Plasma biomarkers of liver injury and inflammation demonstrate a lack of apoptosis during obstructive cholestasis in mice

Abstract

Cholestasis is a pathological common component of numerous liver diseases that results in hepatotoxicity, inflammation, and cirrhosis when untreated. While the predominant hypothesis in cholestatic liver injury remains hepatocyte apoptosis due to direct toxicity of hydrophobic bile acid exposure, recent work suggests that the injury occurs through inflammatory necrosis. In order to resolve this controversy, we used novel plasma biomarkers to assess the mechanisms of cell death during early cholestatic liver injury. C57Bl/6 mice underwent bile duct ligation (BDL) for 6-72 h, or sham operation. Another group of mice were given d-galactosamine and endotoxin as a positive control for apoptosis and inflammatory necrosis. Plasma levels of full length cytokeratin-18 (FL-K18), microRNA-122 (miR-122) and high mobility group box-1 protein (HMGB1) increased progressively after BDL with peak levels observed after 48 h. These results indicate extensive cell necrosis after BDL, which is supported by the time course of plasma alanine aminotransferase activities and histology. In contrast, plasma caspase-3 activity, cleaved caspase-3 protein and caspase-cleaved cytokeratin-18 fragments (cK18) were not elevated at any time during BDL suggesting the absence of apoptosis. In contrast, all plasma biomarkers of necrosis and apoptosis were elevated 6 h after Gal/End treatment. In addition, acetylated HMGB1, a marker for macrophage and monocyte activation, was increased as early as 12 h but mainly at 48-72 h. However, progressive neutrophil accumulation in the area of necrosis started at 6h after BDL. In conclusion, these data indicate that early cholestatic liver injury in mice is an inflammatory event, and occurs through necrosis with little evidence for apoptosis.

Keywords: ALT; Apoptosis; BDL; Bile duct ligation; Biomarkers; Cytokeratin-18; DAMP; FL-K18; GCDC; Gal/End; H&E; HMGB1; High mobility group box-1; ICAM-1; TNF-α; alanine aminotransferase; bile duct ligation; cK18; caspase-cleaved cytokeratin-18 fragment; damage associated molecular pattern; full length cytokeratin-18; galactosamine/endotoxin; glycochenodeoxycholate; hematoxylin and eosin; high mobility group box 1; intercellular adhesion molecule-1; miR-122; microRNA-122; tumor necrosis factor-alpha.

Figure 1

Liver injury in C56Bl/6 mice after treatment with galactosamine/endotoxin (Gal/End), bile duct ligation (BDL) or sham-operation. A) Plasma alanine aminotransferase (ALT) activities after Gal/End, BDL or sham-operation (6–72h). Data represent mean ± SE of n=4–6 animals per group. *P<0.05 (compared to untreated control or sham. B) Representative H&E-stained liver sections at 100x or a 400x enhanced Gal/End picture. Apoptotic cells are marked with black arrows; a necrotic cell is indicated by an asterisk.

Figure 2

Assessment of apoptosis in C56Bl/6 mice after treatment with galactosamine/endotoxin (Gal/End), bile duct ligation (BDL) or sham-operation. A) Caspase-3 activity was measured in plasma after BDL or Gal/End treatment. Data represent mean ± SE of n=3 animals per group. *P<0.05 (compared to untreated control). B) Western blot of procaspase-3 (32 kD) and its active 19 and 17 kD fragments in plasma of an untreated control, 3 or 6 h after Gal/End or 24 h after BDL as a representative time point.

Figure 3

Full length (FL-K18) and caspase-cleaved (cK18) cytokeratin-18 levels in plasma after galactosamine/endotoxin (Gal/End) treatment, bile duct ligation (BDL) or sham-operation. The values of sham-operation (<150 pmol/ml) were subtracted from the BDL values. Data represent mean ± SE of n=4–6 animals per group. *P<0.05 (compared to untreated control); ^#P<0.05 (compared to cK18 values)

Figure 4

Plasma micro-RNA 122 (miR-122) levels were measured after galactosamine/endotoxin (Gal/End) treatment, bile duct ligation (BDL) or sham-operation. The values of miR-122 are expressed as ratio to the reference let-7d and the internal standard, Lin-4. Data represent mean ± SE of n=4–6 animals per group. *P<0.05 (compared to untreated control); ^#P<0.05 (compared to sham-operated control).

Figure 5

Total (A) and acetylated (B) forms of plasma high mobility group box 1 (HMGB1) protein were measured after galactosamine/endotoxin (Gal/End) treatment, bile duct ligation (BDL) or sham-operation. C. Percentage of acHMGB1 compared to the total HMGB1 was calculated. Data represent mean ± SE of n=4–6 animals per group. *P<0.05 (compared to untreated control); ^#P<0.05 (compared to sham-operated control).

Figure 6

Neutrophil accumulation in livers after galactosamine/endotoxin (Gal/End) treatment, bile duct ligation (BDL) or sham-operation. Neutrophils were stained using an anti-neutrophil allotypic antibody against Ly6B2. A) The numbers of neutrophils were counted in 20 randomly selected high power fields (HPF) per section. Data represent mean ± SE of n=4–6 animals per group. *P<0.05 (compared to untreated control); ^#P<0.05 (compared to sham-operated control). B) Representative images (x100) of stained liver sections are shown. After BDL, neutrophils were largely associated with areas of infarct (arrows).