Unraveling the pyroptosis pathway: Key insights into methotrexate-induced liver injury

Methotrexate (MTX)-induced liver injury (MTX-ILI) involves complex mechanisms that remain incompletely understood. This study investigated the role of NLR family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis in MTX-ILI and explored the regulatory involvement of reactive oxygen species (ROS) and mitochondrial permeability transition pore (mPTP) opening. Wistar rats administered MTX and L02 cells exposed to MTX exhibited significant hepatocellular injury and pyroptotic features, as evidenced by elevated aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and Caspase-1 activities; increased interleukin-1β (IL-1β) and interleukin-18 (IL-18) levels; and upregulation of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D (GSDMD), and its N-terminal fragment (GSDMD-N). These effects were attenuated by the NLRP3 inhibitor MCC950. In vitro, scavenging ROS with N-acetylcysteine (NAC) and inhibiting mPTP opening with ciclosporin A (CsA) markedly suppressed pyroptosis by alleviating mitochondrial dysfunction, reducing ROS accumulation, restoring mitochondrial membrane potential, and preserving mitochondrial ultrastructure. Label-free quantitative proteomics and protein-protein interaction analysis identified KNG1 and PARP12 as key proteins associated with ROS/mPTP-mediated pyroptosis, which were validated by Western blotting. Furthermore, Connectivity Map analysis predicted four potential therapeutic agents, including RS-127445, SB-218795, proadifen, and balicatib. Collectively, these findings demonstrate that MTX induces NLRP3-dependent pyroptosis through ROS accumulation and sustained mPTP opening.