Truffle Extract Attenuates TGF-β1-Induced Fibrosis-Associated Responses in HFL-1 Cells and Is Associated with Changes in Smad and MAPK Signaling

Cellular fibrosis is a key pathological feature of chronic disease and is characterized by persistent fibroblast activation and excessive extracellular matrix deposition. In this study, an aqueous truffle extract (TE) was characterized by untargeted LC-ESI(+)-MS/MS and LC-ESI(−)-MS/MS profiling that yielded putative LC-MS/MS annotations, and was evaluated in a TGF-β1-induced HFL-1 fibroblast model. TE attenuated TGF-β1-induced Smad2 phosphorylation, Smad2/3-Smad4 association, nuclear accumulation of Smad2/3 and Smad4, and the expression of the fibrosis-associated proteins α-smooth muscle actin (α-SMA) and fibronectin. TGF-β1 increased MEK1/2 phosphorylation, whereas TE further increased p-MEK1/2 while decreasing Ras expression and p-ERK1/2, revealing a divergent, context-dependent MAPK response rather than uniform activation or inhibition of the Ras/MEK/ERK pathway. shRNA-mediated reduction in TGF-β1, MEK1/2, or ERK1/2 altered fibrosis-associated protein expression in the absence of TE and further modified the protein-expression patterns observed after TE treatment, indicating that the TE response occurred within a signaling context influenced by these components without establishing strict pathway dependence or a linear signaling hierarchy. In the DCFH-DA assay, TGF-β1 increased intracellular oxidation-dependent fluorescence, whereas both TGF-β1 plus TE and TGF-β1 plus N-acetyl-L-cysteine reduced the signal. LC-MS/MS profiling additionally provided exploratory compositional context for the tested aqueous preparation, while the database best matches and putative LC-MS/MS annotation names remain exploratory and unconfirmed. Collectively, these findings show that TE attenuates TGF-β1-induced fibrosis-associated responses in HFL-1 cells in association with suppression of canonical Smad signaling, reduced intracellular oxidative activity, and context-dependent remodeling of MAPK-network readouts. These coordinated changes suggest that the antifibrotic-associated activity of TE involves multiple interconnected cellular processes rather than modulation of a single signaling component. The results support further activity-guided chemical characterization and validation in additional fibroblast and in vivo fibrosis models.