Liqi Huoxue dripping pills alleviate myocardial ischemia-reperfusion injury with improved mitochondrial biogenesis and reduced ferroptosis: Involvement of S100A8/A9-TLR4-ERK signaling

BACKGROUND: S100A8/A9, a critical danger-associated molecular pattern, amplifies inflammatory responses and exacerbates myocardial ischemia-reperfusion injury (MIRI) through Toll-like receptor 4 (TLR4) signaling. Although S100A8/A9-TLR4-related signaling has been implicated in MIRI pathogenesis, effective pharmacological interventions for MIRI remain limited. PURPOSE: This study investigated whether Liqi Huoxue Dripping Pills (Lqhxdw), a traditional Chinese medicine, alleviates MIRI in association with modulation of the S100A8/A9-TLR4-ERK-related signaling axis and elucidated the downstream mechanisms involving mitochondrial dysfunction and ferroptosis. METHODS: A rat MIRI model was established by temporary LAD ligation, followed by 7-day Lqhxdw treatment. An in vitro hypoxia/reoxygenation (H/R) model was established using drug-containing serum. Transcriptomic profiling, network pharmacology, and molecular docking were performed to identify candidate therapeutic targets. Rescue experiments with recombinant S100A8/A9 (rS100A8/A9), pharmacological pathway validation using U0126, ferroptosis inhibitor rescue using ferrostatin-1, and in vivo pharmacological validation using TAK-242 were performed to support the proposed mechanistic framework. Mitochondrial function and ferroptosis were assessed by Seahorse XF analysis, biochemical assays, fluorescent probes, and transmission electron microscopy. RESULTS: Lqhxdw treatment dose-dependently improved cardiac function, attenuated histopathological damage, and reduced myocardial injury biomarkers. Transcriptomic analysis identified S100a8/S100a9 as significantly upregulated genes following MIRI, with negative correlations to mitochondrial Complex I subunits. Molecular docking predicted favorable binding affinity of Lqhxdw components to TLR4. Experimentally, Lqhxdw reduced S100A8/A9-TLR4 co-localization, suppressed TLR4/ERK phosphorylation, and restored the PGC-1α/NRF1/NDUFA9 axis. Seahorse analysis demonstrated restored mitochondrial respiration. Ferroptosis markers were markedly attenuated by Lqhxdw treatment. Critically, exogenous rS100A8/A9 partially reversed these protective effects. Pharmacological validation using U0126 partly restored PGC-1α and NRF1 expression under rS100A8/A9 stimulation, supporting the involvement of ERK upstream of mitochondrial regulatory changes. Ferrostatin-1 rescue experiments provided functional support for the involvement of ferroptosis. In vivo validation with TAK-242 showed regulatory directions broadly consistent with those of Lqhxdw on iron homeostasis markers and NF-κB-associated inflammatory readouts. CONCLUSION: This study provides pharmacological and functional evidence supporting a mechanistic framework in which Lqhxdw attenuates MIRI in association with suppression of S100A8/A9-TLR4-ERK-related signaling, restoration of PGC-1α-mediated mitochondrial biogenesis, and attenuation of ferroptosis. These findings identify S100A8/A9-TLR4-ERK as a biologically relevant signaling framework in MIRI and support the potential clinical application of Lqhxdw in ischemic heart disease.