Shenqi Jiangtang Granules attenuate ferroptosis in diabetic cardiomyopathy via the dihydroorotate dehydrogenase-coenzyme Q pathway

Abstract

BACKGROUND

Shenqi Jiangtang Granules (SQJT), a traditional Chinese patent medicine, have long been prescribed for type II diabetes mellitus. Increasing evidence suggests that SQJT has cardioprotective effects; however, its mechanisms in diabetic cardiomyopathy (DCM), particularly regarding ferroptosis, remain unclear. We hypothesized that SQJT protects against DCM by inhibiting ferroptosis via the dihydroorotate dehydrogenase (DHODH)/coenzyme Q (CoQ) signaling pathway.

AIM

To elucidate the mechanism through which SQJT attenuates ferroptosis in DCM.

METHODS

Active constituents of SQJT were obtained from previous mass spectrometry analyses. Potential targets were predicted using network pharmacology, protein-protein interaction, molecular docking, and dynamics simulation. Cell viability, death, and ferroptosis markers were assessed in high glucose/lipid-induced H9C2 cells. Intracellular iron, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), and reactive oxygen species (ROS) levels were measured, and mitochondrial morphology was examined by MitoTracker staining. The expression of DHODH/CoQ pathway components was analyzed by reverse transcription-quantitative PCR and Western blotting, and pathway dependence was confirmed using the DHODH inhibitor brequinar (BRQ).

RESULTS

The SQJT-DCM-ferroptosis network identified 16 intersecting targets, with DHODH as the primary candidate. SQJT significantly improved cell viability and attenuated ferroptosis. Specifically, treatment with 6% SQJT reduced intracellular free iron levels by 61.8% and lipid peroxidation (MDA) levels by 55.9% (all P < 0.001). Furthermore, SQJT restored antioxidant enzyme activities (SOD and GSH-PX) by over 100%, while maintaining mitochondrial membrane integrity and reducing the proportion of ROS-positive cells from 51.6% to 11.8%. These protective effects, mediated via the DHODH/CoQ signaling axis, were significantly abolished by BRQ.

CONCLUSION

SQJT alleviates ferroptosis and oxidative stress, potentially through modulation of the DHODH/CoQ pathway, while preserving mitochondrial integrity and enhancing cardiomyocyte survival, suggesting its therapeutic potential in DCM. However, the mechanistic findings were based on pharmacological inhibition rather than genetic validation. Therefore, further genetic and in vivo studies are needed.

Key Words: Shenqi Jiangtang Granules; Diabetic cardiomyopathy; Ferroptosis; DHODH/CoQ pathway; Oxidative stress

Core Tip: This study investigates the mechanisms of Shenqi Jiangtang Granules (SQJT) against diabetic cardiomyopathy (DCM). By integrating network pharmacology and experimental validation, we identified dihydroorotate dehydrogenase (DHODH) as a key therapeutic target. We demonstrate that SQJT mitigates ferroptosis and mitochondrial injury in H9C2 cardiomyocytes by activating the DHODH/coenzyme Q axis. These protective effects are significantly abolished by DHODH inhibition. This research provides mechanistic evidence that SQJT exerts cardioprotective effects through ferroptosis regulation and highlights its potential as a therapeutic strategy for mitochondrial dysfunction in DCM.

INTRODUCTION

Diabetic cardiomyopathy (DCM) is a major complication of diabetes mellitus (DM), characterized by structural and functional abnormalities of the myocardium that occur independently of hypertension, coronary artery disease, or other cardiac pathologies[1]. Clinically, DCM markedly increases morbidity and mortality in diabetic patients, posing substantial challenges to public health worldwide[2]. At the molecular level, accumulating evidence indicates that mitochondrial dysfunction, oxidative stress, and abnormal lipid metabolism are key pathogenic mechanisms driving DCM[3]. As central regulators of cellular bioenergetics and redox homeostasis, mitochondria are profoundly impaired under diabetic conditions, leading to excessive reactive oxygen species (ROS) production and lipid peroxidation. Notably, ferroptosis—an iron-dependent form of regulated cell death driven by lipid peroxidation—has recently attracted considerable attention as a key pathogenic process contributing to myocardial injury and cardiac dysfunction in DCM[4]. Distinct from apoptosis, necroptosis, and autophagy, ferroptosis is primarily mediated by iron-catalyzed accumulation of lipid peroxides, highlighting its potential as a promising therapeutic target for DCM[5].

Emerging evidence highlights a critical interplay between mitochondrial dysfunction and ferroptosis in the pathogenesis of DCM. Under diabetic conditions, dysregulated mitochondrial metabolism causes abnormal mitochondrial iron accumulation and impaired antioxidant defenses, thereby driving excessive lipid peroxidation and subsequent ferroptosis activation in cardiomyocytes[6]. Recent studies underscore the importance of mitochondrial dihydroorotate dehydrogenase (DHODH)/coenzyme Q (CoQ) redox cycling in protecting against ferroptosis. DHODH is located on the inner mitochondrial membrane and catalyzes the reduction of ubiquinone to ubiquinol, thereby neutralizing lipid peroxidation and preserving mitochondrial integrity and function[7]. In addition, studies have found that ferroptosis plays a crucial role in the development and progression of cardiomyopathy[8]. Given the central role of mitochondrial ferroptosis in DCM pathogenesis, targeting this pathway represents a promising therapeutic strategy to alleviate cardiac dysfunction and improve clinical outcomes.

Traditional Chinese medicine (TCM), known for its holistic, multi-targeted therapeutic approach, holds considerable promise in addressing multifactorial disorders such as DCM. Shenqi Jiangtang Granules (SQJT; state medical license No. Z10950075), a TCM prescription, were approved by the National Medical Products Administration (NMPA) of China in 2019 for the treatment of type II diabetes[9,10]. Specifically, SQJT is composed of 11 Chinese medicinal herbs: Panax ginseng C.A.Mey., Astragalus mongholicus Bunge, Rehmannia glutinosa (Gaertn.) Libosch. ex DC., Ophiopogon japonicus (Thunb.) Ker Gawl., Schisandra chinensis (Turcz.) Baill., Dioscorea oppositifolia L., Rubus chingii Hu, Wolfiporia extensa (Peck) Ginns, Trichosanthes kirilowii Maxim., Alisma plantago-aquatica subsp. orientale (Sam.) Sam., and Lycium barbarum L. All plant names were provided in full botanical form (refer to www.theplantlist.org) and were verified in 2024 using the Medicinal Plant Names Services database (http://mpns.kew.org) and Index Fungorum (https://www.indexfungorum.org/). Pharmacological studies have shown that SQJT exert “overall regulation” and “receptor modulation” effects, concepts rooted in TCM pharmacology, by modulating the epinephrine-induced hyperglycemic response, protecting pancreatic β-cells, and enhancing insulin receptor function. In experimental studies, SQJT demonstrated significant hypoglycemic effects in streptozotocin-induced diabetic animal models, with potential mechanisms involving improved islet cell function and stimulation of insulin secretion[11]. Strengthened antioxidant defenses in high glucose-injured Schwann cells, including increased total antioxidant capacity and superoxide dismutase (SOD) activity, along with reduced levels of ROS and malondialdehyde (MDA)[11]. Our previous studies demonstrated that SQJT significantly reduced serum glucose levels in rat models of type II DM (T2DM), improved glucose tolerance, and restored circulating insulin levels. SQJT treatment also attenuated oxidative stress and iron overload in pancreatic tissues, thereby alleviating histopathological alterations. These effects may be mediated by inhibition of oxidative stress and ferroptosis signaling, ultimately protecting pancreatic β-cells[12]. Moreover, serum containing SQJT-derived constituents enhanced the viability of rat cardiomyoblast H9C2 cells, improved cellular morphology, reduced apoptosis, and reinforced the antioxidant defenses of cardiomyocytes[13]. However, despite accumulating experimental evidence supporting the therapeutic benefits of SQJT, their precise molecular mechanisms remain to be fully elucidated.

Network pharmacology and computational biology have emerged as powerful approaches for systematically elucidating the multi-component, multi-target mechanisms of TCM formulations[14,15]. Integrating network pharmacology with molecular docking and molecular dynamics (MD) simulations enables the prediction and validation of molecular interactions between bioactive TCM compounds and specific disease targets.

Moreover, this interdisciplinary strategy, in combination with experimental validation, facilitates the systematic elucidation of the pharmacological basis of SQJT, enabling the identification of key active constituents and their molecular targets within ferroptosis-related pathways, thereby providing a rationale for their application in DCM. Accordingly, this study was designed to investigate the protective effects and underlying mechanisms of SQJT against ferroptosis in DCM, with particular emphasis on the DHODH/CoQ pathway. By integrating network pharmacology, molecular docking, MD simulations, and experimental validation, we aimed to elucidate how SQJT regulates ferroptosis and mitochondrial antioxidant defense, thereby providing mechanistic insights into its therapeutic potential for DCM.

MATERIALS AND METHODS

Screening of Active Constituents of SQJT

The major chemical constituents of the 11 herbal medicines comprising SQJT were collected from the TCMSP database (http://ibts.hkbu.edu.hk/LSP/tcmsp.php)[16], the BATMAN-TCM database (http://bionet.ncpsb.org/batman-tcm)[17], and previously published mass spectrometry analyses[18]. Candidate compounds were screened according to absorption, distribution, metabolism, and excretion parameters, using the criteria of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. The Chemical Abstracts Service numbers of the selected compounds were then used to retrieve their Simplified Molecular Input Line Entry System (SMILES) data from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/search/)[19].

Reverse prediction of potential targets of active constituents

The SwissTargetPrediction server (http://www.swisstargetprediction.ch/)[20] was used to predict the molecular targets of bioactive compounds through a reverse molecular docking approach. This method integrates two-dimensional (2D) and three-dimensional (3D) structural similarity analyses with known ligands and supports predictions across five species. In this study, the species was set to Homo sapiens, and the SMILES data obtained in the previous step were submitted to generate the predicted potential targets of the selected constituents.

Identification of DCM and ferroptosisrelated target genes

To identify genes associated with DCM and ferroptosis, data were systematically retrieved from multiple reputable databases. The GeneCards database (http://www.genecards.org/)[21] is a comprehensive, gene-centered resource that automatically integrates genomic, transcriptomic, proteomic, genetic, clinical, and functional information from 125 online sources. Subsequently, DCM-associated genes were retrieved by querying GeneCards with the keyword “Diabetic cardiomyopathy”. Genes with a relevance score greater than or equal to the median were designated as candidate disease-associated target genes. OMIM is a comprehensive resource that documents genetic conditions and their associated genes, offering detailed insights into disease characteristics, clinical manifestations, inheritance patterns, and gene functions. For ferroptosis-related targets, we sourced data from FerrDb (http://www.zhounan.org/ferrdb/), a specialized repository that focuses on genes and proteins implicated in iron metabolism and ferroptosis[22]. FerrDb provides extensive information on iron-related genes, including their biological roles, expression profiles, and links to disease processes. Finally, we used Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/) to integrate and visualize the overlap between DCM-associated genes and ferroptosis-related targets. This approach allowed us to pinpoint shared genes that are potentially central to the interplay between DCM and ferroptosis.

Construction and analysis of a protein-protein interaction network

Drug component-target relationships were derived from the active constituent data and reverse target prediction results, and a component-target network was constructed using Cytoscape 3.7.1 (http://www.cytoscape.org/). Overlapping targets between compounds and disease were identified with a Venn diagram and subsequently imported into STRING (http://string-db.org/), a database for known and predicted protein-protein interactions (PPIs). The “Multiple proteins” option was selected, the species was restricted to Homo sapiens, and the minimum required interaction score was set at 0.40. The resulting PPI data were exported in TSV format, and node1, node2, and combined score information were imported into Cytoscape 3.7.1 to construct the PPI network. Topological analysis was then performed, mapping node degree values to target size and color to establish the final PPI network.

Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis

Taking the intersection of SQJT targets, DCM targets, and ferroptosis targets, we identified the key targets through which SQJT modulates ferroptosis in the context of DCM. To explore the biological roles and pathways of the identified potential targets involved in the action of SQJT against DCM and ferroptosis, official gene symbols were converted into corresponding Entrez gene IDs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were then conducted using R software (version 4.4.3) and several supporting R packages, including clusterProfiler, DOSE, org.Hs.eg.db, ggplot2, enrichplot, pathview, colorspace, stringi, and BiocManager. Enrichment results were filtered by significance threshold and only GO terms and KEGG pathways with P values < 0.05 were included in the final interpretation.

Analysis of binding capacity between active ingredients and key target genes by molecular docking

The 2D structures of small-molecule ligands were retrieved from the PubChem database (http://pubchem.ncbi.nlm.nih.gov/) and converted into 3D structures using ChemOffice, then saved in mol2 format. High-resolution crystal structures of protein targets were obtained from the RCSB PDB database (http://www.rcsb.org/) and used as docking receptors. Protein structures were preprocessed with PyMOL by removing water molecules and phosphate groups and saved as PDB files. Molecular docking was conducted with AutoDock Vina 1.5.6 to evaluate protein-ligand interactions. Prior to docking, proteins were prepared by adding hydrogen atoms and removing water molecules, while ligands were prepared by adding hydrogens and defining torsional degrees of freedom. The docking grid box was defined, and the best binding conformations were identified based on docking scores. PyMOL and Discovery Studio 2019 were then used to visualize the interactions between compounds and key residues in both 2D and 3D formats.

MD simulation

MD simulations were performed using Gromacs 2022. Force field parameters were generated with the pdb2 gmx tool of Gromacs and the AutoFF web server. The CHARMM36 force field[23] was applied to receptor proteins, while ligand parameters were assigned using the CGenFF force field. The system was solvated in a cubic TIP3P water box with a 1.0 nm buffer, and ions were added using the gmx genion tool to neutralize the system. Long-range electrostatic interactions were calculated with the Particle Mesh Ewald method using a 1.0 nm cutoff. All covalent bonds were constrained with the SHAKE algorithm, and the Verlet leapfrog algorithm was applied with a 1 fs integration step.

Before the production run, the system underwent energy minimization consisting of 3000 steps of steepest descent followed by 2000 steps of conjugate gradient minimization. The minimization protocol was conducted in three stages: (1) Constraining solutes while minimizing water molecules; (2) Constraining counterions during minimization; and (3) Minimizing the entire system without constraints.

MD simulations were then performed under NPT ensemble conditions at 310 K and 1 atm for 100 ns. Structural stability and dynamic properties were analyzed using g-rmsd, g-rmsf, g-hbond, g-Rg, and g-sasa to calculate root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonds (H-bonds), radius of gyration (Rg), and solvent-accessible surface area (SASA), respectively.

Reagents and antibodies

SQJT (NMPA Approval No. Z10950075; Batch No. 122003A4) were obtained from Lunan Houpu Pharmaceutical Group Co., Ltd. (Shandong, China).

SQJT is composed of 11 Chinese medicinal herbs: Panax ginseng C.A.Mey. (0.71%), Astragalus mongholicus Bunge (14.71%), Rehmannia glutinosa (Gaertn.) Libosch. ex DC. (22.06%), Ophiopogon japonicus (Thunb.) Ker Gawl. (7.35%), Schisandra chinensis (Turcz.) Baill. (7.35%), Dioscorea oppositifolia L. (7.35%), Rubus chingii Hu (3.7%), Wolfiporia extensa (Peck) Ginns (7.35%), Trichosanthes kirilowii Maxim. (7.35%), Alisma plantago-aquatica subsp. orientale (Sam.) Sam. (7.35%), and Lycium barbarum L. (14.71%).

Panax ginseng, Radix (Panax ginseng C.A. Mey.), 6 g; Astragalus mongholicus, Radix (Astragalus mongholicus Bunge), 124 g; Rehmannia glutinosa, Radix [Rehmannia glutinosa (Gaertn.) Libosch. ex DC.], 186 g; Ophiopogon japonicus, Radix [Ophiopogon japonicus (Thunb.) Ker Gawl.], 62 g; Schisandra chinensis, Fructus [Schisandra chinensis (Turcz.) Baill.], 62 g; Dioscorea oppositifolia, Rhizoma (Dioscorea oppositifolia L.), 62 g; Rubus chingii, Fructus (Rubus chingii Hu), 31 g; Wolfiporia extensa, Sclerotium [Wolfiporia extensa (Peck) Ginns], 62 g; Trichosanthes kirilowii, Radix (Trichosanthes kirilowii Maxim.), 62 g; Alisma plantago-aquatica subsp. orientale, Rhizoma [Alisma plantago-aquatica subsp. orientale (Sam.) Sam.], 62 g; and Lycium barbarum, Fructus (Lycium barbarum L.), 124 g were soaked in 60% ethanol for 1 hour and extracted twice by refluxing for 2 hours. The condensed extracts were mixed with dextrin and sugar powder to make SQJT.

To ensure batch-specific reproducibility and quality control, we employed thin-layer chromatography fingerprinting and marker analysis to detect and identify ginsenosides (Re, Rg1, Rb1) and schisandrin B (Supplementary Figures 1 and 2). In parallel, reversed-phase high performance liquid chromatography was used to obtain a ginsenoside chromatographic fingerprint of SQJT, and peak identities were confirmed by retention-time matching (Supplementary Figures 3 and 4). In addition, previously published ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry and gas chromatography/mass spectrometry profiles of SQJT were consulted to provide chemical context (Supplementary Table 1).

The DHODH inhibitor brequinar (BRQ; also known as DUP-785; Cat No. HY-10419) was purchased from MedChemExpress (Monmouth Junction, NJ, United States). Cell Counting Kit-8 (CK04-500T) was obtained from Dojindo Molecular Technologies, Inc. (Shanghai, China). The mitochondrial ROS probe (S0035S) was obtained from Beyotime Biotechnology (Shanghai, China), and MitoTracker^TM Red CMXRos (A66444) was purchased from Thermo Fisher Scientific (Waltham, MA, United States).

Assay kits, including glutathione peroxidase (GSH-Px; Cat No. A005-1-2), reduced glutathione (GSH; Cat No. A006-2-1), MDA (Cat No. A003-1-2), SOD (Cat No. A001-3-2), tissue iron (Cat No. A039-2-1), and BCA protein (Cat No. A045-4-2), were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Primary antibodies against GAPDH (Cat No. 60004-1-Ig), ACSL4 (Cat No. 22401-1-AP), GPX4 (Cat No. 67763-1-Ig), DHODH (Cat No. 14877-1-AP), and COQ10A (Cat No. 17812-1-AP) were obtained from Proteintech (Wuhan, China) and used at a dilution of 1:1000. HRP-conjugated secondary antibodies were purchased from Pulilai Biotechnology (China). TRIzol^TM reagent (Cat No. 15596026) was obtained from Invitrogen Life Technologies (Carlsbad, CA, United States).

Animals, drug administration, and grouping design

Male specific pathogen-free (SPF) Sprague-Dawley rats were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). A total of 32 eight-week-old rats (300-350 g) were acclimatized for 1 week in the SPF-grade Animal Experimental Center of Beijing University of Chinese Medicine. Environmental conditions were maintained at 22 ± 2 °C, relative humidity of 50%-65%, and a 12-hour light/dark cycle. Bedding and feed were changed daily by trained staff, and animals were provided with standard chow and water ad libitum.

Rats were randomly assigned to two groups: Control and SQJT-containing serum. The SQJT group received intragastric gavage of SQJT (3.6 g/kg) once daily for 7 consecutive days. Control rats received an equal volume of vehicle (distilled water) by oral gavage on the same schedule. One hour after the final gavage on day 7, rats were anesthetized and whole blood was collected from the abdominal aorta. Samples were incubated at room temperature for 2 hours and centrifuged at 3000 rpm for 15 minutes to obtain serum.

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and approved by the Medical and Laboratory Animal Ethics Committee of Beijing Institute of Traditional Chinese Medicine (Approval No. BUCM-2023020604-1150).

Cell experiments and grouping

Rat cardiomyoblast H9C2 cells (passages 5-12) were obtained from the Cell Resource Center, Peking Union Medical College (Beijing, China). To mimic the diabetic milieu in vitro, cells were incubated for 24 hours in a high glucose-high lipid medium containing 25 mmol/L glucose and 0.45 mmol/L palmitic acid (PA). Graded concentrations of SQJT-containing serum were then administered to evaluate its effects on H9C2 cell proliferation under low-glucose (2.5 mmol/L), low-lipid, and high glucose-high lipid conditions.

To further investigate the mechanism of SQJT-containing serum, the DHODH inhibitor BRQ was applied. H9C2 cells exposed to high glucose-high lipid conditions were randomly divided into the following groups: Control (low-glucose medium supplemented with 10% blank serum, serving as the vehicle control), model group (HGHL medium + 10% blank serum), graded-concentration SQJT groups (HGHL medium + 2%, 4%, and 6% SQJT, with the balance being blank serum to maintain a total of 10% serum). All groups were incubated for 24 hours under the specified conditions.

CCK8 assay

After incubation, the culture medium in each well was replaced with fresh medium containing 10% CCK-8 reagent, and cells were incubated for 1 hour in the dark. Absorbance was then measured at 450 nm using a microplate reader. Cell viability was calculated according to the manufacturer's protocol, and the glucose-lipid concentration that produced a half-maximal inhibitory effect on viability was determined.

Flow cytometry - cell death assay and ROS measurement

All cells, including floating dead cells in the supernatant, were collected and stained with propidium iodide (PI; 5 μg/mL). The proportion of PI-positive cells was quantified using a BD Accuri C6 flow cytometer (BD Biosciences) via the FL2 channel. For intracellular ROS measurement, cells were stained with the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime) and incubated at 37°C for 30 minutes. After washing with PBS, fluorescence intensity was quantified through the FL1 (FITC) channel to determine intracellular ROS levels.

Detection of iron ions, MDA, SOD, GSH-PX, and GSH using commercial assay kits

Proteins were extracted from each cell group according to the manufacturer’s instructions, and concentrations were measured using a BCA protein assay kit. The levels of iron ions, MDA, SOD, GSH-PX, and GSH were subsequently measured using the corresponding commercial kits.

Mitochondrial morphology detection (MitoTracker staining with confocal microscopy)

H9C2 cells (1.5 × 10⁵) were seeded into confocal culture dishes and treated according to the experimental design. MitoTracker working solution (2 μL; final concentration, 1 μM) was added to 2 mL culture medium per well, and cells were incubated at 37 °C in the dark for 30 minutes. After staining, cells were gently washed three times with PBS and immediately examined using a confocal laser scanning microscope. Fluorescence images were acquired with Zeiss ZEN 3.3.7 software.

Reverse transcription-quantitative PCR

Total RNA was extracted from H9C2 cells using TRIzol^TM reagent (Invitrogen, United States), and RNA concentration and purity were assessed with a NanoDrop spectrophotometer (Thermo Fisher Scientific, United States). Complementary DNA was synthesized from RNA samples using the PrimeScript^TM RT Reagent Kit (Takara, Japan).

Reverse transcription-quantitative PCR (RT-qPCR) was performed on an Applied Biosystems 7500 system (Thermo Fisher Scientific, United States) with PowerUp^TM SYBR^TM Green Master Mix (Thermo Fisher Scientific, United States). GAPDH was used as the internal control for normalization, and relative gene expression was calculated using the 2^-ΔΔCT method. Primer sequences are listed in Supplementary Table 2.

Western blotting

Cells were lysed on ice for 30 minutes in precooled lysis buffer (RIPA:PMSF:phosphatase inhibitor = 100:1:1). After centrifugation, the supernatant was collected. Protein concentrations were determined using a BCA assay, and samples were denatured for subsequent Western blot analysis. Following SDS-PAGE and transfer to membranes, membranes were blocked and incubated overnight at 4 °C with primary antibodies. The following day, membranes were incubated with appropriate secondary antibodies, and protein bands were visualized. Protein expression levels were quantified with ImageJ software by measuring band intensities and normalized to the loading control, expressed as the ratio of target protein to loading control intensity.

Statistical analysis

All statistical analyses were performed using SPSS 25.0 software. Data are expressed as mean ± SD. Each experiment was independently repeated at least three times (n = 3 biological replicates per group) unless otherwise stated. Normality was first assessed, and normally distributed variables were further tested for homogeneity of variance using Levene’s test. For data with homogeneous variances, one-way analysis of variance (ANOVA) was conducted, followed by least significant difference or Scheffé post hoc tests. For data with heterogeneous variances, Dunnett’s T3 test was applied. For non-normally distributed data, the Kruskal-Wallis rank-sum test was used. Differences were considered statistically significant at P < 0.05 and highly significant at P < 0.01.

RESULTS

According to the drug instructions and the YBZ14962009 standard issued by the China NMPA, SQJT consists of 11 traditional Chinese medicinal herbs, with detailed information presented in Table 1. Based on database searches and screening criteria of OB (≥ 30%) and DL (≥ 0.18), the following numbers of chemical constituents were identified: Astragalus mongholicus Bunge (17), Schisandra chinensis (Turcz.) Baill. (7), Dioscorea oppositifolia L. (8), Rehmannia glutinosa (Gaertn.) Libosch. ex DC. (20), Rubus chingii Hu (7), Ophiopogon japonicus (Thunb.) Ker Gawl. (5), Wolfiporia extensa (Peck) Ginns (15), Trichosanthes kirilowii Maxim. (2), Alisma plantago-aquatica subsp. orientale (Sam.) Sam. (5), and Lycium barbarum L. (22). In SQJT granules, ginseng stem and leaf saponins were used in place of ginseng root, and according to previous mass spectrometry results, 12 prototype blood-absorbed constituents were identified, including ginsenosides Rg1, Rg3, Rg2, Rb1, Rb2, Rh1, Rh2, Rc, Rf, Re, F2, and pseudoginsenoside F11.

Table 1 Components in Shenqi Jiangtang Granules.

Botanical name	Herbal name	Chinese name
Panax ginseng C.A.Mey.	Ginseng stem-leaf saponin	Renshenjingyezaogan
Schisandra chinensis (Turcz.) Baill.	Schisandra	Wuweizi
Astragalus mongholicus Bunge	Astragalus root	Huangqi
Dioscorea oppositifolia L.	Chinese yam	Shanyao
Rehmannia glutinosa (Gaertn.) Libosch. ex DC.	Rehmannia root	Dihuang
Rubus chingii Hu	Red raspberry	Fenpenzi
Ophiopogon japonicus (Thunb.) Ker Gawl.	Dwarf lilyturf tuber	Maidong
Wolfiporia extensa (Peck) Ginns	Indian bread	Fuling
Trichosanthes kirilowii Maxim.	Snake gourd root	Tianhuafen
Alisma plantago-aquatica subsp. orientale (Sam.) Sam.	Water plantain tuber	Zexie
Lycium barbarum L.	Goji berry	Gouqizi

Virtual screening via the SwissTargetPrediction server predicted 292 target genes, all annotated using standardized UniProt nomenclature, resulting in the identification of 949 targets and 6510 compound-target interactions (Supplementary Table 3).

Construction and analysis of the SQJT-ferroptosis-DCM target gene network

A GeneCards database search using the keyword “diabetic cardiomyopathy” identified 1738 related genes, and the OMIM database yielded an additional 514 genes. In total, 2187 candidate disease-associated target genes were integrated. Furthermore, 564 ferroptosis-related targets were retrieved from the FerrDb database.

To investigate the therapeutic mechanisms of SQJT in DCM through ferroptosis modulation, we constructed a comprehensive target network integrating SQJT-related targets, DCM-associated targets, and ferroptosis-specific genes. A Venn diagram revealed 16 shared targets across the three datasets: HRAS, PPARG, SLC16A1, VCP, PHKG2, PIK3CA, TERT, TP53, G6PD, AR, DHODH, IDH1, TGFB1, SNCA, TBK1, and IL6 (Figure 1A). These intersection targets represent critical regulatory nodes through which SQJT may exert therapeutic effects by modulating ferroptosis pathways implicated in DCM.

Figure 1 Shenqi Jiangtang Granules-ferroptosis-diabetic cardiomyopathy target network construction and protein-protein interaction analysis. A: Venn diagram of Shenqi Jiangtang Granules (SQJT), diabetic cardiomyopathy, and ferroptosis-related targets showing 16 overlapping genes; B: Protein-protein interaction network of ferroptosis-associated targets; C: Compound-target network of SQJT illustrating multi-component, multi-target interactions. SQJT: Shenqi Jiangtang Granules; DCM: Diabetic cardiomyopathy.

A PPI network was constructed to visualize the functional connectivity of these targets, with a particular focus on ferroptosis-related genes (Figure 1B). In addition, the active ingredient-target network illustrated direct interactions between SQJT components and therapeutic targets, highlighting its multi-component, multi-target pharmacological profile (Figure 1C). Collectively, these analyses suggest that SQJT may alleviate DCM by selectively modulating key ferroptosis-related nodes, providing novel insights into its mechanisms of action.

GO and KEGG pathway enrichment analysis

To further elucidate the biological roles of the intersection targets shared by SQJT, ferroptosis, and DCM, we performed GO and KEGG enrichment analyses. GO analysis indicated enrichment in biological processes, such as the positive regulation of miRNA metabolic processes, regulation of neuronal apoptosis, and energy derivation from the oxidation of organic compounds. Enriched cellular components included the transferase complex, secretory granule lumen, and cytoplasmic vesicle lumen, while enriched molecular functions involved transcription coregulator binding, protein phosphatase binding, and nuclear receptor activity (Figure 2A). These results suggest that SQJT may influence miRNA regulation, cellular metabolism, apoptosis, and transcriptional modulation in the context of DCM and ferroptosis.

Figure 2 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of intersection targets. A: Gene Ontology enrichment of overlapping targets, including biological processes, cellular components, and molecular functions; B: Kyoto Encyclopedia of Genes and Genomes enrichment pathways, highlighting ferroptosis-related signaling pathways such as AGE-RAGE, PI3K-Akt, AMPK, TNF, HIF-1, and apoptosis.

KEGG pathway analysis revealed several critical signaling cascades that could potentially mediate the therapeutic effects of SQJT, including the AGE-RAGE signaling pathway in diabetic complications, PI3K-Akt signaling, HIF-1 signaling, ferroptosis, AMPK signaling, TNF signaling, and apoptosis (Figure 2B). These pathways are closely linked to cell survival, metabolic regulation, inflammatory responses, and oxidative stress, underscoring their importance as potential therapeutic targets of SQJT in mitigating ferroptosis-induced injury in DCM.

Molecular docking

In general, a binding energy below -5.0 kcal/mol suggests good binding activity, whereas values lower than -7.0 kcal/mol indicate strong binding activity. A lower binding energy corresponds to stronger binding affinity and greater conformational stability. Among the SQJT active compounds docked with ferroptosis-related targets, formononetin showed the lowest binding energy with DHODH (-10.5 kcal/mol), substantially lower than -7.0 kcal/mol (Supplementary Table 4), indicating a strong binding affinity for DHODH.

At the DHODH receptor site, residues PRO52, LEU46, PHE98, LEU359, GLY363, TYR38, PHE62, PRO69, and PRO364 formed van der Waals interactions with formononetin; ALA59 contributed hydrophobic interactions; GLN47 and TYR356 formed H-bonds; MET43 and THR360 established π-σ interactions; HIS56 formed π-π stacking interactions; and ALA55 formed amide-π stacking interactions with formononetin (Figure 3A).

Figure 3 Molecular docking and molecular dynamics simulations of the formononetin- dihydroorotate dehydrogenase complex. A: Molecular docking showing the binding mode of formononetin with dihydroorotate dehydrogenase (DHODH; binding energy = -10.5 kcal/mol); B-G: Molecular dynamics simulation analyses of the formononetin-DHODH complex, including root mean square deviation, radius of gyration, solvent-accessible surface area, number of hydrogen bonds, root mean square fluctuation, and free energy landscape, indicating stable binding and conformational stability. RMSD: Root mean square deviation; Rg: Radius of gyration; SASA: Solvent-accessible surface area; RMSF: Root mean square fluctuation.

MD simulations

RMSD is a key indicator of conformational stability, reflecting deviations of atomic positions from their initial coordinates. Smaller deviations correspond to greater structural stability. Accordingly, we used RMSD to assess the equilibrium of the simulation system. As shown in Figure 3B, the complex reached equilibrium after 80 ns and subsequently fluctuated around 1.8 Å, indicating stable binding of the small molecule to the target protein.

The Rg characterizes overall structural changes and protein compactness, with larger fluctuations suggesting system expansion. During the simulation, the Rg of the complex showed only minor fluctuations before stabilizing, implying conformational adjustments of the small molecule-protein complex during the trajectory (Figure 3C). We calculated the SASA between the target protein and the small molecule, which reflects changes in protein surface exposure. SASA exhibited modest fluctuations (Figure 3D), indicating that small-molecule binding perturbed the local binding microenvironment and induced moderate SASA variations. Hydrogen bonding is critical for ligand-protein interactions. As shown in Figure 3E, the number of H-bonds between the small molecule and the target protein ranged from 0 to 3, with the complex typically maintaining approximately one hydrogen bond, indicating stable hydrogen-bond interactions.

RMSF describes the flexibility of amino acid residues. As shown in Figure 3F, the RMSF values of the complex were relatively low (mostly 0.5-2.1 Å), reflecting limited flexibility and high conformational stability.

The free energy landscape, calculated based on RMSD and Rg values, depicted the distribution of conformational free energy during the simulation. The color gradient represents energy levels from high (red) to low (blue), illustrating the dynamic conformational process of the complex (Figure 3G).

We further estimated binding free energy using the Molecular Mechanics/Poisson-Boltzmann Surface Area method (Figure 4A). The calculated binding free energy of the complex was -14.72 kcal/mol, with the negative value indicating strong affinity and lower values corresponding to stronger binding. Thus, the complex exhibited high binding affinity.

Figure 4 Molecular Mechanics/Poisson-Boltzmann Surface Area binding free energy and residue decomposition analysis. A: Binding free energy decomposition of the formononetin-dihydroorotate dehydrogenase complex, showing major energy components; B: Per-residue free energy contribution analysis, with PHE62, PRO364, LEU42, ALA59, and LEU68 contributing most significantly to binding. MM/PBSA: Molecular Mechanics/Poisson-Boltzmann Surface Area.

Residue decomposition analysis revealed that PHE62, PRO364, LEU42, ALA59, and LEU68 contributed most significantly to binding (Figure 4B), suggesting these residues play critical roles in the binding process. Collectively, the stable binding conformation and low binding free energy support the hypothesis that the small molecule may exert its effects by inhibiting the target protein.

SQJT attenuates H9C2 cardiomyocyte injury and reduces cell death under glucolipotoxicity

We exposed H9C2 cardiomyocytes to a diabetic-like environment characterized by high glucose and lipid levels. With increasing lipid concentration and prolonged exposure time, cellular injury progressively worsened (Figure 5A and B). Based on these observations, we selected treatment with 25 mmol/L glucose and 0.45 mmol/L PA for 24 hours to establish the H9C2 injury model. Administration of increasing concentrations (2%, 4%, and 6%) of SQJT-containing serum significantly alleviated injury (Figure 5C). Notably, treatment with 6% SQJT-containing serum increased cell viability by approximately 73.5% compared with the model group (P < 0.001). The cardioprotective effect of 6% SQJT-containing serum was significantly attenuated by the DHODH inhibitor BRQ (50 μM), which abolished approximately 65.1% of the observed improvement in cell viability (Figure 5C, P < 0.001).

Figure 5 Shenqi Jiangtang Granules significantly attenuate H9C2 cell injury. A: CCK-8 assay showing the effects of high glucose combined with different concentrations of palmitic acid (PA) on H9C2 cells (0-0.5 mmol/L); B: Effects of high glucose combined with 0.45 mmol/L PA for 6-, 12-, and 24-hour on H9C2 cell injury; C: Dose-dependent protective effects of Shenqi Jiangtang Granules (SQJT)-containing serum against glucolipotoxicity-induced H9C2 cell injury. Low-, medium-, and high-dose groups correspond to 2%, 4%, and 6%, respectively; D and E: Flow cytometry analysis of propidium iodide-positive cells (n = 3). ^aP < 0.05 vs control; ^bP < 0.01 vs control; ^cP < 0.05 vs model group; ^dP < 0.01 vs model group; ^eP < 0.01 vs SQJT-high + brequinar; ^fP < 0.01 vs 6 hours control; ^gP < 0.01 vs 12 hours control; ^hP < 0.01 vs 24 hours control.

PI staining followed by flow cytometric analysis further confirmed the dose-dependent protective effect of SQJT-containing serum against cardiomyocyte death. Increasing concentrations of SQJT-containing serum progressively reduced cell death, with the 6% SQJT group showing a 57.5% reduction in the proportion of PI-positive cells relative to the model group (Figure 5D and E, P < 0.05). Furthermore, the protective effect was significantly attenuated by co-treatment with high-dose SQJT and BRQ, which abolished approximately 29.2% of the observed improvement in cell survival, but this effect did not reach statistical significance (Figure 5D and E).

SQJT reduces iron deposition and enhances antioxidant defenses in H9C2 cells under glucolipotoxicity

To evaluate whether the protective effects of SQJT on H9C2 cardiomyocyte injury involve the regulation of iron metabolism and antioxidant capacity, we assessed the relevant indicators using colorimetric assays.

We quantified intracellular free iron (Fe) by colorimetry (Figure 6A). Compared with the control group (mean = 0.911 ± 0.07), free Fe levels were markedly elevated in the model group (high glucose-high lipid, mean = 5.437 ± 0.17, P < 0.001). Treatment with 6% SQJT-containing serum significantly reduced intracellular Fe levels to 2.078 ± 0.09, representing a 61.8% reduction compared with the model group (Figure 6A, P < 0.001). Furthermore, this protective effect was significantly attenuated by co-treatment with the DHODH inhibitor BRQ (mean = 3.443 ± 0.10), which abolished approximately 40.6% of the observed improvement in iron homeostasis (P < 0.001). These results suggest that SQJT regulates iron homeostasis in a DHODH-dependent manner.

Figure 6 Shenqi Jiangtang Granules enhance antioxidant defense of H9C2 cells under glucolipotoxicity. A: Intracellular free iron quantified by colorimetric assay; B: Malondialdehyde (MDA), the end product of lipid peroxidation, measured using the thiobarbituric acid-MDA condensation assay; C: Superoxide dismutase activity quantified by the WST-1 method; D: Glutathione peroxidase activity quantified by colorimetric assay; E: Glutathione quantified by the 5,5′-dithiobis-(2-nitrobenzoic acid) colorimetric/spectrophotometric assay; F and G: Flow cytometric detection of reactive oxygen species-positive cells using fluorescent probes (n = 3). ^aP < 0.05 vs control; ^bP < 0.01 vs control; ^cP < 0.05 vs model group; ^dP < 0.01 vs model group; ^eP < 0.05 vs Shenqi Jiangtang Granules-high (SQJT-H) + brequinar (BRQ); ^fP < 0.01 vs SQJT-H + BRQ. Con: Control; Mod: Model; SQJT: Shenqi Jiangtang Granules; MDA: Malondialdehyde; SOD: Superoxide dismutase; GSH: Glutathione; GSH-Px: Glutathione peroxidase; ROS: Reactive oxygen species.

We measured MDA levels to assess lipid peroxidation (Figure 6B). Compared with the control group (mean = 0.997 ± 0.08), MDA levels were significantly elevated in the model group (mean = 4.596 ± 0.24, P < 0.001). Treatment with 6% SQJT-containing serum markedly reduced MDA levels to 2.028 ± 0.17, representing a 55.9% reduction relative to the model group (P < 0.001). This antioxidant effect was significantly reversed by BRQ (mean = 3.080 ± 0.05), which abolished approximately 41.0% of the observed improvement in MDA levels (P < 0.001). These findings indicate that SQJT suppresses lipid peroxidation through a DHODH-dependent mechanism.

We next evaluated antioxidant enzyme activities and GSH levels (Figure 6C-E). Compared with the control group, SOD, GSH-PX, and GSH levels were markedly reduced in the model group (P < 0.001). SQJT treatment significantly and dose-dependently restored all three parameters; notably, antioxidant enzyme activities (SOD and GSH-PX) increased by over 100% in the 6% SQJT group relative to the model group (P < 0.001). These beneficial effects were significantly abolished by BRQ, which reversed the observed improvement (P < 0.05). These findings indicate that SQJT alleviates glucolipotoxicity-induced oxidative stress by enhancing intracellular antioxidant defenses in a DHODH-dependent manner. Given that DHODH is a key mitochondrial inner membrane enzyme linking the electron transport chain (ETC) and NAD⁺/NADH balance, SQJT may exert its protective effects by modulating mitochondrial function via DHODH.

We further measured mitochondrial superoxide production using a mitochondrial superoxide probe kit followed by flow cytometry (Figure 6F and G). The proportion of ROS-positive cells was significantly higher in the model group compared with controls. SQJT treatment dose-dependently reduced ROS levels, whereas BRQ reversed this effect, confirming that the antioxidant activity of SQJT is mediated through DHODH and closely linked to the regulation of mitochondrial ROS production.

SQJT regulates the DHODH/CoQ pathway to improve mitochondrial morphology and modulate ferroptosis

RT-qPCR analysis showed that, compared with the control group, the model group had increased mRNA expression of ACSL4 and reduced expression of DHODH and GPX4 (P < 0.01). Treatment with SQJT-containing serum significantly reversed these changes, decreasing ACSL4 while increasing DHODH and GPX4 expression. Notably, these effects were attenuated by DHODH inhibition (Figure 7A).

Figure 7 Shenqi Jiangtang Granules-containing serum modulates the dihydroorotate dehydrogenase/coenzyme Q signaling pathway and mitochondrial morphology in H9C2 cells under glucolipotoxicity. A: Reverse transcription-quantitative PCR analysis of ferroptosis-related marker genes and dihydroorotate dehydrogenase (DHODH)/coenzyme Q10A pathway genes; B and C: Western blot analysis of ferroptosis-related marker proteins and DHODH/coenzyme Q pathway proteins, with densitometric quantification; D: Confocal microscopy of mitochondrial morphology following MitoTracker staining (n = 3). ^aP < 0.05 vs control; ^bP < 0.01 vs control; ^cP < 0.05 vs model group; ^dP < 0.01 vs model group; ^eP < 0.01 vs Shenqi Jiangtang Granules-high + brequinar. Con: Control; Mod: Model; SQJT: Shenqi Jiangtang Granules; SQJT-H: Shenqi Jiangtang Granules-high; BRQ: Brequinar.

Western blot analysis further corroborated the regulatory effect of SQJT on the DHODH/CoQ signaling axis (Figure 7B and C). Administration of 6% SQJT-containing serum increased DHODH and COQ10A protein expression while reducing ACSL4 levels. These effects were diminished after BRQ treatment, indicating that SQJT acts through a DHODH-dependent mechanism.

MitoTracker staining provided additional evidence for the mitochondrial protective effect of SQJT (Figure 7D). In the model group, mitochondria were markedly fragmented, displaying predominantly ring-shaped or punctate structures consistent with mitochondrial fission. SQJT-containing serum improved mitochondrial morphology, partially restoring elongated and irregularly branched structures. However, this effect was abolished by BRQ treatment, leading to renewed fragmentation and punctate distribution. Collectively, these findings indicate that the DHODH pathway plays a pivotal role in mediating the mitochondrial protective effects of SQJT.

DISCUSSION

Mechanistic insights into SQJT-mediated cardioprotection in DCM

DCM markedly increases the risk of heart failure, and strict glycemic control alone is insufficient to prevent disease progression. Persistent metabolic abnormalities in diabetes promote oxidative stress and inflammatory injury, which are important drivers of diabetic complications[24]. As terminally differentiated cells, cardiomyocytes have limited regenerative capacity and are highly susceptible to injury. Once cardiomyocytes are lost, irreversible myocardial damage and structural remodeling occur, accelerating the onset of heart failure.

In this study, SQJT treatment significantly improved H9C2 cardiomyocyte viability and reduced cell death after exposure to high glucose and lipid conditions. These cardioprotective effects were mechanistically associated with the DHODH/CoQ pathway. Previous studies on SQJT have primarily focused on its hypoglycemic effects, enhancement of antioxidant defenses, and alleviation of oxidative stress. Our findings provide novel insights into the molecular mechanisms by which SQJT exerts cardioprotective effects in DCM, particularly through the regulation of ferroptosis.

SQJT regulates iron metabolism and ferroptosis in DCM

Previous studies have reported elevated iron content and increased expression of ferroptosis markers in the hearts of diabetic patients, suggesting a potential role of ferroptosis in DCM pathogenesis[25]. Chronic hyperglycemia impairs fatty acid oxidation and reduces the tolerance of cardiomyocytes to iron overload, thereby triggering ferroptosis and accelerating DCM progression[8,25]. In addition, diabetic patients frequently present with elevated ferritin and hepcidin levels, indicative of systemic iron overload[26].

Consistent with these observations, our study demonstrated that intracellular iron levels were elevated in H9C2 cardiomyocytes exposed to high-glucose and lipid conditions. Consistently, ACSL4 expression was increased, whereas GPX4 expression was decreased. SQJT significantly reduced intracellular iron accumulation, downregulated ACSL4 expression, and restored GPX4 expression at both the mRNA and protein levels. These results suggest that SQJT may exert cardioprotective effects, at least in part, by inhibiting ferroptosis.

SQJT alleviates ferroptosis and mitochondrial dysfunction in DCM

Ferroptosis is a pathological cascade encompassing initiators (e.g., ROS), core features (lipid peroxidation and mitochondrial structural collapse), and key mechanisms (dysregulation of the oxidative stress-antioxidant defense system). ROS generation is central to this process. Under chronic hyperglycemia and lipotoxicity, cardiomyocytes undergo metabolic reprogramming that promotes excessive mitochondrial ROS production, increased activity of nicotinamide adenine dinucleotide phosphate oxidase, and impaired antioxidant defenses, collectively driving DCM development[3]. Oxidative stress plays a pivotal role in the pathogenesis of diabetic vascular complications, including cardiovascular diseases and nephropathy[24]. In myocardial tissue from patients with diabetic heart failure, GPX4 expression and activity are markedly reduced, accompanied by downregulation of ATF4 and NRF2, indicative of impaired antioxidant defenses and activation of ferroptotic pathways[25]. Furthermore, antioxidant defenses have been reported in diabetic myocardium, including reduced endogenous antioxidant capacity and NQO1-related protective signaling, further promoting lipid peroxide accumulation and ROS-mediated injury[27].

The heart is an organ with exceptionally high metabolic demands, relying heavily on mitochondria—which occupy nearly one-third of cardiomyocyte volume—for continuous ATP generation. Mitochondrial energy metabolism disorders are widely recognized as a central mechanism in DCM[28]. Diamant et al[29] reported impaired diastolic function in diabetic patients without hypertension, and 31P magnetic resonance spectroscopy demonstrated a significant reduction in the phosphocreatine-to-ATP ratio, suggesting that inadequate mitochondrial energy supply contributes to early cardiac dysfunction. Anderson et al[30] observed heightened sensitivity of mitochondria to calcium-induced mitochondrial permeability transition pore opening, along with increased hydrogen peroxide production in cardiomyocytes from diabetic patients, providing clinical evidence of mitochondria-dependent cell death in diabetes. Similarly, Croston et al[31] showed that subsarcolemmal mitochondria isolated from atrial appendages of T2DM patients had impaired respiration, with reduced activity and expression of ETC complexes I and IV. Collectively, these findings establish mitochondrial dysfunction as a consistent hallmark of DCM in both experimental models and clinical samples.

Consistent with this evidence, our study demonstrated that SQJT treatment attenuated high glucose and lipid exposure-induced ROS overproduction and improved mitochondrial morphology by reducing fragmentation and partially restoring elongated and irregularly branched structures. In H9C2 cardiomyocytes exposed to high glucose and lipid conditions, the oxidative stress marker MDA was elevated, whereas antioxidant defenses—including SOD, GSH-PX, and GSH—were reduced, reflecting compromised antioxidant capacity. SQJT treatment reversed these changes, downregulating oxidative stress markers and restoring antioxidant enzyme activities and GSH levels.

Taken together, these findings indicate that SQJT mitigates multiple pathological aspects of ferroptosis—including initiation, core features, and key mechanisms—thereby underscoring its therapeutic potential for the treatment of DCM.

SQJT modulates ferroptosis via the DHODH/CoQ axis

Although mitochondria are key regulators of ferroptosis, GPX4 knockout of in tumor cell lines induces ferroptosis without mitochondrial lipid peroxidation, suggesting that mitochondria may not be indispensable in GPX4-related ferroptotic pathways. Subsequent studies revealed that when mitochondrial GPX4 is inactivated, DHODH assumes a critical role in counteracting ferroptosis[7]. DHODH, a flavin-dependent enzyme located on the inner mitochondrial membrane, is essential for de novo pyrimidine synthesis[32]. Pyrimidines serve as fundamental precursors for DNA, RNA, phospholipids, and glycoproteins. DHODH catalyzes the conversion of dihydroorotate to orotate while reducing CoQ to CoQH₂[33], thereby participating in the CoQ redox cycle. Inhibition of DHODH impairs ETC activity and oxidative phosphorylation[34]. Within mitochondria, CoQ10 functions not only as an electron carrier between complexes I/II and III but also as a regulator of uncoupling protein metabolism and mitochondrial permeability transition pore opening, making it indispensable for mitochondrial function[35]. In addition, CoQ10 acts as an endogenous lipophilic antioxidant, suppressing oxidative stress by limiting ROS generation, particularly oxygen radicals[36]. On the outer surface of the inner mitochondrial membrane, DHODH contributes to the production of the radical-trapping antioxidant CoQ10, thereby preventing mitochondrial lipid peroxidation and inhibiting ferroptosis.

Accumulating evidence highlights a cardioprotective role of DHODH through ferroptosis inhibition and mitochondrial preservation. In heart failure models, DHODH downregulation aggravated mitochondrial dysfunction and cell death, whereas pharmacological activation of DHODH suppressed ferroptosis, improved ventricular remodeling, and enhanced cardiac function, underscoring its therapeutic potential[37]. In cardiac transplantation models under hypothermic preservation and reperfusion, loss or inhibition of DHODH compromised ferroptosis defense and impaired graft function, whereas maintenance of DHODH activity markedly enhanced hypothermic cardioprotection[38]. In DCM cardiomyocytes, elevated PACS2 expression has been shown to reduce DHODH activity, contributing to ferroptotic injury via the PACS2/CPT1A/DHODH axis, likely through modulation of mitochondrial function[39].

Consistent with these findings, our study demonstrated that high glucose-high lipid exposure suppressed DHODH expression in H9C2 cardiomyocytes, accompanied by reduced intracellular CoQ10 levels and mitochondrial dysfunction. SQJT treatment restored DHODH expression, elevated CoQ10 content, and alleviated mitochondrial injury and ferroptosis. These results reveal a novel mechanism by which SQJT modulates ferroptosis through the DHODH/CoQ axis in the context of DCM.

Moreover, growing evidence suggests that bioactive constituents of SQJT play pivotal roles in regulating oxidative stress, inhibiting ferroptosis, and preserving mitochondrial integrity. For instance, formononetin has been reported to exert cardioprotective effects in H9C2 cardiomyocytes by attenuating oxidative stress, supporting its potential as a representative candidate for further validation in our DCM-like in vitro setting[40]. Astragaloside IV attenuates myocardial dysfunction in DCM rats by downregulating CD36 expression, thereby inhibiting ferroptosis, preserving mitochondrial function, and improving cardiac performance[41]. AS-IV inhibited bleomycin-induced ferroptosis by reducing iron and lipid ROS accumulation, attenuating cellular senescence, and upregulating SLC7A11 and GPX4, while restoring mitochondrial morphology and limiting ferroptosis[42]. Astragalus polysaccharides protect mitochondrial ultrastructure in high glucose-stimulated H9C2 cells, reduce apoptosis, inhibit superoxide production, and alleviate oxidative DNA damage (8-OH-dG), while inducing SOD2 activity and expression[43]. Schisandrol B alleviates diabetic cardiac injury in mice by suppressing ferroptosis and ameliorating lipid metabolic disturbances, leading to reduced lipid peroxidation and enhanced cardiac function[44]. Kong et al[45] reported that ginsenoside Rg1 alleviated neuronal ferroptosis in chronic LPS-induced mice by activating Nrf2 signaling, upregulating GPX4, and inhibiting inflammasome activation. Ginsenoside Rb1 has been shown to reduce mitochondrial ROS, decrease infarct size, protect cardiac function, and attenuate fibrosis in ischemia/reperfusion models. Mechanistically, Rb1 binds to the ND3 subunit to inhibit NADH dehydrogenase activity, thereby trapping mitochondrial complex I in an inactive state during reperfusion and suppressing ROS bursts[46].

Findings from both in vivo and in vitro studies suggest that the bioactive constituents of SQJT provide a multi-layered defense to maintain mitochondrial integrity. Although these individual components have been demonstrated to alleviate DCM by mitigating oxidative stress, iron deposition, and ROS generation, their specific synergistic roles in regulating the DHODH/CoQ axis remain to be fully elucidated.

The therapeutic superiority of SQJT over single-target interventions likely arises from the synergy among its diverse bioactive constituents. Within the “component-target-effect” framework, various molecules (such as astragaloside IV and ginsenosides) function as a coordinated system. While they may interact with distinct upstream signaling nodes, their biological effects converge on the preservation of the DHODH/CoQ axis. This collective modulation ensures a more resilient defense against ferroptosis and mitochondrial dysfunction in the complex pathological environment of DCM.

Notably, combination therapy strategies have demonstrated therapeutic advantages over monotherapy in similar DCM contexts. Combined treatment with Tilianin + Syringin (from Dracocephalum moldavica and Panax ginseng) significantly improved cardiac pathology and more effectively attenuated inflammation, oxidative stress, apoptosis, and mitochondrial dysfunction compared with either compound alone. These effects were accompanied by concurrent modulation of the TLR4/MyD88/NF-κB/NLRP3 inflammatory axis and the PGC-1α/SIRT3-mitochondrial ROS signaling pathway. Notably, although tilianin or syringin monotherapy exerted partial protective effects on selected molecular and functional parameters, neither agent consistently improved all assessed endpoints. By contrast, the combined intervention conferred robust cardioprotection in diabetic rats and hyperglycemic H9C2 cells[47].

Toxicological and dose-translation considerations

SQJT is a NMPA-approved Chinese patent medicine (Approval No. Z10950075) that has been clinically used for the treatment of T2DM for more than two decades, and post-marketing reports to date have not identified clear signals of severe adverse reactions.

According to an expert consensus document on its clinical application, pre-marketing safety evaluations included an acute toxicity test in animals (up to 150 × the clinical equivalent dose for 1 week) and a 6-month repeated-dose toxicity study in rats (4 g/kg, 3 g/kg, and 2 g/kg), in which no observable toxic effects were reported, supporting an overall favorable safety profile[11]. In the present study, SQJT-containing serum at 2%-6% showed no detectable cytotoxicity in H9C2 cells, suggesting that the observed cellular effects were pharmacological rather than nonspecific toxicity. Nevertheless, further studies are warranted to better define the translational potential of SQJT, including its pharmacokinetics and long-term safety.

In addition, while our data support DHODH/CoQ pathway involvement primarily based on pharmacological inhibition and in vitro experiments using SQJT-containing serum, further in vivo validation and genetic modulation of DHODH will be important to strengthen mechanistic causality. Future in vivo studies should further evaluate myocardial ferroptosis-related markers, oxidative stress indices, mitochondrial structural and functional alterations, and DHODH/CoQ pathway-associated molecules to better validate and extend the translational relevance of our current in vitro findings. Moreover, individual validation of representative candidate compounds identified by network pharmacology/molecular docking is warranted to clarify the “component-target-effect” relationship and the material basis of SQJT.

Taken together, these findings underscore the therapeutic potential of SQJT in DCM. This study provides preliminary evidence that the bioactive constituents of SQJT contribute to the regulation of oxidative stress, mitochondrial function, and ferroptosis in DCM. Nevertheless, these mechanisms remain incompletely defined and warrant further investigation. Future studies should focus on clarifying the roles of individual bioactive components to provide deeper mechanistic insights into the cardioprotective potential of SQJT.

CONCLUSION

Taken together, this study elucidates a novel mechanism by which SQJT regulates ferroptosis in the context of DCM. We demonstrated that SQJT modulates the DHODH/CoQ axis, thereby reducing cardiomyocyte injury, limiting iron deposition, and restoring the balance of antioxidant enzymes and small-molecule antioxidants to alleviate oxidative stress. These actions suppressed ROS overproduction, improved mitochondrial morphology, and ultimately prevented ferroptosis. Collectively, our findings highlight the cardioprotective potential of SQJT and provide experimental evidence to guide the clinical management of DCM. They also suggest a promising translational value for SQJT as a therapeutic candidate in DCM.