Zha-Chong Shi-San-Wei Pill inhibits ferroptosis in cerebral ischemia via bioinformatics and experimental confirmation...

Ischemic stroke (IS), accounting for approximately 85% of all stroke cases, is a cerebrovascular catastrophe that inflicts cerebral parenchymal damage through multifactorial mechanisms, resulting in millions of deaths and long-term neurological disabilities worldwide. Zha-Chong Shi-San-Wei Pill (ZCSSW), a compound formulation derived from a synergistic blend of Mongolian medicinal herbs, has demonstrated significant therapeutic efficacy in managing IS.

This study aims to investigate whether ZCSSW inhibits ferroptosis in cerebral ischemia (CI) via the hypoxia-inducible factor-1α (HIF-1α)/heme oxygenase-1 (HO-1) pathway.

A network pharmacology analysis integrating component identification and database screening was conducted to explore the anti-CI effects of ZCSSW. Molecular docking was performed to validate interactions between crucial bioactive constituents and core targets. A permanent middle cerebral artery occlusion (pMCAO, 12 h) model in rats and an oxygen-glucose deprivation (OGD, 12 h) model in PC12 cells were established to elucidate the molecular mechanisms. Pharmacodynamic evaluations in pMCAO rats included tetrazolium red staining for cerebral infarction quantification, neurological deficit scoring and hematoxylin-eosin staining for histopathological assessment. Cellular viability under OGD was measured using the cell counting kit-8. Mechanistic investigations employed transmission electron microscopy for ultrastructural analysis. Protein expression levels of HIF-1α, HO-1, ferritin heavy (FTH1), transferrin (TF) and transferrin receptor (TFRC) were quantified via immunohistochemistry, Western blot and immunofluorescence. Pharmacological modulation using erastin (a ferroptosis inducer) and lificiguat (YC-1, a HIF-1α inhibitor) to assess the role of the HIF-1α/HO-1 pathway in CI-associated ferroptosis. Molecular interaction techniques including molecular dynamics simulation, cellular thermal shift assay, drug affinity responsive target stability and co-immunoprecipitation (Co-IP) assays were used to validate the binding of core ZCSSW components to the HIF-1α/HO-1 pathway and their regulatory effects on ferroptosis.

Network analysis identified 84 bioactive constituents in ZCSSW targeting 1000 CI-related proteins, with the ferroptosis pathway as the central regulatory mechanism. Liquiritigenin showed strong binding affinity to HIF-1α. Optimal doses of ZCSSW (162 mg/kg in vivo and 2.5% medicated serum in vitro) significantly reduced cerebral infarction volume, neuronal loss and neurological scores while attenuating mitochondrial ultrastructural damage. ZCSSW intervention modulated differential metabolite profiles, also attenuated reactive oxygen species and ferrous ion accumulation. This correlated with downregulation of HIF-1α, HO-1, TF and TFRC and upregulation of FTH1. Erastin counteracted ZCSSW's therapeutic effects while YC-1 synergistically enhanced its efficacy. Molecular interaction experiments confirmed tight binding of crucial ZCSSW components to the HIF-1α/HO-1 pathway, enhancing protein stability. Co-IP assays verified a direct HIF-1α/HO-1 interaction. Further experiments demonstrated that these vital components (liquiritigenin and glycyrrhizin) alleviated ferroptosis in rat CI injury and in OGD-injured PC12 cells by modulating the HIF-1α/HO-1 pathway.

This study demonstrates through pharmacological and mechanistic validation that ZCSSW exerts anti-ferroptosis effects in CI through inhibiting HIF-1α/HO-1 pathway. The findings highlight the therapeutic synergy between antioxidative stress modulation and enhanced mitophagy in mitigating CI pathogenesis.