Shexiang Baoxin Pill alleviates atherosclerosis by inhibiting macrophage-mediated inflammation via suppressing KMT5A mediated Irf7 transcription

Abstract

Ethnopharmacological relevance

Shexiang Baoxin Pill (SBP) is a traditional compound formulation composed of seven Chinese medicinal ingredients. Although SBP has shown promising clinical outcomes in the treatment of cardiovascular diseases, its role and underlying mechanisms in alleviating atherosclerosis remain insufficiently studied.

Aim of the study

This study aims to investigate the effects and mechanisms of SBP in epigenetic modulating macrophage inflammatory responses to mitigate atherosclerosis.

Materials and methods

ApoE^−/− mice were treated with high fat diet (HFD) following varying concentrations of SBP. Oil Red O staining, hematoxylin-eosin (HE) staining, and ELISA were used to assess the anti-atherosclerotic and anti-inflammatory efficiency of SBP. Subsequently, RNA sequencing (RNA-seq), RT-PCR, Western blot (WB), immunofluorescence (IF) and chromatin immunoprecipitation (ChIP) were employed in bone marrow derived macrophages (BMDMs) to elucidate the epigenetic mechanisms of SBP in alleviating macrophage inflammatory responses. Lysine methyltransferase 5A (KMT5A) was overexpressed in vivo and in vitro for further validation.

Results

SBP significantly attenuated atherosclerosis in HFD treated ApoE^−/− mice by decreasing plaque areas, serum inflammation levels and macrophages infiltration in the aortic root and plaques. SBP treatment reduced BMDMs inflammatory responses following oxidized low-density lipoprotein (oxLDL) treatment. Mechanistically, SBP inhibited interferon regulatory factor 7 (IRF7) expression by reducing KMT5A-mediated mono-methylation of histone H4 lysine 20 (H₄K₂₀), thus decreasing the secretion of multiple pro-inflammatory cytokines, including interferon (IFN)-α, IFN-β, TNF-α. Overexpression of KMT5A abolished the anti-atherosclerotic and anti-inflammatory effects of SBP, further confirming that KMT5A/H₄K₂₀me/IRF7 axis is a key target for SBP exerting therapeutic effect.

Conclusion

SBP exerts anti-atherosclerotic effects by inhibiting macrophage inflammatory responses through downregulation of the H₄K₂₀ methylase KMT5A, thereby suppressing the transcription of Irf7. Our findings provide a novel epigenetic mechanism by which SBP alleviates atherosclerosis.