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

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.

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.

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.

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.

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.

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.

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.