Abstract

Background: Progressive familial intrahepatic cholestasis (PFIC) 3 is a life-threatening hereditary disease caused by adenosine triphosphate-binding cassette subfamily B member 4 (ABCB4) gene mutations. The murine bile acid composition is different from that of humans. Mice are less likely to develop cholestasis due to the predominance of hydrophilic bile acids such as muricholic acids, which are synthesized by enzymes including Cyp2c70. In this study we utilized Cyp2a12/Cyp2c70 double-knockout (DKO) mice which have human-like hydrophobic bile acid composition. We induced liver-specific Abcb4 deletion using adeno-associated virus (AAV)-mediated Cas9 and gRNA expression vectors in the DKO mice and investigated whether this mouse model would represent human PFIC3.

Methods: We constructed an AAV vector containing SaCas9 under a liver-specific promoter. The DKO and wild type (WT) mice were infected with AAV containing SaCas9 and gRNAs against Abcb4. Liver injury, bile acid metabolism and gene expression were analyzed in 5 weeks after infection.

Results: Abcb4 deficient DKO mice (Abcb4-DKO) showed a significant increase in the body-liver weight ratio compared with Abcb4 deficient WT mice (Abcb4-WT). Liver enzymes were significantly elevated in Abcb4-DKO compared with WT and Abcb4-WT; AST: 35.3 ± 31.5, 323.5 ± 173.7 and 740.2 ± 358.1, ALT: 33.5 ± 51.2, 393 ± 65.9 and 1284.8 ± 416.4, and ALP: 129.0 ± 53.2, 260 ± 63.3 and 614.8 ± 342.5 IU/L (mean ± SD), in WT, Abcb4-WT and Abcb4-DKO, respectively, suggesting the presence of severe liver injury in Abcb4-DKO. Serum total bile acid levels in Abcb4-DKO were also higher than Abcb4-WT (9.1 ± 6.1 vs. 5.8 ± 0.9 µmol/L). Abcb4-DKO showed more intense inflammatory cell infiltration in the portal region and scattered CK19 positive cells representing ductal reaction than Abcb4-WT. The mRNA expressions of mCol1a1 and mTimp-1 were significantly increased in Abcb4-DKO, indicating the progression of liver fibrosis. Analysis of bile acid composition in the liver demonstrated increase of TCDCA and decrease of TDCA in Abcb4-DKO.

Conclusion: Abcb4 deficient mice with human-like bile acid composition showed severe liver inflammation compared with Abcb4 deficient WT mice, suggesting the importance of hydrophobic bile acid composition to induce liver injury. This mouse could be a useful tool for cholestasis research as a PFIC3 animal model.