A Novel Murine Model for Studying Alcohol‐associated Biliary Dysfunction

Abstract

Alcoholic hepatitis (AH) is a severe, potentially life‐threatening form of alcohol‐associated liver disease (ALD) with limited therapeutic options. Existing evidence indicates that cholestasis and biliary dysfunction are common in AH patients and can worsen the prognosis. However, most ALD murine models used today can only recapitulate hepatic steatosis with limited features of AH, which significantly curbs research related to ALD pathogenesis. To develop a murine model that is feasible and less time‐consuming, we included a subtoxic dose of α‐naphthylisothiocyanate (ANIT), a well‐studied intrahepatic cholestasis inducer, in an alcohol feeding model, as a second hit. Mice were fed with an ethanol diet (EtOH) for 10 days or 4 weeks, followed by ANIT administration 48 hours before sample collection. Our results showed that ANIT did not cause overt liver injury in pair‐fed mice, but an observable increase in serum levels of AST in mice fed EtOH for 10 days. Remarkably, in the 4‐week ALD model, serum levels of ALT, AST, total bile acids (TBA), and hepatic levels of TBA were all significantly increased in mice given EtOH and ANIT, indicating a synergistic effect of EtOH and ANIT on liver injury that is dependent on EtOH feeding. In the liver, 4‐week EtOH‐fed mice displayed cholangitis, lytic cell death, and neutrophil infiltration around the periportal area following ANIT treatment as shown by H&E and LY6G staining. In contrast, pair‐fed mice did not show abnormal liver histology following ANIT treatment. Expression of several inflammation‐related genes that are increased in livers from AH patients were analyzed. Consistent with histological observations, hepatic expression of genes like Cxcl1, Ccl2, Ccl20, and Lcn2 were significantly increased in 4‐week EtOH‐fed mice following ANIT treatment, suggesting that ANIT synergistically enhanced EtOH‐induced immune response in the liver. Taken together, our results suggest that EtOH‐dependent changes in the liver can cause higher susceptibility to biliary injury and that a subtoxic dose of ANIT pushes alcohol‐associated liver damage towards a more severe phenotype in mice. Overall, our results support a treatment scheme for alcohol‐associated cholestasis that is feasible, less time‐consuming, cost‐effective, and with modulable severity of phenotypes.