PAX5 alleviates atherosclerosis by transcriptionally regulating GSDMD and inhibiting STAT3-mediated pyroptosis

OBJECTIVE: This study aims to elucidate the role of PAX5 in the pathogenesis of atherosclerosis (AS) and its underlying mechanisms, with a particular focus on the transcriptional regulation of GSDMD and STAT3-mediated pyroptosis. METHODS: Serum PAX5 and GSDMD levels were detected in AS patients and healthy controls. In vitro, human umbilical vein endothelial cells (HUVECs) were stimulated with oxidized low-density lipoprotein (ox-LDL) and transfected with PAX5 overexpression vectors, followed by assays for cell viability, tube formation, lipid accumulation, LDH release, and pro-inflammatory cytokine (IL-1β, IL-18) secretion. Immunofluorescence and dual-luciferase reporter assays were performed to verify the co-localization of PAX5 and GSDMD, and the transcriptional interaction between PAX5 and the GSDMD promoter. Co-immunoprecipitation (Co-IP) ubiquitination assays were conducted to determine whether PAX5 promotes GSDMD poly-ubiquitination. In vivo, ApoE⁻/⁻ AS mouse models were treated with AAV-mediated PAX5 overexpression, followed by histopathological assessment of atherosclerotic lesions, lipid metabolism detection, and Western blot analysis of pyroptosis and STAT3 signaling-related proteins. RESULTS: Serum PAX5 was significantly downregulated in AS patients, while GSDMD was upregulated. In vitro, PAX5 overexpression reversed ox-LDL-induced viability reduction, lipid accumulation, LDH release, and IL-1β/IL-18 hypersecretion in HUVECs. Dual-luciferase assay confirmed that PAX5 directly binds to and activates the GSDMD promoter. Ubiquitination assays demonstrated that PAX5 promotes both K48-linked and K63-linked poly-ubiquitination of GSDMD, with the degradation pathway predominating over transcriptional activation, resulting in a net decrease in GSDMD protein abundance. In vivo, PAX5 overexpression reduced atherosclerotic lesion size, lipid deposition, and systemic inflammatory levels, improved serum lipid profiles, and inhibited STAT3 phosphorylation, GSDMD-N generation, and pyroptosis-related protein expression. CONCLUSION: PAX5 alleviates AS progression through a dual regulatory mechanism involving transcriptional activation and ubiquitin-proteasome-mediated degradation of GSDMD, thereby inhibiting STAT3-associated pyroptosis. PAX5 represents a promising therapeutic target for AS intervention.