MPTP mediated Ox-mtDNA release inducing macrophage pyroptosis and exacerbating MCD-induced MASH via promoting the ITPR3/Ca(2+)/NLRP3 pathway.

BACKGROUND: Metabolic Dysfunction-Associated Steatohepatitis (MASH) is a severe and progressive form of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), with approximately 25% of adults worldwide suffering from MASLD, of which 20%-30% progress to MASH, and the global incidence continues to rise. Oxidized mitochondrial DNA (Ox-mtDNA) release is a key contributor to MASH. However, its underlying mechanism remains unclear. Clarifying this process may provide a theoretical foundation for MASH treatment. METHODS: In this study, we separately established MASH models using methionine- and choline deficient diet (MCD) fed mice in vivo and free fatty acid (FFA)-stimulated THP-1 derived macrophages in vitro. Cyclosporin A (CsA: mitochondrial permeability transition pore, mPTP, channel inhibitor) was used to inhibit the release of Ox-mtDNA. 8-OH-dG detection and fluorescent probe were used to evaluate Ox-mtDNA release. Liver lipid deposition was analyzed by Triglyceride (TG) and Oil Red O, and tissue damage were analyzed by aspartate transaminase and alanine aminotransferase (ALT, AST) and H&E staining. Pyroptosis markers, such as cleaved-Caspase1, GSDMD-N, and inflammatory cytokines, such as interleukin - 1β, interleukin 18 (IL-1β, IL-18), were detected by WB, ELISA and transmission electron microscopy (TEM) experiments, and the key pyroptosis pathways activated by Ox-mtDNA were screened by RNA-seq. Finally, ITPR3 was silenced by siRNA in vitro and by Adeno-associated virus (AAV) in vivo respectively, which confirmed the role of ITPR3/Ca(2+)/NLRP3 axis in Ox-mtDNA regulating macrophage pyroptosis mediated MASH. RESULTS: The cytosolic Ox-mtDNA level was significantly increased during MASH. Inhibition of Ox-mtDNA release alleviated macrophage pyroptosis to improve the pathological phenotype of MASH. RNA-seq analysis showed that cytosolic Ox-mtDNA triggered an inflammatory response by activating the NOD-like receptor pathway, in which FFA induced upregulation of inositol 1,4,5-Trisphosphate Receptor Type 3 (ITPR3, IP3R) expression, and Inhibition of Ox-mtDNA release could relieve this effect. ITPR3 silencing significantly reduced Ca²⁺ release, which in turn inhibited nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome activation and macrophage pyroptosis. Cytosolic Ox-mtDNA promotes Ca²⁺ release by upregulating ITPR3, activates NLRP3-dependent macrophage pyroptosis, and ultimately exacerbates liver injury and MASH progression. CONCLUSIONS: This study demonstrates that Ox-mtDNA drives MASH progression by promoting macrophage pyroptosis via the ITPR3/Ca²⁺/NLRP3 axis, providing a novel therapeutic strategy for targeted intervention.