Fufang Danshen Pill improves mitochondrial homeostasis by regulating the S100a9/TLR4 axis to alleviate myocardial ischemia-reperfusion injury.

BACKGROUND

S100a9 protein participates in regulating mitochondrial damage during Myocardial ischemia-reperfusion injury(I/R), but its cell type-specific mechanisms remain unclear. Fufang Danshen Pill (FDP), a traditional Chinese medicine for I/R treatment, requires further investigation to elucidate its therapeutic targets.

PURPOSE: Investigate the macrophage-specific role of S100a9 in I/R pathogenesis, and clarifies the molecular pathways through which FDP improves mitochondrial function by modulating S100a9.

METHODS

The murine I/R model was established and subjected to determine the optimal therapeutic dosage of FDP. By integrating Single-cell transcriptomics(scRNA-seq)with spatial transcriptomics, we conducted comprehensive analyses of myocardial tissues from sham, I/R, and FDP groups, including cellular subtyping, intercellular communication analysis, and spatial transcriptomic mapping. Mechanistic validation was performed using Adeno-associated virus serotype 2/9(AAV2/9)-mediated macrophage-specific S100a9 overexpression models to confirm key regulatory targets.

RESULTS

The medium-dose FDP (122.85 mg/kg) exhibited the best efficacy in improving cardiac function. FDP intervention markedly attenuated macrophage-cardiomyocyte cellular crosstalk. In I/R models, cardiomyocytes displayed significant alterations in mitochondrial-associated gene expression, whereas S100a9 emerged as the predominant differentially expressed gene in macrophages. Spatial transcriptomic mapping localized S100a9 predominantly within myocardial injury regions. Functional investigations revealed that AAV2/9-mediated macrophage-specific S100a9 overexpression exacerbated I/R pathological manifestations, including increased infarct size, reduced ejection fraction, elevated serum cardiac troponin levels, TLR4 signaling activation, and mitochondrial dysfunction. Notably, FDP treatment substantially reversed these pathological alterations.

CONCLUSION

This study demonstrates that macrophage-derived S100a9 mediates mitochondrial damage in cardiomyocytes during I/R injury. FDP effectively maintains mitochondrial functional homeostasis in cardiomyocytes by targeting and inhibiting macrophage S100a9 expression. These findings provide a important scientific basis for developing intervention strategies targeting the S100a9/TLR4 axis.