Abstract
Rationale: Biliary complications after liver transplantation persistently affect patient prognosis and graft survival. Neutrophil-mediated immune injury is an important factor leading to biliary injury. However, the mechanism by which neutrophils reach the periphery of the bile duct and further mediate bile duct injury is not fully understood. Methods: First, we obtained hepatic tissue samples from grafted rats subjected to warm and nonwarm ischemic injury. We constructed a protein map via proteomics and analyzed the correlations between neutrophil extracellular traps (NETs) and biliary injury. HuCCT1 cells were cocultured with NETs isolated from the peripheral blood of grafted rats in vitro to evaluate the role of NETs in bile duct injury. Next, we assessed NET extravasation through the PIEZO1/SRC pathway in liver samples from rats with liver grafts via proteomic analysis, immunohistochemical staining and immunofluorescence. Finally, we evaluated the correlations between hepatic arterial blood flow and the PIEZO1/SRC pathway in a liver graft model. Results: The results revealed a close correlation between NET formation by activated neutrophils and bile duct injury. Low hepatic arterial blood flow leads to NET extravasation through the activation of the mechanosensitive ion channel PIEZO1 and its downstream signaling events, including phosphorylation of tyrosine kinases sarcoma (SRC) protein. The extravasated NETs accumulate around the bile ducts and subsequently mediate biliary cell apoptosis. Verapamil was further used to increase hepatic artery blood flow to inhibit the PIEZO1/SRC axis, which reduced bile duct injury caused by extravasated NETs. Conclusions: Suppressing NET extravasation by increasing hepatic arterial blood flow is a potential strategy for the treatment of biliary complications after liver transplantation.