Gualou Huoxue Jiedu Decoction inhibits VSMC phenotypic switching to alleviate atherosclerosis via promoting Mfap4 DNA methylation

BACKGROUND: Atherosclerosis (AS) is a chronic disease characterized by lipid deposition in the vascular intima. As the pathological basis of cardiovascular diseases, AS represents a major contributor to global morbidity and mortality. While Gualou Huoxue Jiedu Decoction (GHJD) has been widely used in clinical practice for the treatment of AS, the molecular mechanisms remain unclear. PURPOSE: To investigate the anti-atherosclerotic effects and underlying mechanisms of GHJD. METHODS: mice were treated with GHJD to evaluate its effects on plaque formation, lipid profiles, and inflammatory responses. The main compounds in GHJD were identified using UHPLC-Q-Orbitrap HRMS. Integrated analyses of network pharmacology, methyl-capture sequencing (MC-seq), and RNA-seq were performed to elucidate the molecular mechanisms of GHJD. In vitro experiments were conducted to validate its mechanism of action. RESULTS: GHJD alleviated plaque formation, improved lipid metabolism, and suppressed inflammation in vivo. Multi-omics analysis revealed that DNA methylation of Mfap4 could be a pivotal target of GHJD efficacy. In vitro assays confirmed that GHJD suppressed Mfap4 transcription and translation, leading to downregulation of integrin receptor family expression and inhibition of VSMC phenotypic switching. CONCLUSION: GHJD exerts anti-atherosclerotic effects through epigenetic modulation of Mfap4 and downstream integrin/FAK signaling pathway, thereby inhibiting VSMC phenotypic switching. These findings provide pharmacological evidence supporting GHJD as a potential therapy for AS and, for the first time, validate MFAP4 as a pharmacological target, offering new insights into AS prevention and treatment.