All-trans retinoic acid mitigates ethanol-induced human PSC activation and type I collagen synthesis by downregulating TGF-β1/pSmad2/3 pathway

Background: Pancreatic stellate cell (PSC) activation drives fibrogenesis in alcoholic chronic pancreatitis (ACP). While cytokines and growth factors are known regulators of PSC activation. This study investigated the role of TGF-β1/Smad pathway in ethanol (EtOH)-induced human PSC activation and collagen type I alpha 1 chain ( Col1A1) synthesis, as well as the mechanism through which all‑trans retinoic acid (ATRA) antagonizes this pathway. Methods Using human activated PSC line (HP-1 cells), we quantified the mRNA and protein levels of TGF-β1, IL-6, α-SMA and Col1A1 by qRT-PCR and ELISA respectively. Phospho-Smad2/3 and RARα/RARγ levels were analyzed by Western blot, RARα/α-SMA localization by double immunofluorescence, and serum retinol by HPLC. Results EtOH significantly upregulated TGF-β1 and IL-6 mRNA and protein levels. EtOH-induced TGF-β1 upregulation was partially inhibited by si-IL-6R or IL-6 antibody, indicating that IL-6 acts as an intermediate. Crucially, both TGF-β1 siRNA and a neutralizing antibody largely abolished EtOH-induced Smad2/3 phosphorylation and the subsequent production of α-SMA and Col1A1, underscoring the central role of TGF-β1 in this pathway. ATRA suppressed α-SMA expression and prevented Col1A1 synthesis in EtOH-treated HP-1 cells without affecting RARα. Mechanistically, these effects occurred via partial inhibition of TGF-β1 and marked downregulation of Smad2/3 phosphorylation. Clinically, a high vitamin A deficiency rate (44.7%) and elevated serum TGF-β1/Col1A1 levels were observed in 38 ACP patients. Conclusion ATRA mitigates EtOH-induced fibrogenesis in PSCs through inhibition of the TGF-β1/Smad2/3 axis. This mechanistic insight, together with the observed vitamin A deficiency in ACP patients, suggests that restoring retinoid signaling may be a viable antifibrotic strategy.