Melatonin Alleviates Graft Biliary Fibrosis by Inhibiting VIM + Cholangiocyte Subcluster via Hypoxia/TGF‐β‐CREM‐VIM Axis

ABSTRACT Non‐anastomotic stricture (NAS), a severe liver transplantation (LT) complication, is pathologically defined by biliary fibrosis, with unclear mechanisms and limited pharmacological therapeutic strategies. Single‐cell RNA sequencing was performed on the bile duct tissues of NAS patients, ongoing (fibrosis persistent group) or succeeding (fibrosis remission group) in endoscopic treatment. Integrated analysis indicated that biliary epithelial‐mesenchymal transition (EMT) and fibrosis were reduced in the fibrosis remission group than in the fibrosis persistent group. VIM + biliary epithelial cell (BEC), a novel EMT‐related BEC subcluster, exhibited robust activation of the EMT pathway and decreased during biliary fibrosis remission. Mechanistically, extracellular hypoxia and TGF‐β signaling enhanced CREM transcriptional activity in VIM + BEC, which promotes EMT by regulating the VIM expression. CREM knockdown in BEC significantly suppressed hypoxia‐ and TGF‐β‐induced VIM expression. BEC‐specific Crem knockout (Crem fl/fl ; Sox9‐Cre +/− ) suppressed Vim expression, EMT, and fibrosis in rats post‐LT. Melatonin administration reduced Crem expression, biliary EMT, and fibrosis in rats post‐LT. An explorative clinical trial preliminarily confirmed that some NAS patients showed improved liver function after melatonin treatment. Our findings revealed that the hypoxia/TGF‐β‐CREM‐VIM axis modulates the VIM + BEC subcluster, which in turn drives biliary EMT and fibrosis, and melatonin emerges as a promising drug candidate for NAS‐related biliary fibrosis.