Integrated transcriptomic and experimental validation reveal that N‑acetylcysteine ameliorates acute liver failure through multi‑target synergistic regulation of pyroptosis and related signaling networks.

Pyroptosis is a major contributor to the pathophysiology of acute liver failure (ALF), a condition with high mortality. Although N‑acetylcysteine (NAC) is used clinically for ALF, its mechanisms of regulating pyroptosis and multiple signaling pathways to achieve hepatoprotection remain incompletely understood. We integrated public transcriptomic data (GSE14668, GSE96851, GSE38941) and performed differential expression analysis, weighted gene co‑expression network analysis, protein-protein interaction network construction, and machine learning to identify ALF‑related pyroptosis signature genes. Network pharmacology and molecular docking were used to predict the core therapeutic targets and mechanisms of NAC. An in vitro cellular inflammation model (LPS/D‑GalN‑treated L02 hepatocytes) was established, and the effects of NAC on target proteins and signaling pathways were validated by CCK‑8, LDH release, Western blot, ELISA, and qPCR. Six core targets-IL18, BCL2, TLR4, CASP1, CCNB1, and CAV1-were identified. Molecular docking predicted binding affinities between NAC and these targets in the moderate range (- 4.2 to - 5.3 kcal/mol). In vitro, NAC (10 mM) significantly reduced total CASP1 and GSDMD protein levels. More importantly, we further assessed NLRP3 expression, cleaved caspase‑1 (p20 subunit), and the N‑terminal cleavage fragment of GSDMD (GSDMD‑NT); NAC markedly suppressed all three markers, providing direct evidence that it inhibits the canonical NLRP3/caspase‑1/GSDMD pyroptotic axis. NAC also restored BCL2, CCNB1, and CAV1 expression, reversed TLR4 overexpression, and normalized the LC3B‑II/I ratio and p62 levels, indicating restoration of autophagic flux. Compared with a pyroptosis inhibitor (Ac‑YVAD‑CMK), NAC showed superior efficacy in reversing TLR4 upregulation and cell‑cycle‑related protein abnormalities. NAC exerts a comprehensive hepatoprotective effect that surpasses that of a simple pyroptosis inhibitor by synergistically modulating pyroptosis, apoptosis, cell cycle, inflammatory recognition, and endocytosis pathways through multiple targets. These findings provide a theoretical foundation for expanding the clinical application of NAC in ALF.