Fluorofenidone mitigates liver fibrosis through GSK-3β modulation and hepatocyte protection in a 3D tissue-engineered model

Liver fibrosis, a critical stage in chronic liver disease progression, presents a significant global health challenge. This study investigates the antifibrotic and hepatoprotective properties of fluorofenidone (AKF-PD) using a 3D tissue-engineered model. A 3D in vitro liver fibrosis model was developed using decellularized rat liver scaffolds seeded with hepatocytes, hepatic stellate cells (HSCs), and sinusoidal endothelial cells to replicate the multicellular liver microenvironment. The model was stimulated with carbon tetrachloride (CCl₄) to induce fibrotic conditions, resulting in collagen deposition, HSC activation, and elevated fibrosis markers. Parallel in vivo studies employed C57BL/6J mice with CCl₄-induced liver fibrosis. The antifibrotic and hepatoprotective effects of AKF-PD were evaluated by assessing collagen deposition, fibrosis markers, and hepatocyte apoptosis. Oxidative stress markers and inflammation-related proteins were also measured. Molecular docking identified GSK-3β as a target protein of AKF-PD, and subsequent analyses explored the GSK-3β/β-catenin and Nrf2/HO-1 signaling pathways. AKF-PD demonstrated significant efficacy in reducing fibrosis markers and protecting hepatocytes by inhibiting apoptosis and oxidative stress. Mechanistically, AKF-PD targets the GSK-3β/β-catenin pathway, suppressing β-catenin-mediated pro-fibrotic gene expression, while activating the Nrf2/HO-1 pathway to mitigate oxidative stress, thereby reducing hepatocyte apoptosis. These findings are consistent with results from CCl₄-induced mouse fibrosis models, validating the 3D model's applicability for preclinical drug evaluation. This 3D liver fibrosis model provides a physiologically relevant platform for studying fibrosis and anti-fibrotic mechanisms, highlighting AKF-PD's promise as a therapeutic agent and advancing liver fibrosis research.