Recombinant C-terminal heparin-binding domain of fibronectin polypeptide protects against liver damage, reduces serum inflammatory cytokines, and decreases mortality in acute liver failure

Abstract

This study aimed to evaluate the effect of recombinant C-terminal heparin-binding domain of fibronectin (FNCHBD) polypeptide on live-damage protection, inflammation, and mortality in acute liver failure (ALF) mice. 25 mice were randomly divided into five groups: normal controls, lipopolysaccharide (LPS)/D-galactosamine (GalN), 5 mg/kg FNCHBD, 10 mg/kg FNCHBD and 20 mg/kg FNCHBD groups. Blood samples were obtained at 9 h after treatment for measurement of liver indexes and inflammatory cytokine levels, and livers were acquired for H&E and TUNEL staining assays. 90 mice (18 mice in each group) were randomly divided into five groups for mortality assessment after LPS/GalN administration at 48 h. Compared to LPS/GalN group, levels of blood liver indexes including AST, ALT and TBIL were decreased in FNCHBD polypeptide-treated groups. H&E staining disclosed FNCHBD polypeptide protected cell morphology and histomorphology, and necrosis rates in FNCHBD polypeptide-treated groups were lower compared to LPS/GalN group. TUNEL staining assay revealed cell apoptosis was inhibited in FNCHBD polypeptide-treated groups compared to LPS/GalN group. Serum inflammatory cytokines including TNF-α, IL-1β, and IL-6 were reduced in FNCHBD polypeptide-treated groups compared to LPS/GalN group. As to mortality rate, it was only decreased in 10 mg/kg FNCHBD and 20 mg/kg FNCHBD groups but not in 5 mg/kg FNCHBD compared to LPS/GaIN group. In addition, most effects of FNCHBD presented in a dose-dependent manner. FNCHBD polypeptide protects against liver damage, inhibits elevation of serum inflammatory cytokines, and decreases mortality in ALF.

Keywords: C-terminal heparin-binding domain; acute liver failure; fibronectin; liver damage; mortality; serum inflammatory cytokines.

Figure 1

Assessments of blood liver indexes in FNCHBD polypeptide-treated groups and LPS/GalN group. AST level was decreased in 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group and 20 mg/kg FNCHBD group respectively compared to LPS/GalN group (A). ALT level in 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group and 20 mg/kg FNCHBD group was lower than that in LPS/GalN group respectively (B). TBIL level was reduced in 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group, and 20 mg/kg FNCHBD group respectively compared to LPS/GalN group (C). Comparison between groups was performed by one-way ANOVA followed by Tukey’s multiple comparisons test. FNCHBD, C-terminal heparin-binding domain of fibronectin; LPS/GalN, lipopolysaccharide/D-galactosamine; AST, aspartate transaminase; ALT, alanine transaminase; TBIL, total bilirubin. P<0.05 was considered significant. **, FNCHBD polypeptide-treated groups vs. LPS/GalN group, P<0.01; ***, FNCHBD polypeptide-treated groups vs. LPS/GalN group, P<0.001; ###, LPS/GalN group vs. normal control group, P<0.001.

Figure 2

Histological alterations of the liver by H&E staining. Decreased degree of liver damage in a dose-dependent manner was observed in FNCHBD polypeptide-treated groups including 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group and 20 mg/kg FNCHBD group compared to LPS/GalN group (A). As to necrosis rate, it was lower in 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group and 20 mg/kg FNCHBD group respectively compared to LPS/GalN group (B). Comparison between two groups was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. H&E, hematoxylin-eosin; FNCHBD, FNCHBD, C-terminal heparin-binding domain of fibronectin; LPS/GalN, lipopolysaccharide/D-galactosamine. P<0.05 was considered significant. **, FNCHBD polypeptide-treated groups vs. LPS/GalN group, P<0.01; ***, FNCHBD polypeptide-treated groups vs. LPS/GalN group, P<0.001; ###, LPS/GalN group vs. normal controls group, P<0.001.

Figure 3

TUNEL staining assay. Compared to LPS/GaIN group, the number of TUNEL-positive hepatocytes was much lower in 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group, and 20 mg/kg FNCHBD group (A). Apoptosis rates in 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group and 20 mg/kg FNCHBD group were reduced compared to LPS/GaIN group (B). Comparison between two groups was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; LPS/GaIN, lipopolysaccharide/D-galactosamine; FNCHBD, C-terminal heparin-binding domain of fibronectin. P<0.05 was considered significant. ***, FNCHBD polypeptide-treated groups vs. LPS/GalN group, P<0.001; ###, LPS/GalN group vs. normal controls group, P<0.001.

Figure 4

Assessments of serum inflammatory cytokines. Compared to LPS/GaIN group, TNF-α level was decreased in 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group and 20 mg/kg FNCHBD group (A); IL-1β level was reduced in 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group and 20 mg/kg FNCHBD group (B); IL-6 level was lower in 5 mg/kg FNCHBD group, 10 mg/kg FNCHBD group and 20 mg/kg FNCHBD group (C). Comparison between two groups was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. LPS/GaIN, lipopolysaccharide/D-galactosamine; TNF-α, tumor necrosis factor α; FNCHBD, C-terminal heparin-binding domain of fibronectin; IL-1β, Interleukin 1β. P<0.05 was considered significant. **, FNCHBD polypeptide-treated groups vs. LPS/GalN group, P<0.01; ***, FNCHBD polypeptide-treated groups vs. LPS/GalN group, P<0.001; ###, LPS/GalN group vs. normal control group, P<0.001.

Figure 5

Mortality assessment. The mortality rate was unchanged in 5 mg/kg FNCHBD group compared to LPS/GaIN group, whereas it was decreased in 10 mg/kg FNCHBD group and 20 mg/kg FNCHBD group respectively compared to LPS/GaIN group. Comparison between two groups was determined by Chi-square test. FNCHBD, C-terminal heparin-binding domain of fibronectin; LPS/GaIN, lipopolysaccharide/D-galactosamine. P<0.05 was considered significant. **, FNCHBD polypeptide-treated groups vs. LPS/GalN group, P<0.01; ***, FNCHBD polypeptide-treated groups vs. LPS/GalN group, P<0.001; ###, LPS/GalN group vs. normal control group, P<0.001.