METTL3 inhibition attenuates AFB1-induced hepatic fibrosis by suppressing m6A-mediated hepatic stellate cell activation

Abstract Background Aflatoxin B 1 (AFB 1 ) is a potent hepatotoxic mycotoxin and a major environmental risk factor for hepatocellular carcinoma (HCC). Hepatic fibrosis is a critical intermediate stage in this process, and METTL3-mediated m 6 A modification may represent an important post-transcriptional mechanism linking AFB 1 -induced liver injury to fibrogenic progression. Methods AFB 1 -induced hepatic fibrosis was evaluated using in vivo mouse models and in vitro cultured hepatic stellate cells (HSC). Global m 6 A methylation and methyltransferase-like 3 (METTL3) expression were assessed by liquid chromatography-mass spectrometry, Western blotting, single-nucleus RNA sequencing, and quantitative real-time PCR. METTL3 was inhibited using small interfering RNA or the selective inhibitor STM2457. Molecular docking was performed to identify potential METTL3-binding compounds, followed by functional validation. Results AFB 1 exposure promoted hepatic fibrosis and HSC activation, accompanied by global m 6 A hypermethylation and upregulation of METTL3. METTL3 increased the m 6 A modification of collagen-related transcripts, enhancing their stability and promoting extracellular matrix production in a YTHDF1 -dependent manner. Inhibition of METTL3 suppressed HSC activation and fibrotic gene expression both in vitro and in vivo. Molecular docking identified saxagliptin as a potential METTL3-binding compound, which reduced AFB 1 -induced HSC activation and extracellular matrix accumulation, consistent with the effects of STM2457. Conclusions These findings indicate that METTL3 functions as a post-transcriptional regulator in AFB 1 -induced liver fibrosis via m 6 A modification. METTL3 inhibition, achieved via genetic knockdown or selective inhibitors, affects HSC activation and fibrotic gene expression, supporting its role as a therapeutic target in AFB 1 -induced liver fibrosis.