slightly lighter in color,and the sarcoplasmic reticulum（SPR）is not dilated.The Z lines are arranged neatly and continuously.Compared to the Ctrl group,the Model group shows moderate edema and sparsity in the cytoplasm of cardiomyocytes.There are more fractures and reductions in myofibrils,with many sarcomeres disintegrating.The nuclei（N）appear elongated.Most mitochondria（M）are mildly swollen with an uneven matrix.Some cristae are fractured and shortened,and some membranes are bulging or thinning.A few mitochondria are shrunken with relatively high electron density and smaller volume.The SPR is mildly dilated,and there are numerous fractures in the Z lines.After treatment with Ershen Decoction,the structure of cardiomyocytes is well maintained.No edema is observed in the cytoplasm,and myofibrils are neatly arranged without obvious fractures.The sarcomeres are intact and uniformly wide.The nuclei（N）are irregular in shape,and the mitochondria（M）are relatively well-structured with no swelling.The cristae are arranged in parallel,and the SPR is not dilated.The Z lines are relatively neat and continuous.（5）Masson’s trichrome staining:In the Ctrl group,the morphology and structure of myocardial fibers showed a regular arrangement with good connections between muscle fibers,and clear transverse striations were visible.In the Model group,there was significant proliferation of collagen fibers in the myocardial tissue,disordered arrangement of muscle fibers,and an increased number of collagen fibers.After treatment with Ershen Decoction,the proliferation of collagen fibers in the myocardial tissue was relatively reduced,the arrangement of myocardial cells was regular,and the disordered tissue structure was effectively improved.（6）Levels of plasma TG,TC,LDL-C,and HDL-C:Compared to the Ctrl group,the levels of TC,TG,and LDL-C in the Model group were significantly increased（P<0.01）,while the level of HDL-C was significantly decreased（P<0.01）.After treatment with Ershen Decoction,compared to the Model group,the levels of TG,TC,and LDL-C were significantly reduced（P<0.05）,and the level of HDL-C was significantly increased（P<0.01）.（7）Lipid peroxidation-related indicators:Compared to the Model group,after treatment with Ershen Decoction,the activities of GSH and SOD were significantly increased（P<0.05,P<0.01,P<0.001）.The levels of ROS,LPO,MDA,and 4-HNE were significantly reduced（P<0.05,P<0.001）.（8）Fe²⁺concentration and FTH1 expression:Compared to the Ctrl group,the Model group showed a significant increase in Fe²⁺concentration（P<0.001）and significant inhibition of FTH1 m RNA and protein expression（P<0.001）.After Ershen Decoction treatment,Fe²⁺levels significantly decreased（P<0.001）,and significantly promoted FTH1 m RNA and protein expression（P<0.001）.（9）Expression of Nrf2/GPX4 signaling pathway-related genes and proteins:Compared to the Ctrl group,the Model group showed significant increases in Keap-1 m RNA and protein levels（P<0.01,P<0.001）,and decreases in Nrf2,GPX4,HO-1,and SLC7A11 m RNA and protein levels（P<0.05,P<0.01,P<0.001）.Compared to the Model group,Ershen Decoction inhibited Keap-1 m RNA and protein expression（P<0.05,P<0.01,P<0.001）,and promoted the expression of Nrf2,GPX4,HO-1,and SLC7A11 m RNA and proteins（P<0.05,P<0.01,P<0.001）.2.2 In Vitro Experiments（1）Establishment of Ferroptosis Model:Treatment of H9C2 cells with 10μM Erastin for24 hours significantly reduced cell viability（P<0.001）and promoted intracellular Fe²⁺deposition（P<0.001）,confirming the successful establishment of the ferroptosis model.（2）H9C2 Cell Viability and LDH Activity:Compared to the Model group,Ershen Decoction effectively restored the viability of H9C2 cells（P<0.01,P<0.001）and effectively reduced LDH activity in H9C2 cells（P<0.01）.（3）ROS and Lipid Peroxidation:Compared to the Model group,Ershen Decoction intervention significantly inhibited ROS activity in H9C2 cells（P<0.01,P<0.001）,reduced the contents of MDA and 4-HNE（P<0.05,P<0.01,P<0.001）,and significantly increased the activity of SOD and GSH（P<0.05,P<0.01,P<0.001）.（4）Expression of Fe²⁺and FTH1:Compared to the Model group,Ershen Decoction intervention significantly reduced the content of Fe²⁺in H9C2 cells（P<0.05,P<0.001）and significantly increased the expression levels of FTH1 m RNA and protein（P<0.01,P<0.001）.（5）Expression of Nrf2/GPX4 Signaling Pathway-Related Genes and Proteins:Compared to the Model group,Ershen Decoction intervention significantly increased the expression levels of Nrf2,HO-1,SLC7A11,and GPX4 m RNA and proteins（P<0.01,P<0.001）and significantly downregulated the expression levels of Keap-1 m RNA and protein（P<0.01,P<0.001）.（6）Regulation of Ferroptosis-Mediated H9C2 Cell Damage by the Nrf2/GPX4 Signaling Pathway:（1）Compared to the Model group,both ES and Fer-1 groups significantly improved H9C2 cell viability（P<0.001）and significantly reduced LDH activity（P<0.001）.Compared to the ES group,the ES+ML385 group weakened the aforementioned effects（P<0.05）;（2）Compared to the Model group,the ES and Fer-1 groups significantly reduced ROS（P<0.001）,significantly increased the activity of SOD and GSH（P<0.001）,and significantly decreased the contents of MDA and 4-HNE（P<0.001）.Compared to the ES group,the ES+ML385group significantly reduced the protective effects of Ershen Decoction（P<0.05,P<0.01）;（3）Compared to the Model group,the Fe²⁺content in H9C2 cells was significantly reduced in both ES and Fer-1 groups（P<0.001）.Compared to the ES group,the Fe²⁺content in the ES+ML385 group was significantly increased（P<0.05）;（4）Compared to the Model group,ES and Fer-1 groups significantly inhibited the expression of Keap-1 protein in H9C2 cells（P<0.001）and promoted the expression of Nrf2,SLC7A11,GPX4 proteins（P<0.05,P<0.01,P<0.001）.Compared to the ES group,ML385 weakened Ershen Decoction’s effect on the expression of key proteins in the Nrf2/GPX4 signaling pathway induced by Erastin in H9C2cells（P<0.05,P<0.001）.Conclusion:1.Literature Research1.1 At present,research on ferroptosis is undergoing rapid development.China has the highest number of publications in this field,followed by the United States,with Central South University being the institution with the most publications.This indicates that China is at the forefront of research on ferroptosis.The current research hotspots are mainly focused on reactive oxygen species（ROS）,oxidative stress,lipid peroxidation,and the Nrf2 signaling pathway.1.2 Research on cardiovascular diseases and ferroptosis has been increasing year by year,with China having the highest number of publications in this field.Ferroptosis holds great potential in the treatment of cardiovascular diseases,with oxidative stress and lipid peroxidation being the core mechanisms linking ferroptosis to cardiovascular conditions.Traditional Chinese medicine（TCM）is experiencing rapid development in the study of cardiovascular diseases and ferroptosis.This progress can offer new strategies for further exploring the treatment of cardiovascular diseases and provide new ideas and methods for TCM in the treatment and experimental research of related diseases.2.Experimental studies2.1 Ershen Decoction can effectively improve myocardial injury in rats with coronary heart disease by inhibiting lipid peroxidation in myocardial tissue and regulating iron ion balance.Its mechanism may involve the regulation of the Nrf2/GPX4 signaling pathway to inhibit ferroptosis,thereby exerting a protective effect on myocardial tissue.2.2 Ershen Decoction improves Erastin-induced H9C2 cell survival,reduces LDH release,decreases lipid peroxidation,and regulates Fe²⁺level,the mechanism of which is to inhibit the ferroptosis of cardiomyocytes by regulating the Nrf2/GPX4 signalling pathway to achieve the alleviation of cell damage.