Qi-Blood Harmony Formula mediates post-MI cardioprotection via FTO-driven dual modulation of energy metabolism and fibrosis: a multi-omics validation

Figures

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  Fig. 1. Protective effects of QBHF on cardiac function and structure in MI mice. A Schematic illustration of the experimental workflow. B Representative M-mode traces and C statistical evaluation of LVEF, LVFS, LVIDd, LVIDs, LVVold, and LVVols after 4-week intervention (n ≥ 6). D Representative images of Masson’s trichrome staining. E Representative TTC staining image. F Quantification of fibrosis (n = 6). G Quantification of infarct size (n = 6). H HW/TL ratio (n ≥ 6). I Myocardial ATP levels (n = 5). J Serum CK-MB levels by ELISA in sham, model, QBHF group (n = 6). K Representative H&E of the myocardial tissues (n = 6). L Representative TEM images of sham, model, and QBHF group hearts (n = 3). Red arrows indicate the missing cristae and vacuole inside the mitochondrion. Yellow arrows indicate the faded and disarrayed sarcomeric structure. M Mitochondrial cristae count and N cristae integrity score (> 50 mitochondria from 3 mice per group). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 vs. sham group; #P < 0.05, ##P < 0.01 vs. model group

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  Fig. 2. Transcriptomic profiling reveals QBHF-induced reversal of disrupted metabolic and fibrotic programs in the post-MI heart. A Schematic workflow of the bulk RNA-sequencing experiment. B Volcano plot of DEGs between QBHF‑treated and model mice (criteria: |log₂FC|> 1, adjusted P < 0.05); significantly up‑regulated and down‑regulated genes are shown in orange and blue, respectively. C Heatmap of DEGs in sham, model, and QBHF groups. D GO terms for significantly enriched biological processes. E Significantly enriched KEGG pathways. F Significantly enriched GSEA pathways in MI model and QBHF-treated mice. G GSVA pathway analysis across groups. H FTO expression levels from bulk RNA‑seq data (n = 3). I RT‑qPCR validation of FTO mRNA levels (n = 6). J Western blot analysis of FTO protein expression. Representative images show FTO levels in sham, model, and QBHF groups, with GAPDH as the loading control (n = 4). Data are presented as the mean ± SEM. *P < 0.05 vs. sham group; ##P < 0.01 vs. model group

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  Fig. 3. QBHF ameliorates MI by restoring metabolic homeostasis. A Experimental workflow for LC–MS‑based non‑targeted metabolomics analysis of myocardial tissue. B PLS‑DA score plot. C PLS‑DA permutation test plot (200 permutations). D Volcano plot of differential metabolites between groups. E Correlation heatmap of differential metabolites. F Hierarchical clustering heatmap of 29 significantly altered metabolites (red and blue denote increased and decreased levels in the QBHF group compared with the MI group, respectively). G KEGG pathway enrichment analysis of differential metabolites; pathways are ranked by − log₁₀(P‑value)

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  Fig. 4. Experimental pipeline and snRNA-seq landscape of QBHF intervention in MI mice. A Schematic of the experimental and analytical workflow for snRNA‑seq. B, C t-SNE (B) and UMAP (C) visualizations of all sequenced nuclei, colored by major cell type. D Bar plot of relative cellular abundance across the sham, model, and QBHF groups. E Dot plot of canonical marker-gene expression used to assign cluster identity. F Heatmap summarizing sub-cluster annotation, top marker genes per subset, and representative GO terms. G Cell-type-specific metabolic pathway enrichment profiles

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  Fig. 5. snRNA-seq analysis uncovers the molecular mechanisms of QBHF’s therapeutic effects in MI. A Rose chart of DEGs counts between QBHF and MI model groups across major cardiac cell types. B Volcano plots of DEGs for each cell type between QBHF and MI model groups. C–F Enrichment analyses of DEGs: C GO terms, D KEGG pathways, E GSEA of hallmark pathways, and F GSVA activity scores. G Comparison of pathway activity scores for key biological processes: myocardial contraction, fibrosis, fatty acid oxidation, and oxidative phosphorylation. H–K Expression of genes linked to H cardiac contraction, I myocardial fibrosis, J fatty acid oxidation, and K oxidative phosphorylation in MI and QBHF-treated hearts

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  Fig. 6. SnRNA-seq analysis demonstrates that QBHF intervention effectively suppresses fibroblast activation and fibrosis. A GO enrichment of genes up-regulated or down-regulated by QBHF in fibroblasts. B KEGG pathway enrichment of genes regulated by QBHF in fibroblasts. C UMAP (left) and t‑SNE (right) visualizations identifying three transcriptionally distinct fibroblast subpopulations. D Relative abundance of the fibroblast subpopulations across experimental groups. E Dot plots of subpopulation-specific marker genes across the three fibroblast subpopulations. F Bubble plots of GO terms enriched in DEGs from fibroblast subpopulations. G Functional comparison of collagen formation, TGF-β signaling, and inflammatory response scores among fibroblast subpopulations. H Pseudotime trajectory analysis of fibroblasts, colored by pseudotime (top) and subpopulations (bottom). I Heatmap of representative gene expression dynamics along the pseudotime trajectory. J GO enrichment of DEGs from fibroblast trajectory analysis. K Dynamic expression profiles of collagen-related genes (Col1a1, Col1a2, Col3a1, Col5a2, Meg3, and Postn) during the transition period from FB2 to FB1 fibroblasts. L Expression…

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  Fig. 7. SnRNA-seq reveals transcriptional reprogramming of cardiomyocytes after QBHF intervention. A UMAP visualization of cardiomyocytes, colored by the four identified transcriptional subpopulations. B UMAP projection of cardiomyocytes, colored by experimental group (sham, model, QBHF). C Relative abundance of the cardiomyocyte subpopulations across groups. D GO enrichment of subpopulation-specific DEGs. E Dot plot of subpopulation-specific marker genes across the four identified cardiomyocyte subpopulations. F FeaturePlot visualization of representative marker genes on the UMAP coordinates. G Bar plot of average functional scores for myocardial contraction, cardiac stress, and metabolism across cardiomyocyte subpopulations. H GO terms enriched in cardiomyocyte DEGs comparing QBHF with model group. I KEGG enrichment of DEGs. J Functional comparison of myocardial contraction and fatty acid metabolism scores between QBHF and model groups. K Bubble plot of FTO expression in cardiomyocytes across the sham, model, and QBHF groups

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  Fig. 8. Heterozygous FTO knockout attenuates QBHF-mediated cardioprotective effects after MI. A Experimental workflow. B Representative M‑mode echocardiograms. C HW/TL ratio (n = 6). D–I Echocardiographic parameters of left ventricular function and geometry: LVEF, LVFS, LVIDd, LVIDs, LVVols, and LVVold (n = 6). J Quantitative analysis of myocardial fibrosis area. K Representative Masson’s trichrome‑stained sections (collagen shown in blue). L Representative H&E-stained myocardial sections (n = 6). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 vs. WT sham group; #P < 0.05, ##P < 0.01 vs. WT model group; &P < 0.05, &&P < 0.01 vs. FTO⁺/− sham group; $P < 0.05, $$P < 0.01 vs. FTO⁺/− QBHF group

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  Fig. 9. QBHF enhances cardiomyocyte energy metabolism via FTO upregulation in vitro. A Myocardial ATP levels across groups (n = 6). B FTO mRNA expression in cardiac tissue of FTO+/− mice determined by RT-qPCR (n = 6). C Representative western blot of FTO protein expression in heart; GAPDH loading control (n = 4). D Cell viability assay of OGD-injured cardiomyocytes treated with graded concentrations of QBHF-serum. E FTO mRNA expression in cardiomyocytes treated with different doses of QBHF-serum measured by RT-qPCR (n = 6). F Efficiency of FTO overexpression via lentiviral transduction assessed by RT-qPCR (n = 6). G FTO expression across experimental groups determined by RT-qPCR. H ATP levels in cardiomyocytes across groups (n = 6). I–L ATP production rates determined by Seahorse extracellular flux analysis in AC16 cardiomyocytes under the indicated conditions (n ≥ 4). I Representative OCR traces. Gray lines indicate the addition of mitochondrial functional modulators (oligomycin, rotenone/antimycin A [Rot/AA]). Vertical axis: oxygen consumption rate (OCR); horizontal axis: time. J Total ATP production rate. K Mitochondrial ATP production rate (aerobic respiration). L Glycolytic ATP…

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  Fig. 10. Summary: The traditional Chinese medicine QBHF provides comprehensive cardioprotection after MI via FTO-mediated amelioration of both metabolic dysfunction and fibrosis, thereby providing a multi-dimensional therapeutic approach to prevent MI progression