The antifibrinolytic drug tranexamic acid reduces liver injury and fibrosis in a mouse model of chronic bile duct injury

Abstract

Hepatic fibrin deposition has been shown to inhibit hepatocellular injury in mice exposed to the bile duct toxicant α-naphthylisothiocyanate (ANIT). Degradation of fibrin clots by fibrinolysis controls the duration and extent of tissue fibrin deposition. Thus, we sought to determine the effect of treatment with the antifibrinolytic drug tranexamic acid (TA) and plasminogen activator inhibitor-1 (PAI-1) deficiency on ANIT-induced liver injury and fibrosis in mice. Plasmin-dependent lysis of fibrin clots was impaired in plasma from mice treated with TA (1200 mg/kg i.p., administered twice daily). Prophylactic TA administration reduced hepatic inflammation and hepatocellular necrosis in mice fed a diet containing 0.025% ANIT for 2 weeks. Hepatic type 1 collagen mRNA expression and deposition increased markedly in livers of mice fed ANIT diet for 4 weeks. To determine whether TA treatment could inhibit this progression of liver fibrosis, mice were fed ANIT diet for 4 weeks and treated with TA for the last 2 weeks. Interestingly, TA treatment largely prevented increased deposition of type 1 collagen in livers of mice fed ANIT diet for 4 weeks. In contrast, biliary hyperplasia/inflammation and liver fibrosis were significantly increased in PAI-1(-/-) mice fed ANIT diet for 4 weeks. Overall, the results indicate that fibrinolytic activity contributes to ANIT diet-induced liver injury and fibrosis in mice. In addition, these proof-of-principle studies suggest the possibility that therapeutic intervention with an antifibrinolytic drug could form a novel strategy to prevent or reduce liver injury and fibrosis in patients with liver disease.

Figures

Fig. 1.

Hepatic fibrin deposition and antifibrinolytic activity of TA in mice fed ANIT diet. Male, WT C57Bl/6J mice were fed a control diet (AIN-93M) or an identical diet containing 0.025% ANIT for 4 weeks. (A) Representative photomicrographs (100×) show liver sections stained for fibrin (red) and counterstained with 4′,6-diamidino-2-phenylindole to visualize nuclei (blue). (B) Comparison of clot formation and lysis by turbidity (absorbance [A] 450 nm) in ANIT diet–fed mice treated with vehicle (n = 5 mice) or TA (1200 mg/kg i.p.) (n = 5 mice) twice daily for 2 weeks. Recalcified human pooled plasma was spiked with 10% mouse plasma from each group and clotting was initiated with thrombin (1 U/ml final) in the presence of 250 ng/ml human tPA. Data are expressed as mean ± S.E.M. An increase in absorbance implies fibrin clot formation and a decrease indicates lysis.

Fig. 2.

Effect of TA on liver injury in mice fed ANIT diet for 2 weeks. Male, WT C57Bl/6J mice were fed a control diet (AIN-93M) or an identical diet containing 0.025% ANIT for 2 weeks. Mice were treated with vehicle (sterile water) or TA twice daily (1200 mg/kg i.p.) for the duration of ANIT exposure. (A) and (B) Representative photomicrographs showing (A) low magnification (40×) and (B) high magnification (200×) hematoxylin and eosin–stained liver sections. Arrow indicates area of coagulative necrosis. Asterisk indicates area of biliary hyperplasia and portal inflammation. (C) Serum ALT activity and (D) bile acid concentration were determined as described under Materials and Methods. Data are expressed as mean ± S.E.M.; n = 3 mice per group for control diet and 6 or 7 mice per group for mice fed ANIT diet. *P < 0.05; significantly different from respective treatment fed a control diet.

Fig. 3.

Effect of TA on early hepatic profibrogenic changes in mice fed ANIT diet for 2 weeks. Male, WT C57Bl/6J mice were fed a control diet (AIN-93M) or an identical diet containing 0.025% ANIT for 2 weeks. Mice were treated with vehicle (sterile water) or TA twice daily (1200 mg/kg i.p.) for the duration of ANIT exposure. Hepatic levels of mRNAs encoding (A) TIMP1, (B) ITGB6, (C) TGFβ1, (D) TGFβ2, and (E) COL1A1 were determined using real-time quantitative PCR. (F) Deposition of type 1 collagen protein in liver was quantified as described under Materials and Methods. Data are expressed as mean ± S.E.M.; n = 3 mice per group for control diet and six or seven mice per group for mice fed ANIT diet. *P < 0.05; significantly different from respective treatment fed a control diet. #P < 0.05; significantly different from vehicle-treated mice fed the same diet.

Fig. 4.

Effect of TA treatment on liver injury in mice fed ANIT diet for 4 weeks. Male, WT C57Bl/6J mice were fed a control diet (AIN-93M) or an identical diet containing 0.025% ANIT for 4 weeks. Mice were treated with vehicle (sterile water) or TA twice daily (1200 mg/kg i.p.) beginning in week 3. Representative photomicrographs show low magnification (40×) (A) and high magnification (200×) (B) hematoxylin and eosin–stained liver sections. Arrow indicates area of coagulative necrosis. Asterisk indicates area of biliary hyperplasia and portal inflammation. Serum ALT activity (C) and bile acid concentration (D) were determined as described under Materials and Methods. Data are expressed as mean ± S.E.M.; n = 5 mice per group for control diet and 10 mice per group for mice fed ANIT diet. *P < 0.05; significantly different from respective treatment fed a control diet.

Fig. 5.

Effect of TA treatment on biliary hyperplasia in mice fed ANIT diet for 4 weeks. Male, WT C57Bl/6J mice were fed a control diet (AIN-93M) or an identical diet containing 0.025% ANIT for up to 4 weeks. Mice were treated with vehicle (sterile water) or TA twice daily (1200 mg/kg i.p.) beginning in week 3. (A) and (B) Representative photomicrographs showing CK19 staining in liver sections of mice fed a control and ANIT diet fed mice for 2 weeks (A) and 4 weeks (B). Images were converted to grayscale and inverted such that CK19 staining is dark. (C) Quantification of CK19 staining area as described under Materials and Methods. Data are expressed as mean ± S.E.M.; n = 3–5 mice per group for control diet and 7–10 mice per group for mice fed ANIT diet. *P < 0.05; significantly different from respective treatment fed a control diet. #P < 0.05, significantly different from vehicle-treated mice fed the same diet.

Fig. 6.

Effect of TA treatment on profibrogenic gene induction in livers of mice fed ANIT diet for 4 weeks. Male, WT C57Bl/6J mice were fed a control diet (AIN-93M) or an identical diet containing 0.025% ANIT for 4 weeks. Mice were treated with vehicle (sterile water) or TA twice daily (1200 mg/kg i.p.) beginning in week 3. Hepatic expression of mRNAs encoding ITGB6 (A), TGFβ1 (B), TGFβ2 (C), and TIMP1 (D) were determined using real-time quantitative PCR. Data are expressed as mean ± S.E.M.; n = 5–10 mice per group. *P < 0.05; significantly different from respective treatment fed a control diet. #P < 0.05; significantly different from vehicle-treated mice fed the same diet.

Fig. 7.

Effect of TA treatment on type 1 collagen expression and deposition in livers of mice fed ANIT diet for 4 weeks. Male, WT C57Bl/6J mice were fed a control diet (AIN-93M) or an identical diet containing 0.025% ANIT for 4 weeks. Mice were treated with vehicle (sterile water) or TA twice daily (1200 mg/kg i.p.) beginning in week 3. (A) Representative photomicrographs showing type 1 collagen (100×) and Sirius red–stained (200×) liver sections. Type 1 collagen images were converted to grayscale and inverted such that collagen staining is dark. (B) Collagen staining was quantified as described under Materials and Methods. (C) Hepatic mRNA expression levels of COL1A1 were determined using real-time quantitative PCR. Data are expressed as mean ± S.E.M.; n = 5–10 mice per group. *P < 0.05; significantly different from respective treatment fed a control diet. #P < 0.05; significantly different from vehicle-treated mice fed the same diet.

Fig. 8.

Effect of PAI-1 deficiency on liver injury and fibrosis in mice fed ANIT diet for 4 weeks. Male, WT and PAI-1−/− mice were fed a diet containing 0.025% ANIT for 4 weeks. Representative photomicrographs showing low magnification (40×) (A) and high magnification (100×) (B) hematoxylin and eosin–stained liver sections. Asterisks mark areas of biliary hyperplasia and lymphocytic inflammation. Arrowheads mark necrotic foci (bile infarcts). (C) Serum ALT activity was determined as described under Materials and Methods. (D) Hepatic mRNA expression levels of COL1A1 and α-SMA were determined using real-time quantitative PCR. Data are expressed as mean ± S.E.M.; n = 5 mice per group. (E) Representative photomicrographs showing type I collagen staining (red) on 4′,6-diamidino-2-phenylindole–counterstained (blue) liver sections of mice fed ANIT diet for 4 weeks. Collagen protein levels were quantified as described under Materials and Methods. Data are expressed as mean ± S.E.M.; n = 5 mice per group. *P < 0.05 vs. WT mice fed the same diet.