Tetrathiomolybdate protects against bile duct ligation-induced cholestatic liver injury and fibrosis

Abstract

Tetrathiomolybdate (TM), a potent copper-chelating drug, was initially developed for the treatment of Wilson's disease. Our working hypothesis is that the fibrotic pathway is copper-dependent. Because biliary excretion is the major pathway for copper elimination, a bile duct ligation (BDL) mouse model was used to test the potential protective effects of TM. TM was given in a daily dose of 0.9 mg/mouse by means of intragastric gavage 5 days before BDL. All the animals were killed 5 days after surgery. Plasma liver enzymes and total bilirubin were markedly decreased in TM-treated BDL mice. TM also inhibited the increase in plasma levels of tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 seen in BDL mice. Cholestatic liver injury was markedly attenuated by TM treatment as shown by histology. Hepatic collagen deposition was significantly decreased, and it was paralleled by a significant suppression of hepatic smooth muscle alpha-actin and fibrogenic gene expression in TM-treated BDL mice. Although the endogenous antioxidant ability was enhanced, oxidative stress as shown by malondialdehyde and 4-hydroxyalkenals, hepatic glutathione/oxidized glutathione ratio, was not attenuated by TM treatment, suggesting the protective mechanism of TM may be independent of oxidative stress. In summary, TM attenuated BDL-induced cholestatic liver injury and fibrosis in mice, in part by inhibiting TNF-alpha and TGF-beta1 secretion. The protective mechanism seems to be independent of oxidative stress. Our data provide further evidence that TM might be a potential therapy for hepatic fibrosis.

Fig. 1

Plasma ceruloplasmin levels after 5-day bile duct ligation. BDL or sham surgery (Sham) was performed in male C57BL/6J mice as described under Materials and Methods. Some mice were pretreated with tetrathiomolybdate (TM) at 0.9 mg/mouse/day by intragastric gavage 5 days before BDL until 5 days after BDL (BDL + TM), and some mice received the same amount of tetrathiomolybdate from the day of sham surgery (sham + TM). Ceruloplasmin levels were determined in plasma samples. Data represent means ± S.E.M. (n = 5). *, significantly different from sham group; †, significantly different from BDL group.

Fig. 2

Effect of TM on plasma liver enzymes, total bilirubin, and liver/body weight ratio on sham-operated and BDL mice. The animals were subjected to the same treatment protocol as described in Fig. 1. ALT, AST, γ-GTP, ALP, and total bilirubin were determined in plasma samples by colorimetric assay. Data represent means ± S.E.M. (n = 5). *, significantly different from sham group; †, significantly different from BDL group.

Fig. 3

BDL-induced histopathological changes in the livers 5 days after surgery. Representative photomicrographs of liver sections processed for Masson’s trichrome staining: sham (A), sham + TM (B), BDL (C), and BDL + TM (D). Extensive bile infarcts, bile duct proliferation, and bridging fibrosis in untreated BDL mice is shown in C. All these lesions were markedly attenuated in TM-treated BDL mice (D). No pathological changes were observed in liver tissues with sham operation and sham + TM (A and B). Original magnification, 100×.

Fig. 4

Plasma TNF-α and TGF-β1 levels after 5 days BDL or sham surgery. Plasma TNF-α and TGF-β1 levels were determined by enzyme-linked immunosorbent assay. There is no significant difference between sham and sham + TM group in both TNF-α and TGF-β1 levels. Plasma TGF-β1 in BDL + TM group is significantly decreased compared with BDL group. There is no significant difference among BDL + TM, sham, and sham +TM group. Data represent means ± S.E.M. (n = 5). *, significantly different from sham group; †, significantly different from BDL group.

Fig. 5

Hepatic collagen accumulation after 5 days BDL. Representative photomicrographs of liver sections processed for Sirius red staining: sham (A), sham + TM (B), BDL (C), and BDL + TM (D). In sham-operated and sham-operated plus TM mouse livers (A and B), only normal staining around vessels was observed, whereas mild bridging fibrosis was seen in livers from BDL mice (C), and it was markedly reduced in TM-treated BDL mouse livers (D). Original magnification, 100×. Quantification of Sirius red-positive staining showed that collagen content in BDL + TM group is significantly decreased compared with BDL group (E). There is no significant difference among BDL + TM, sham, and sham + TM group. Data represent means ± S.E.M. (n = 5). *, significantly different from sham group; †, significantly different from BDL group.

Fig. 6

Hepatic α-SMA expression after 5 days BDL. Representative photomicrographs of immunohistochemistry staining for liver α-SMA. A, sham. B, sham + TM. C, BDL. D, BDL + TM. α-SMA expression was significantly increased in the liver of mice with BDL (C) compared with that in sham-operated mice (A and B), and it was markedly diminished in the liver of BDL mice treated with TM (D). Original magnification, 100×.

Fig. 7

Hepatic fibrogenic gene expression after 5 days BDL. Real-time RT-PCR was performed as described under Materials and Methods to determine hepatic TIMP-1, MMP-9, procollagen I α1, and PAI-1 mRNA expression. The expression was normalized as a ratio using GAPDH as housekeeping gene. A value of 1 for this ratio was arbitrarily assigned to the data obtained from sham-operated mice. Data represent means ± S.E.M. (n = 5). *, significantly different from BDL group.

Fig. 8

Oxidative Stress after 5 days BDL. Lipid peroxidation was assessed by MDA + 4-HAE in liver homogenate. Liver GSH/GSSG ratio was determined by HPLC. SOD1 expression was examined by Western blot analysis using whole liver extract, and optical density of band was quantified by ImageJ software. A value of 1 was arbitrarily assigned to the data obtained from sham-operated mice. MDA + 4-HAE and liver GSH/GSSG ratio are significantly increased in both BDL and BDL + TM group compared with sham and sham + TM group. There is no significant difference between BDL and BDL + TM group. Data represent means ± S.E.M. (n = 5). *, significantly different from sham group.