Carbon tetrachloride does not promote hepatic fibrosis in ob/ob mice via downregulation of lipocalin-2 protein

Abstract

Although leptin-deficient ob/ob mice have been investigated to determine whether hepatic steatosis promotes susceptibility to hepatotoxic insults, carbon tetrachloride (CCl₄)-induced hepatic fibrosis in ob/ob mice remains largely unknown. In this study, we evaluate the pathogenic mechanisms of hepatic fibrosis in CCl₄-treated wild-type (WT) and ob/ob mice and analyze some parameters related to lipogenesis, inflammation, fibrosis, oxidative stress, apoptosis, and autophagy. CCl₄ treatment attenuated liver weight and lipogenesis in ob/ob mice. Increased hepatic fibrosis-related proteins were reduced in CCl₄-treated ob/ob mice compared with CCl₄-treated WT mice. Specifically, the expression of lipocalin-2 (LCN2) was markedly reduced in CCl₄-treated ob/ob mice versus CCl₄-treated WT mice. Compared with CCl₄-treated WT mice, CCl₄-treated ob/ob mice had reduced expression of neutrophil-related inflammatory genes and proteins. Hepatic heme oxygenase-1 protein was reduced in CCl₄-treated ob/ob mice compared with CCl₄-treated WT mice. However, CCl₄ did not promote hepatic apoptosis in ob/ob mice. Therefore, these findings highlight LCN2 as a key signaling factor in CCl₄-induced hepatic fibrosis.

Keywords: Carbon tetrachloride; Hepatic fibrosis; Lipocalin-2; Ob/ob mouse.

Fig. 1

Effects of CCl₄on metabolic parameters in WT and ob/ob mice. (A) Weekly body weight changes over 4 weeks after CCl₄ treatment. Levels of serum (B) leptin, (C) insulin, (D) and glucose, and (E) liver weight. (F) Representative macroscopic pictures of livers. Scale bar, 1 cm. (G) Serum ALT and AST and (H) total cholesterol concentrations. Significance is determined using two-way ANOVA. ∗P < 0.05 vs. oil-treated WT. †P < 0.05 vs. CCl₄-treated WT.

Fig. 2

Effects of CCl₄on hepatic lipid accumulation in WT and ob/ob mice. (A) Representative Nile Red with DAPI staining of liver sections. Scale bar, 50 μm. (B) Nile Red-positive area (%). (C) Electron micrographs of hepatocytes. LD indicates lipid droplet. Scale bar, 2㎛. (D) Hepatic TG levels. (E) Western blot analysis and quantification of CD36, perilipin-2, PPAR-γ, FAS, and SCD1 proteins in liver lysates. β-actin is used as a loading control. Significance is determined using two-way ANOVA. ∗P < 0.05 vs. oil-treated WT. †P < 0.05 vs. CCl₄-treated WT.

Fig. 3

Effects of CCl₄on hepatic fibrosis in WT and ob/ob mice. (A) Representative images of Sirius Red and Masson trichrome staining of liver sections. Scale bar, 60 μm. (B–C) Quantification of Sirius Red (B) and Masson trichrome (C) staining. (D) Hepatic hydroxyproline concentration. (E) Western blot analysis and quantification of TGF-β1, α-SMA, lumican, and vimentin proteins in liver lysates. β-actin is used as a loading control. (F) Height images in liver sections. Scale bar, 1 μm. (G) Young's modulus (%) was obtained from force-distance curves using AFM measurements. Significance is determined by two-way ANOVA. ∗P < 0.05 vs. oil-treated WT. †P < 0.05 vs. CCl₄-treated WT.

Fig. 4

Effects of CCl₄on hepatic LCN2 protein expression in WT and ob/ob mice. (A) Serum LCN2 levels. (B) Western blot analysis and quantification of LCN2 protein in epididymal fat pad lysates. α-Tubulin is used as a loading control. (C) LCN2 mRNA in liver lysates quantified using qPCR. (D) Western blot analysis and quantification of LCN2, MMP9, pSTAT3, and STAT3 proteins in liver lysates. β-actin is used as a loading control. (E) Representative LCN2 immunostaining of liver sections. PV and CV indicate portal vein and central vein, respectively. The Arrowhead and arrow indicate LCN2-positive Kupffer cells and hepatocytes, respectively. Scale bar, 100 μm (upper panel), 10 μm (middle and lower panels). Significance is determined using two-way ANOVA. ∗P < 0.05 vs. oil-treated WT. †P < 0.05 vs. CCl₄-treated WT.

Fig. 5

Effects of CCl₄on hepatic inflammation in WT and ob/ob mice. (A) Representative images of H&E staining of liver sections. Scale bars, 60 μm (upper panel), 30 μm (lower panel). Yellow arrows indicate neutrophils. (B) MASLD activity score. (C) Western blot analysis and quantification of MPO protein in liver lysates. β-actin is used as a loading control. (D) Representative MPO and F4/80 immuno-staining of liver sections. Nuclei were counterstained with DAPI. Scale bar, 100 μm. (E) Western blot analysis and quantification of nuclear NF-kBp65 protein in liver lysates. p84 is used as a loading control. Significance is determined using two-way ANOVA. ∗P < 0.05 vs. oil-treated WT. †P < 0.05 vs. CCl₄-treated WT.

Fig. 6

Effects of CCl₄on hepatic cytokine and chemokines expression in WT and ob/ob mice. (A) Proinflammatory and anti-inflammatory cytokine and chemokine mRNA expression in liver lysates quantified using qPCR. (B) Cytokine and chemokine expression in liver lysates were determined using mouse cytokine arrays. (C) Bar graphs for each cytokine with mean pixel density. Significance is determined using two-way ANOVA. ∗P < 0.05 vs. oil-treated WT. †P < 0.05 vs. CCl₄-treated WT.

Fig. 7

Effects of CCl₄on hepatic oxidative stress in WT and ob/ob mice. (A) Western blot analysis and quantification of Nrf2, NQO-1, GPX-4, HO-1, iNOS, and 4-HNE proteins in liver lysates. Nuclear p84 and total β-actin are used as loading controls. (B) Western blot analysis and quantification of catalase protein in liver lysates. β-actin is used as a loading control. (C) Representative catalase-immunostaining of liver sections. DAPI was stained as the nucleus. CV indicates central vein. Scale bar, 50 μm. (D) Electron micrographs of hepatocytes. N, Nucleus; M, mitochondria; P, peroxisome; LD, lipid droplet. Scale bar, 1 μm. Significance is determined using two-way ANOVA. ∗P < 0.05 vs. oil-treated WT. †P < 0.05 vs. CCl₄-treated WT.

Fig. 8

Effects of CCl₄on hepatic apoptosis and autophagy flux in WT and ob/ob mice. (A) Western blot analysis and quantification of GS and cleaved caspase-3 proteins in liver lysates. β-actin is used as a loading control. (B) Representative GS-immuno-staining and TUNEL staining of liver sections. Yellow arrows indicate TUNEL-positive cells. DAPI was stained as the nucleus. Scale bar, 100 μm. (C) GS-positive area and TUNEL-positive cells. (D) Western blot analysis and quantification of LC3B and p62 proteins in liver lysates. β-actin is used as a loading control. (E) Electron micrographs of hepatocytes. N, nucleus; M, mitochondria; ML, mitolysosome; LD, lipid droplet; LL, lipolysosome. Scale bar, 1 μm. Significance is determined using two-way ANOVA. ∗P < 0.05 vs. oil-treated WT. †P < 0.05 vs. CCl₄-treated WT.