Role of fibrinogen and protease-activated receptors in acute xenobiotic-induced cholestatic liver injury

Abstract

Alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury causes tissue factor (TF)-dependent coagulation in mice, and TF deficiency reduces ANIT-induced liver injury. However, the mechanism whereby TF contributes to hepatotoxicity in this model is not known. Utilizing pharmacological and genetic strategies, we evaluated the contribution of fibrinogen and two distinct receptors for thrombin, protease-activated receptor-1 (PAR-1) and PAR-4, in a model of acute ANIT hepatotoxicity. ANIT administration (60 mg/kg, po) caused a marked induction of the genes encoding the three fibrinogen chains (α, β, and γ) in liver, an increase in plasma fibrinogen, and concurrent deposition of thrombin-cleaved fibrin in liver. Partial depletion of circulating fibrinogen with ancrod did not impact ANIT hepatotoxicity. However, complete fibrin(ogen) deficiency significantly reduced serum alanine aminotransferase activity and hepatocellular necrosis in ANIT-treated mice. ANIT-induced hepatocellular necrosis was similar in PAR-1(-/-) mice compared with PAR-1(+/+) mice. Interestingly, the progression of ANIT-induced hepatocellular necrosis was significantly reduced in PAR-4(-/-) mice and by administration of an inhibitory PAR-4 pepducin (P4Pal-10, 0.5 mg/kg, sc) to wild-type mice 8 h after ANIT treatment. Interestingly, a distinct lesion, parenchymal-type peliosis, was also observed in PAR-4(-/-) mice treated with ANIT and in mice that were given P4Pal-10 prior to ANIT administration. The results suggest that fibrin(ogen), but not PAR-1, contributes to the progression of ANIT hepatotoxicity in mice. Moreover, the data suggest a dual role for PAR-4 in ANIT hepatotoxicity, both mediating an early protection against peliosis and contributing to the progression of hepatocellular necrosis.

FIG. 1.

Induction of fibrinogen and SAA1 expression in ANIT-treated mice. Wild-type C57Bl/6J mice were treated with vehicle (corn oil) or ANIT (60 mg/kg, po). Hepatic (A) fbgβ, (B) fbgγ, and (C) fbgα mRNA levels, (D) plasma fibrinogen concentration, and (E) SAA1 mRNA expression were determined 24 and 48 h later. Levels of all biomarkers in mice given vehicle were not different between 24 and 48 h and were pooled. Data are expressed as mean ± SEM. n = 5 mice per group. *Significantly different from vehicle-treated mice.

FIG. 2.

Effect of fibrinogen depletion on ANIT-induced liver injury. ANIT-treated (60 mg/kg, po) wild-type C57Bl/6J mice were given multiple injections of ancrod (1.75 U/mouse, ip) or PBS as described in “Materials and Methods” section. (A) Plasma fibrinogen levels and (B) hepatic fibrin deposition (representative Western blot shown) were determined 48 h after ANIT administration. (C) Serum ALT, (D) ALP activity, (E) and bile acid levels were determined 24 and 48 h after ANIT administration. For analysis of liver histopathology, the (F) average number of necrotic lesions per ×40 field, (G) average lesion size, and (H) percentage of necrotic tissue were determined as described in “Materials and Methods” section. Data are expressed as mean ± SEM. n = 10 mice per group for circulating biomarkers in the blood and 5 mice per group for analysis of liver histopathology. *Significantly different from ANIT-treated mice given PBS.

FIG. 3.

Effect of complete fibrinogen deficiency on ANIT-induced liver injury. Fbg^+/− mice and Fbg^−/− mice were treated with ANIT (60 mg/kg, po), and (A) serum ALT, (B) ALP activity, (C) and bile acid levels were determined 24 and 48 h later. For analysis of liver histopathology, the (D) average number of necrotic lesions per ×40 field, (E) average lesion size, and (F) percentage of necrotic tissue were determined as described in “Materials and Methods” section. Data are expressed as mean ± SEM. n = 7 Fbg^+/− mice and 5 Fbg^−/− mice per group at 24 h and 11 Fbg^+/− mice and 7 Fbg^−/− mice per group at 48 h. *Significantly different from ANIT-treated Fbg^+/− mice. Representative photomicrographs showing hematoxylin- and eosin-stained liver sections from ANIT-treated (G) Fbg^+/− mice and (H) ANIT-treated Fbg^−/− mice.

FIG. 4.

Effect of PAR-1 deficiency on ANIT-induced liver injury. PAR-1^+/+ mice and PAR-1^−/− mice were treated with ANIT (60 mg/kg, po), and (A) serum ALT, (B) ALP activity, and (C) bile acid levels were determined 48 h later. For analysis of liver histopathology, the (D) average number of necrotic lesions per ×40 field, (E) average lesion size, and (F) percentage of necrotic tissue were determined as described in “Materials and Methods” section. Data are expressed as mean ± SEM. n = 8 PAR-1^+/+ mice and 7 PAR-1^−/− mice per group.

FIG. 5.

Effect of PAR-4 deficiency on ANIT-induced liver injury. PAR-4^+/+ mice and PAR-4^−/− mice were treated with ANIT (60 mg/kg, po), and (A) serum ALT, (B) ALP activity, and (C) bile acid levels were determined 24 and 48 h later. For analysis of liver histopathology, the (D) average number of necrotic lesions per ×40 field, (E) average lesion size, and (F) percentage of necrotic tissue were determined as described in “Materials and Methods” section. Representative photomicrographs of hematoxylin- and eosin-stained liver sections from ANIT-treated PAR-4^+/+ mice at (G) 24 and (I) 48 h and ANIT-treated PAR-4^−/− mice at (H) 24 h and (J) 48 h are shown. Bar = 10 μm. Data are expressed as mean ± SEM. n = 4 mice per group at 24 h and 6 mice per group at 48 h. *Significantly different from mice of the same genotype at 24 h. #Significantly different from ANIT-treated PAR-4^+/+ mice at that time.

FIG. 6.

Effect of delayed PAR-4 inhibitor treatment on ANIT-induced liver injury. ANIT-treated (60 mg/kg, po) wild-type C57Bl/6J mice were treated with P4Pal-10 (0.5 mg/kg, sc) or PBS 8 and 24 h after ANIT, as described in “Materials and Methods” section. (A) Serum ALT, (B) ALP activity, and (C) bile acid levels were determined 48 h after ANIT administration. For analysis of liver histopathology, the (D) average number of necrotic lesions per ×40 field, (E) average lesion size, and (F) percentage of necrotic tissue were determined as described in “Materials and Methods” section. Representative photomicrographs of hematoxylin- and eosin-stained liver sections from ANIT-treated mice given (G) PBS or (H) P4Pal-10 are shown. Data are expressed as mean ± SEM. n = 10 mice treated with PBS and 10 mice treated with P4Pal-10. *Significantly different from ANIT-treated mice given PBS.

FIG. 7.

Effect of PAR-4 inhibitor pretreatment on ANIT-induced liver injury. ANIT-treated (60 mg/kg, po) wild-type C57Bl/6J mice were given P4Pal-10 (0.5 mg/kg, sc) or PBS 2 h prior to and 8 h after ANIT, as described in “Materials and Methods” section. (A) Serum ALT, (B) ALP activity, and (C) bile acid levels were determined 24 h later. Representative photomicrographs of hematoxylin- and eosin-stained liver sections from ANIT-treated mice given (D) PBS or (E) P4Pal-10 are shown. n = 6 mice per group. *Significantly different from ANIT-treated mice given PBS.