This study aims to explore the specific mechanism by which puerarin inhibits ferroptosis and improves the myocardial contractile function in myocardial hypertrophy through the nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE)/heme oxygenase-1(HO-1) signaling pathway. The hypertrophic cardiomyocyte model was established using phenylephrine, and H9c2 cells were divided into control group, model group, puerarin group, and puerarin+ML385 group. Cell viability and surface area were detected by cell counting kit-8(CCK-8) and immunofluorescence experiments. The mitochondrial membrane potential and Ca~(2+) concentration were measured. The ferroptosis-related indicators were detected by biochemical and fluorescence staining methods. The expression of proteins related to ferroptosis and the Nrf2/ARE/HO-1 signaling pathway was detected by Western blot. A myocardial hypertrophy model was established, and 40 rats were randomly divided into sham group, model group, puerarin group, and puerarin+Nrf2 inhibitor(ML385) group, with 10 rats in each group. Echocardiogram, hemodynamic parameters, and myocardial hypertrophy parameters were measured. Histopathological changes of myocardial tissues were observed by hematoxylin and eosin(HE) staining and Masson staining. Biochemical methods, enzyme-linked immunosorbent assay(ELISA), and fluorescence staining were used to detect inflammatory factors and ferroptosis-related indicators. Immunohistochemistry was used to detect the expression of proteins related to ferroptosis and the Nrf2/ARE/HO-1 signaling pathway. Cell experiments showed that puerarin intervention significantly enhanced the viability of hypertrophic cardiomyocytes, reduced their surface area, and restored mitochondrial membrane potential and Ca~(2+) homeostasis. Mechanism studies revealed that puerarin promoted Nrf2 nuclear translocation, upregulated the expression of HO-1, solute carrier family 7 member 11(SLC7A11), and glutathione peroxidase 4(GPX4), and decreased malondialdehyde(MDA), reactive oxygen species(ROS), and iron levels. These protective effects were reversed by ML385. In animal experiments, puerarin improved cardiac function in rats with myocardial hypertrophy, alleviated myocardial hypertrophy and fibrosis, inhibited inflammatory responses and ferroptosis, and promoted nuclear Nrf2 translocation and HO-1 expression. However, combined intervention with ML385 led to deterioration of hemodynamics and a rebound in ferroptosis marker levels. In conclusion, puerarin may inhibit cardiomyocyte ferroptosis through the Nrf2/ARE/HO-1 signaling pathway, thereby improving myocardial contractile function in myocardial hypertrophy.
Animals ; NF-E2-Related Factor 2/genetics* ; Rats ; Ferroptosis/drug effects* ; Signal Transduction/drug effects* ; Isoflavones/pharmacology* ; Male ; Rats, Sprague-Dawley ; Cardiomegaly/genetics* ; Myocytes, Cardiac/metabolism* ; Antioxidant Response Elements/drug effects* ; Myocardial Contraction/drug effects* ; Heme Oxygenase-1/genetics* ; Cell Line

Objective To investigate the impact of Astragaloside-IV (AS-IV) on the progression of myocardial infarction (MI) through macrophage-dependent mechanisms by regulating Snail1 lactylation and acetylation, as well as the transforming growth factor β (TGF-β) pathway. Methods Oxygen glucose deprivation (OGD) was used to establish an in vitro myocardial ischemia model in rat cardiomyocytes (H9c2), which were then treated with AS-IV. Cell viability was assessed using CCK-8, apoptosis was evaluated by flow cytometry, and LDH levels were measured to assess cellular damage. RAW246.7 cells were treated with LPS, and lactate levels in the supernatant were measured using ELISA, while expression of macrophage phenotype markers was evaluated using Western blot. RAW246.7 cell-conditioned medium (CM) was co-cultured with H9c2 cells to assess the protective effects of AS-IV on macrophage CM-mediated H9c2 damage. RAW246.7 cells were induced to differentiate into M1-like macrophages using LPS (100 ng/mL) + IFN-γ (20 ng/mL), and Snail1 was overexpressed in M1 macrophages. Transfected M1 macrophage CM was co-cultured with H9c2 cells to validate the mechanisms of AS-IV in MI. An MI rat model was established by ligation of the left anterior descending coronary artery (LAD), and was treated with AS-IV. Cardiac function, myocardial cell apoptosis, and cardiac tissue pathology were studied using echocardiography, TUNEL, and HE staining, respectively. Results Compared to the OGD group, AS-IV treatment promoted cell viability, reduced apoptosis and decreased LDH release. LPS upregulated lactate levels in the supernatant of RAW246.7 cell cultures and induced polarization of RAW246.7 cells to the M1 phenotype. AS-IV attenuated the damaging effects of RAW246.7 cell CM on H9c2 cells . Overexpression of Snail1 in M1 macrophages weakened the protective effects of AS-IV on H9c2 cells . In vivo study, results showed that, compared to the MI group, AS-IV treatment reduced lactate levels in the hearts of MI rats, improved cardiac function and myocardial injury and attenuated myocardial cell apoptosis. Conclusion AS-IV inhibits TGF-β pathway activation through the suppression of Snail1 lactylation and acetylation in a macrophage-dependent manner, thereby mitigating myocardial cell damage following MI.
Animals ; Myocardial Infarction/drug therapy* ; Rats ; Snail Family Transcription Factors/metabolism* ; Macrophages/cytology* ; Myocytes, Cardiac/metabolism* ; Triterpenes/pharmacology* ; Saponins/pharmacology* ; Acetylation/drug effects* ; Apoptosis/drug effects* ; Mice ; Cell Line ; RAW 264.7 Cells ; Transforming Growth Factor beta/metabolism*

OBJECTIVE: To explore the molecular mechanism of Shenmai Injection (SMI) against doxorubicin (DOX) induced cardiomyocyte apoptosis. METHODS: A total of 40 specific pathogen-free (SPF) male Sprague Dawley (SD) male rats were divided into 5 groups based on the random number table, including the control group, the model group, miR-30a agomir group, SMI low-dose (SMI-L) group, and SMI high-dose (SMI-H) group, with 8 rats in each group. Except for the control group, the rats were injected weekly with DOX (2 mg/kg) in the tail vein for 4 weeks to induce myocardial injury, and were given different regimens of continuous intervention for 2 weeks. Cardiac function was detected by echocardiography and myocardial pathological changes were observed by Van Gieson (VG) staining. Myocardial injury serum markers, including creatine kinase (CK), lactate dehydrogenase (LDH), troponin T (cTnT), N-terminal pro-brain natriuretic peptide (NT-proBNP), soluble ST2 (sST2), and growth differentiation factor-15 (GDF-15) were detected by enzyme linked immunosorbent assay (ELISA). Cardiomyocyte apoptosis was observed by terminal deoxynucleotidyl transferase-mediated biotinylated dUTP triphosphate nick end labeling (TUNEL) and transmission electron microscopy, and the expressions of target proteins and mRNA were detected by Western blot and quantitative real time polymerase chain reaction (qRT-RCR), respectively. RESULTS: The treatment with different doses of SMI reduced rat heart mass index and left ventricular mass index (P<0.05), significantly improved the left ventricular ejection fraction (P<0.05), decreased the levels of serum CK, LDH, cTnT, and NT-proBNP (P<0.05 or P<0.01), reduced the levels of serum sST2 and GDF-15 (P<0.05 or P<0.01), decreased the collagen volume fraction, reduced the expressions of rat myocardial type I and type III collagen (P<0.05 or P<0.01), and effectively alleviated myocardial fibrosis. And the study found that SMI promoted the expression levels of miR-30a and Bcl-2 in myocardium, and down-regulated the expression of Bax, which inhibited the activation of Caspase-3 and Caspase-9 (P<0.05 or P<0.01), and improved myocardial cell apoptosis. CONCLUSIONS SMI can alleviate myocardial injury and apoptosis caused by DOX, and its mechanism possibly by promoting the targeted expression of myocardial Bcl-2 protein through miR-30a.
Animals ; Myocytes, Cardiac/metabolism* ; Apoptosis/drug effects* ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Doxorubicin/pharmacology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Drug Combinations ; Injections ; Rats

BACKGROUND: Salvianolate is a compound mainly composed of salvia magnesium acetate, which is extracted from the Chinese herb Salvia miltiorrhiza . In recent years, salvianolate injection has been widely used in the treatment of cardiovascular diseases, but the mechanism of how it can alleviate cardiotoxicity remains unclear. METHODS: The cardiac injury model was constructed by treatment with doxorubicin (Dox) or azithromycin (Azi) in zebrafish larvae. Heart phenotype, heart rate, and cardiomyocyte apoptosis were observed in the study. RNA-sequencing (RNA-seq) analysis was used to explore the underlying mechanism of salvianolate treatment. Moreover, cardiomyocyte autophagy was assessed by in situ imaging. In addition, the miR-30a/becn1 axis regulation by salvianolate was further investigated. RESULTS: Salvianolate treatment reduced the proportion of pericardial edema, recovered heart rate, and inhibited cardiomyocyte apoptosis in Dox/Azi-administered zebrafish larvae. Mechanistically, salvianolate regulated the lysosomal pathway and promoted autophagic flux in zebrafish cardiomyocytes. The expression level of becn1 was increased in Dox-induced myocardial tissue injury after salvianolate administration; overexpression of becn1 in cardiomyocytes alleviated the Dox/Azi-induced cardiac injury and promoted autophagic flux in cardiomyocytes, while becn1 knockdown blocked the effects of salvianolate. In addition, miR-30a, negatively regulated by salvianolate, partially inhibited the cardiac amelioration of salvianolate by targeting becn1  directly. CONCLUSION This study has proved that salvianolate reduces cardiomyopathy by regulating autophagic flux through the miR-30a/becn1 axis in zebrafish and is a potential drug for adjunctive Dox/Azi therapy.
Animals ; Zebrafish ; MicroRNAs/genetics* ; Autophagy/drug effects* ; Myocytes, Cardiac/metabolism* ; Cardiomyopathies/metabolism* ; Beclin-1/genetics* ; Apoptosis/drug effects* ; Plant Extracts/therapeutic use* ; Doxorubicin

OBJECTIVE: Baicalein has been reported to have wide therapeutic effects that act through its anti-inflammatory activity. This study examines the effect and mechanism of baicalein on sepsis-induced cardiomyopathy (SIC). METHODS: A thorough screening of a small library of natural products, comprising 100 diverse compounds, was conducted to identify the most effective drug against lipopolysaccharide (LPS)-treated H9C2 cardiomyocytes. The core target proteins and their associated signaling pathways involved in baicalein's efficacy against LPS-induced myocardial injury were predicted by network pharmacology. RESULTS: Baicalein was identified as the most potent protective agent in LPS-exposed H9C2 cardiomyocytes. It exhibited a dose-dependent inhibitory effect on cell injury and inflammation. In the LPS-induced septic mouse model, baicalein demonstrated a significant capacity to mitigate LPS-triggered myocardial deficits, inflammatory responses, and ferroptosis. Network pharmacological analysis and experimental confirmation suggested that hypoxia-inducible factor 1 subunit α (HIF1-α) is likely to be the crucial factor in mediating the impact of baicalein against LPS-induced myocardial ferroptosis and injury. By combining microRNA (miRNA) screening in LPS-treated myocardium with miRNA prediction targeting HIF1-α, we found that miR-299b-5p may serve as a regulator of HIF1-α. The reduction in miR-299b-5p levels in LPS-treated myocardium, compared to the control group, was reversed by baicalein treatment. The reverse transcription quantitative polymerase chain reaction, Western blotting, and dual-luciferase reporter gene analyses together identified HIF1-α as the target of miR-299b-5p in cardiomyocytes. CONCLUSION Baicalein mitigates SIC at the miRNA level, suggesting the therapeutic potential of it in treating SIC through the regulation of miR-299b-5p/HIF1-α/ferroptosis pathway. Please cite this article as: Zhou WY, Du JK, Liu HH, Deng L, Ma K, Xiao J, Zhang S, Wang CN. Baicalein attenuates lipopolysaccharide-induced myocardial injury by inhibiting ferroptosis via miR-299b-5p/HIF1-α pathway. J Integr Med. 2025; 23(5):560-575.
Flavanones/pharmacology* ; Animals ; MicroRNAs/genetics* ; Lipopolysaccharides ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Ferroptosis/drug effects* ; Mice ; Myocytes, Cardiac/metabolism* ; Signal Transduction/drug effects* ; Rats ; Male ; Mice, Inbred C57BL ; Cardiomyopathies/etiology* ; Cell Line ; Sepsis/complications*

Astragali Radix (AR) and Notoginseng Radix et Rhizoma (NR) are frequently employed in cardiovascular disease treatment. However, the efficacy of the AR-NR medicine pair (AN) in improving cardiac remodeling and its underlying mechanism remains unclear. This study aimed to evaluate AN's cardioprotective effect and potential mechanism on cardiac remodeling using transverse aortic constriction (TAC) in mice and angiotensin II (Ang II)-induced neonatal rat cardiomyocytes (NRCMs) and fibroblasts in vitro. High-performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (HPLC-Q-TOF-MS/MS) characterized 23 main components of AN. AN significantly improved cardiac function in the TAC-induced mice. Furthermore, AN considerably reduced the serum levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), cardiac troponin T (CTn-T), and interleukin-6 (IL-6) and mitigated inflammatory cell infiltration. Post-AN treatment, TAC-induced heart size approached normal. AN decreased cardiomyocyte cross-sectional area and attenuated the upregulation of cardiac hypertrophy marker genes (ANP, BNP, and MYH7) in vivo and in vitro. Concurrently, AN alleviated collagen deposition in TAC-induced mice. AN also reduced the expression of fibrosis-related indicators (COL1A1 and COL3A1) and inhibited the activation of the transforming growth factor-β1 (TGF-β1)/mothers against decapentaplegic homolog 3 (Smad3) pathway. Thus, AN improved TAC-induced cardiac remodeling. Moreover, AN downregulated p-dynamin-related protein (Drp1) (Ser616) expression and upregulated mitogen 2 (MFN-2) and optic atrophy 1 (OPA1) expression in vivo and in vitro, thereby restoring mitochondrial fusion and fission balance. In conclusion, AN improves cardiac remodeling by regulating mitochondrial dynamic balance, providing experimental data for the rational application of Chinese medicine prescriptions with AN as the main component in clinical practice.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Myocytes, Cardiac/metabolism* ; Mice ; Rats ; Male ; Mitochondrial Dynamics/drug effects* ; Ventricular Remodeling/drug effects* ; Astragalus Plant/chemistry* ; Mice, Inbred C57BL ; Rhizome/chemistry* ; Panax notoginseng/chemistry* ; Rats, Sprague-Dawley ; Natriuretic Peptide, Brain/genetics* ; Humans ; Angiotensin II ; Astragalus propinquus

Zishen Huoxue decoction (ZSHX) enhances cardiomyocyte viability following hypoxic stress; however, its upstream therapeutic targets remain unclear. Network pharmacology and RNA sequencing analyses revealed that ZSHX target genes were closely associated with mitophagy and apoptosis in the mitochondrial pathway. In vitro, ZSHX inhibited pathological mitochondrial fission following hypoxic stress, regulated FUN14 domain-containing protein 1 (FUNDC1)-related mitophagy, and increased the levels of mitophagy lysosomes and microtubule-associated protein 1 light chain 3 beta II (LC3II)/translocase of outer mitochondrial membrane 20 (TOM20) expression while inhibiting the over-activated mitochondrial unfolded protein response. Additionally, ZSHX regulated the stability of beta-tubulin through Sirtuin 5 (SIRT5) and could modulate FUNDC1-related synergistic mechanisms of mitophagy and unfolded protein response in the mitochondria (UPR^mt) via the SIRT5 and -β-tubulin axis. This targeting pathway may be crucial for cardiomyocytes to resist hypoxia. Collectively, these findings suggest that ZSHX can protect against cardiomyocyte injury via the SIRT5-β-tubulin axis, which may be associated with the synergistic protective mechanism of SIRT5-β-tubulin axis-related mitophagy and UPR^mt on cardiomyocytes.
Mitophagy/drug effects* ; Tubulin/genetics* ; Animals ; Myocytes, Cardiac/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Sirtuins/genetics* ; Unfolded Protein Response/drug effects* ; Myocardial Ischemia/genetics* ; Rats ; Humans ; Rats, Sprague-Dawley ; Apoptosis/drug effects* ; Male

OBJECTIVES: To investigate the mechanism through which N-acetylneuraminic acid (Neu5Ac) exacerbates hypoxia/reoxygenation (H/R) injury in rat cardiomyocytes (H9C2 cells). METHODS: H9C2 cells were cultured in hypoxia and glucose deprivation for 8 h followed by reoxygenation for different durations to determine the optimal reoxygenation time. Under the optimal H/R protocol, the cells were treated with 0, 5, 10, 20, 30, 40, 50, and 60 mmol/L Neu5Ac during reoxygenation to explore the optimal drug concentration. The cells were then subjected to H/R injury followed by treatment with Neu5Ac, Fer-1 (a ferroptosis inhibitor), or both. The changes in SOD activity, intracellular Fe²⁺ and lipid ROS levels in the cells were evaluated, and the cellular expressions of Nrf2, GPX4, HO-1, FSP1, and xCT proteins were detected using Western blotting. RESULTS: Following hypoxia and glucose deprivation for 8 h, the cells with reoxygenation for 6 h, as compared with other time lengths of reoxygenation except for 9 h, showed the lowest expression levels of Nrf2, GPX4, HO-1, and FSP1 proteins (P<0.001). Neu5Ac treatment of dose-dependently decreased the viability of the cells with H/R injury with an IC₅₀ of 30.07 mmol/L. Reoxygenation for 3 h with normal glucose supplementation and a Neu5Ac concentration of 30 mmol/L were selected as the optimal conditions in the subsequent experiments. The results showed that Neu5Ac could significantly increase SOD activity, Fe²⁺ and lipid ROS levels and reduce Nrf2, GPX4, HO-1, and FSP1 protein expressions in H9C2 cells with H/R injury, but its effects were significantly attenuated by treatment with Fer-1. CONCLUSIONS Neu5Ac exacerbates ferroptosis of myocardial cells with H/R injury by inhibiting the Nrf2 axis to promote the production of ROS and lipid ROS.
Ferroptosis/drug effects* ; Myocytes, Cardiac/cytology* ; Animals ; NF-E2-Related Factor 2/metabolism* ; Rats ; N-Acetylneuraminic Acid/pharmacology* ; Cell Hypoxia ; Reactive Oxygen Species/metabolism* ; Cell Line ; Myocardial Reperfusion Injury/metabolism*

OBJECTIVES: To explore the mechanism that mediate the therapeutic effect of quercetin on heart failure. METHODS: We searched the TCMSP and Swiss ADME databases for the therapeutic targets of quercetin and retrieved heart failure targets from the Genecards and OMIM databases. The intersecting targets were analyzed with GO and KEGG pathway analysis using DAVID database, and the key genes were identified via PPI analysis. Molecular docking between the core targets and quercetin was performed using PyMOL and AutoDock Tools. In a heart failure model established in H9C2 cardiomyocytes by treatment with isoproterenol, the effect of quercetin on the expressions of the MAPK signaling pathway was tested. RESULTS: A total of 60 intersecting targets were identified. Enrichment analysis revealed that quercetin may inhibit heart failure through the MAPK signaling pathway. The core genes, including AMPK3 and BCL-2, were identified as potential key regulators in quercetin-mediated improvement of heart failure. Cellular experiments demonstrated that quercetin significantly reduced isoproterenol-induced apoptosis of cardiomyocytes in a dose-dependent manner and obviously decreased the Bax/Bcl-2 ratio and the expression levels of caspase-3, ERK and p38 in the cells. CONCLUSIONS Quercetin improves heart failure possibly by inhibiting cardiomyocyte apoptosis through the MAPK signaling pathway.
Quercetin/pharmacology* ; Myocytes, Cardiac/drug effects* ; Heart Failure/metabolism* ; Apoptosis/drug effects* ; MAP Kinase Signaling System/drug effects* ; Rats ; Animals ; Isoproterenol

OBJECTIVES: To investigate the effects of quercetin on cuproptosis and L-type calcium currents in the myocardium of diabetic rats. METHODS: Forty SD rats were randomized into control group and diabetic model groups. The rat models of diabetes mellitus (DM) induced by high-fat and high-sugar diet combined with streptozotocin (STZ) injection were further divided into DM model group, quercetin treatment group, and empagliflozin treatment group (n=10). Blood glucose and body weight were measured every other week, and cardiac function of the rats was evaluated using echocardiography. HE staining, Sirius red staining, and wheat germ agglutinin (WGA) analysis were used to observe the changes in myocardial histomorphology, and serum copper levels and myocardial FDX1 expression were detected. In cultured rat cardiomyocyte H9c2 cells with high-glucose exposure, the effects of quercetin and elesclomol, alone or in combination, on intracellular CK-MB and LDH levels and FDX1 expression were assessed, and the changes in L-type calcium currents were analyzed using patch-clamp technique. RESULTS: The diabetic rats exhibited elevated blood glucose, reduced body weight, impaired left ventricular function, increased serum copper levels and myocardial FDX1 expression, decreased L-type calcium currents, and prolonged action potential duration. Quercetin and empagliflozin treatment significantly lowered blood glucose, improved body weight, and restored cardiac function of the diabetic rats, and compared with empagliflozin, quercetin more effectively reduced serum copper levels, downregulated FDX1 expression, and enhanced myocardial L-type calcium currents in diabetic rats. In H9c2 cells, high glucose exposure significantly increased myocardial expressions of FDX1, CK-MB and LDH, which were effectively lowered by quercetin treatment; Elesclomol further elevated FDX1, CK-MB and LDH levels in the exposed cells, and these changes were not significantly affected by the application of quercetin. CONCLUSIONS Quercetin ameliorates myocardial injury in diabetic rats possibly by suppressing myocardial cuproptosis signaling and restoring L-type calcium channel activity.
Animals ; Quercetin/pharmacology* ; Calcium Channels, L-Type/metabolism* ; Diabetes Mellitus, Experimental/metabolism* ; Rats, Sprague-Dawley ; Rats ; Myocytes, Cardiac/drug effects* ; Myocardium/pathology* ; Male