Roles Of Cathelicidin In Acetaminophen-induced Acute Liver Injury In Mice

Objective: Acetaminophen(APAP)is a widely used antipyretic and analgesic drug.APAP overdose leads to acute liver injury,which has been the most common cause of acute liver failure in western countries.In China,APAP poisoning is also one of the main causes of drug-induced acute liver injury.N-acetylcysteine(NAC)is the only clinically therapeutic option against APAP-induced acute liver injury,but it is effective within 24 h after APAP poisoning.If the most treatable stage is missed,liver transplantation serves as the only choice to rescue patients with acute liver failure.Therefore,it is of great scientific and clinical significance to elucidate the pathogenesis of APAP-induced liver injury and find the intervention target to prevent the progression of liver injury.The antimicrobial peptide cathelicidin is an important component of the innate immune system.Its precursor/mature forms in human and mouse are h CAP18/LL-37(encoding gene CAMP)and pro-CRAMP/CRAMP(encoding gene Camp),respectively.In addition to its direct antibacterial effect,cathelicidin can also promote wound healing and angiogenesis,and participate in autoimmune diseases,metabolic diseases and tumors as a key immune regulator.Current studies have shown that cathelicidin can protect chronic liver diseases in mice.However,it is still unclear whether cathelicidin is involved in the pathological processes of acute liver injury and acute liver failure.In this study,we used genetic and pharmacological approaches to interfere with the levels of cathelicidin to explore the role of cathelicidin in APAP-induced acute liver injury and its underlying mechanisms in vivo and in vitro,and to evaluate the therapeutic potential of cathelicidin in preventing APAP-induced liver injury and liver failure.Methods: 1.Western blot and immunofluorescence staining were used to examine the protein expression and cell source of pro-CRAMP and CRAMP in the liver of C57BL/6J wild-type(WT)mice after APAP overdose.2.Camp knockout and exogenous supplementation of cathelicidin synthetic peptide were applied to interfere with the levels of cathelicidin.The degree of liver damage was evaluated by hepatic H&E staining and serum alanine transaminase(ALT),and the hepatocyte proliferation was evaluated by Ki67 immunohistochemical staining after APAP overdose.3.Camp knockout and exogenous supplementation of cathelicidin synthetic peptide were applied to interfere with the levels of cathelicidin.The number of liver-infiltrating neutrophils and macrophages after APAP overdose was determined by flow cytometry and the role of cathelicidin in the inflammation resolution at the late stage of liver injury was evaluated.4.In murine APAP toxicity model and human peripheral blood neutrophils cultured in vitro,the role of cathelicidin in the phagocytic function of neutrophils was examined by flow cytometry.5.The APAP-induced acute liver injury and acute liver failure models were established in WT mice,and CRAMP,CRAMP(1-39)(the extended form of CRAMP)or LL-37 at the late stage of liver injury were injected to mice,respectively.The therapeutic effect comparison between three different active forms of cathelicidin and NAC was evaluated by hepatic H&E staining.Results: 1.An increase in hepatic pro-CRAMP and CRAMP was observed in APAP-intoxicated mice,and the up-regulated cathelicidin was derived from liver-infiltrating neutrophils.2.Camp deficiency did not affect initial liver injury but delayed liver recovery in the late phase of APAP intoxication.CRAMP administration reversed impaired liver recovery observed in APAP-challenged Camp KO mice.3.Camp KO or LL-37 treatment significantly increased or decreased the number of liver-infiltrating neutrophils and macrophages during the repair phase after APAP-induced liver injury,respectively.4.Camp KO or LL-37 treatment significantly inhibited or promoted the phagocytic function of liver-infiltrating neutrophils during the repair phase after APAP-induced liver injury,respectively.LL-37 enhanced the phagocytic function of cultured human peripheral blood neutrophils in a concentration-dependent manner.5.In the APAP-induced acute liver injury model,CRAMP,CRAMP(1-39)or LL-37 treatment exhibited reduced liver damage in the late phase of APAP intoxication.The combined treatment of cathelicidin at the late stage and NAC at the early stage displayed a stronger hepatoprotective effect than NAC alone.Likewise,in the APAP-induced acute liver failure model,an additive protective effect of CRAMP(1-39)or LL-37 and NAC co-treatment in the late phase of liver injury was observed.Conclusions: The pro-reparative role of cathelicidin in the APAP-injured liver is attributed to an accelerated resolution of inflammation,possibly through enhanced phagocytic function of liver-infiltrating neutrophils in an autocrine manner.This study for the first time reveals that neutrophil-derived cathelicidin is a key regulatory mediator for liver repair after APAP overdose,and provides novel evidence for understanding the role of neutrophils in promoting tissue regeneration and repair.These results also shed light on potential therapeutic strategies for late-presenting APAP-induced acute liver injury patients.