ALYREF drives NLRP3 inflammasome-dependent macrophage pyroptosis to promote sepsis-associated acute liver injury by enhancing GADD45A mRNA stability.

Sepsis-associated acute liver injury (SALI) is an independent risk factor that significantly worsens sepsis prognosis, underscoring the need for mechanistic insights to guide targeted therapy. Accordingly, we established a sepsis mouse model via cecum ligation and puncture (CLP) and performed in vitro experiments using murine hepatic Kupffer cells stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Here, we found that growth arrest and DNA damage 45A (GADD45A) was highly expressed in liver tissues of CLP-induced septic mice, and its knockdown ameliorated liver injury and systemic inflammation. Mechanistically, GADD45A knockdown upregulated ATP synthase F1 subunit alpha (ATP5A1) expression by disrupting the interaction between tripartite motif-containing 25 (TRIM25) and ATP5A1, thereby suppressing pyroptosis and pro-inflammatory cytokine secretion in LPS + ATP-stimulated murine Kupffer cells. Additionally, Aly/REF export factor (ALYREF), a 5-methylcytosine (m5C) reader, was upregulated in septic liver tissues and LPS + ATP-treated Kupffer cells. ALYREF likely stabilized GADD45A mRNA in an m5C-dependent manner, thereby promoting its expression. ALYREF knockdown inhibited NLRP3 inflammasome-dependent macrophage pyroptosis and alleviated SALI, likely by modulating the GADD45A/TRIM25/ATP5A1 axis. Notably, the increased expression of ALYREF in SALI appeared to be driven by activated STAT3 signaling. In conclusion, ALYREF, likely driven by activated STAT3 signaling, appears to activate the GADD45A/TRIM25/ATP5A1 axis in an m5C-dependent manner to promote NLRP3 inflammasome-dependent macrophage pyroptosis, thereby contributing to the development of SALI and suggesting a promising therapeutic strategy for targeted SALI intervention.