Longitudinal assessment activation of TGF-β1/pSmad2/3 signaling promotes hepatocyte reprogramming and fibrosis via Oct4, Sox9, and RunX2 in experimentally induced rats.

This study aimed to determine whether hepatocytes acquire stemness properties during their dedifferentiation toward myofibroblast-like phenotype and to evaluate the role of TGF-β1 signaling in mediating this process during liver fibrosis (LF) progression. LF was induced in male Sprague-Dawley (SD) rats, and tissues were analyzed at 4, 8, and 12 weeks using histological staining, immunohistochemistry, immunofluorescence, and biochemical approaches. In parallel, in-vitro experiments were performed in HepG2 cells to further investigate fibrosis-related signaling mechanisms. TA administration resulted in progressive hepatocellular injury, characterized by macrophage infiltration/inflammation and extensive collagen deposition in the periportal and central vein areas. Persistent oxidative stress was evidenced by increased NOX2 and malondialdehyde (MDA) levels, together with reduced antioxidant defenses. These alterations were associated with sustained activation of the Smad2/Smad3 pathway downstream of TGF-β1. Concurrently, hepatocytes showed induction of stemness-associated transcription factors, including Oct4, Runx2, and Sox9, along with partial loss of hepatocyte identity markers such as albumin and HNF4α, suggesting the acquisition of partial myofibroblast-like characteristics, including α-SMA and Col I and Col III expression. Dysregulated extracellular matrix turnover was further indicated by increased TIMP1 and reduced MMP9 expression. In-vitro inhibition of TGF-β1 signaling and suppression of Oct4 significantly attenuated TA-induced fibrotic responses in HepG2 cells, supporting the role of TGF-β1-Oct4 signaling in hepatocyte partial differentiation and LF remodeling. This dual mechanism underscores the role of hepatocyte differentiation in LF progression and broadens the therapeutic landscape.