Thrombomodulin-mediated Inhibition of Neutrophil Extracellular Trap Formation Alleviates Hepatic Ischemia-reperfusion Injury by Blocking TLR4 in Rats Subjected to Liver Transplantation

Abstract

Background: Hepatic ischemia/reperfusion injury (IRI) is an unavoidable outcome of liver transplantation, during which neutrophil extracellular traps (NETs) may play a critical role in the IRI-induced immune response to inflammation. The purpose of this study was to identify the function of recombinant human thrombomodulin (rTM) in the remission of hepatic IRI after liver transplantation and elucidate the specific mechanism. Methods: NET formation was detected in the serum of liver transplantation patients and rats following liver transplantation. Hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, immunohistochemistry and immunofluorescence were used to assess the effect of rTM on NET formation in vitro and in vivo. Results: We found that rTM markedly inhibited neutrophil formation in NETs, reduced apoptosis in hepatocytes, alleviated rat hepatic IRI and improved liver function. In vitro, rTM inhibited neutrophil formation in NETs, and lipopolysaccharide (LPS) (a Toll-like receptor (TLR)-4 agonist) reversed the inhibitory effect of rTM on NET formation. rTM blocked TLR-4 and the downstream extracellular signal-regulated kinase (ERK)/c-Jun NH2 terminal kinase (JNK) and nicotinamide adenine dinucleotide phosphate (NADPH)/ROS/peptidylarginine deiminase 4 (PAD4) signaling pathways to protect against hepatic IRI and inhibit NET formation. In addition, we demonstrated that combined treatment with rTM and an NADPH oxidative inhibitor had a better effect than either treatment alone. Conclusions: NETs are a potential therapeutic target in hepatic IRI, and rTM could be used to prevent IR-induced hepatic injury. In addition, cotargeting NETosis-related signaling pathways might be a novel therapeutic strategy for hepatic IRI treatment.