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. 2014 Jun;33(6):1498-506.
doi: 10.3892/ijmm.2014.1730. Epub 2014 Apr 7.

Nature and mechanisms of hepatocyte apoptosis induced by D-galactosamine/lipopolysaccharide challenge in mice

Yi-Hang Wu  1 , Shao-Qing Hu  1 , Jun Liu  1 , Hong-Cui Cao  2 , Wei Xu  2 , Yong-Jun Li  2 , Lan-Juan Li  2
Affiliations

Nature and mechanisms of hepatocyte apoptosis induced by D-galactosamine/lipopolysaccharide challenge in mice

Yi-Hang Wu et al. Int J Mol Med. 2014 Jun.

Abstract

Apoptosis plays a role in the normal development of liver. However, overactivation thereof may lead to hepatocellular damage. The aim of this study was to assess D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptotic changes in mice and clarify the mechanisms involved in this process. DNA ladder detection was employed to determine the induction condition of hepatic apoptosis. An initial test indicated that typical hepatocyte apoptosis was observed at 6-10 h after the intraperitoneal injection of D-GalN (700 mg/kg) and LPS (10 µg/kg). Subsequently, we evaluated hepatocyte apoptosis at 8 h after administering D-GalN/LPS by histopathological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end‑labeling (TUNEL) detection, flow cytometry and electron microscopy analysis. To clarify the apoptosis-related gene expression, the expression levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), caspase-3, and Fas/Fas ligand (FasL) were determined by serum enzyme immunoassay, immunohistochemistry and western blot analysis. Strong apoptotic positive signals following D-GalN/LPS injection were observed from the results of the serum analysis, histopathological and immunohistochemical analyses, DNA ladder detection, TUNEL detection, flow cytometry and electron microscopy analysis. Additionally, apoptotic hepatocytes were mainly at the late stage of cell apoptosis. The expression of TNF-α, TGF-β1, caspase-3 and Fas/FasL was significantly increased. In conclusion, this study evaluated the D-GalN/LPS-induced hepatocyte apoptotic changes and clarified the apoptosis-related gene expression in mice. The hepatocyte apoptosis induced by D-GalN/LPS may be mainly regulated by the death receptor pathway. TGF-β signaling pathway may also play a vital role in this process of hepatocyte apoptosis.

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Figures

Figure 1
Figure 1
Analysis of DNA ladder from the liver tissues at 6–24 h after the d-galactosamine (d-GalN)/lipopolysaccharide (LPS) injection. Lane M, marker (molecular weight standard); lanes 1–3, isolated liver cells at 6 h; lanes 4–6, isolated liver cells at 8 h; lanes 7–9, isolated liver cells at 10 h; lanes 10–12, isolated liver cells at 12 h; and lanes 13–15, isolated liver cells at 24 h after the d-GalN/LPS treatment.
Figure 2
Figure 2
Histopathological analysis of d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptosis model in mice [hematoxylin and eosin (H&E) ×400]. (A) Normal central vein and hepatocyte structure in untreated control mouse; (B) d-GalN/LPS-treated mouse showing apoptotic liver cells (arrowhead) and hepatic necrosis with leukocytes infiltration etc.
Figure 2
Figure 2
Histopathological analysis of d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptosis model in mice [hematoxylin and eosin (H&E) ×400]. (A) Normal central vein and hepatocyte structure in untreated control mouse; (B) d-GalN/LPS-treated mouse showing apoptotic liver cells (arrowhead) and hepatic necrosis with leukocytes infiltration etc.
Figure 3
Figure 3
Immunohistochemical analysis of caspase-3 expression of the apoptotic hepatocyte (magnification, ×400). (A) Untreated control mouse; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse showing the positive expression of caspase-3 (brown stain).
Figure 3
Figure 3
Immunohistochemical analysis of caspase-3 expression of the apoptotic hepatocyte (magnification, ×400). (A) Untreated control mouse; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse showing the positive expression of caspase-3 (brown stain).
Figure 4
Figure 4
Immunohistochemical analysis of Fas expression of the apoptotic hepatocyte (magnification, ×400). (A) Untreated control mouse; (B) d-GalN/lipopolysaccharide (LPS)-treated mouse showing the positive expression of Fas receptor (brown stain).
Figure 4
Figure 4
Immunohistochemical analysis of Fas expression of the apoptotic hepatocyte (magnification, ×400). (A) Untreated control mouse; (B) d-GalN/lipopolysaccharide (LPS)-treated mouse showing the positive expression of Fas receptor (brown stain).
Figure 5
Figure 5
Immunohistochemical analysis of Fas ligand (FasL) expression of the apoptotic hepatocyte (magnification, ×400). (A) Untreated control mouse; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse showing the positive expression of FasL (brown stain).
Figure 5
Figure 5
Immunohistochemical analysis of Fas ligand (FasL) expression of the apoptotic hepatocyte (magnification, ×400). (A) Untreated control mouse; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse showing the positive expression of FasL (brown stain).
Figure 6
Figure 6
Analysis of DNA ladder from the liver tissues at 8 h after the d-galactosamine (d-GalN)/lipopolysaccharide (LPS) injection. Lane M, marker (molecular weight standard); lanes 1 and 2 to the left of lane M, normal liver cells; lanes 1–4 to the right of lane M, isolated liver cells at 8 h after d-GalN/LPS treatment.
Figure 7
Figure 7
TUNEL detection of apoptotic hepatocytes (magnification, ×400). (A) Untreated control mouse; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse showing apoptotic liver cells (arrowhead).
Figure 7
Figure 7
TUNEL detection of apoptotic hepatocytes (magnification, ×400). (A) Untreated control mouse; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse showing apoptotic liver cells (arrowhead).
Figure 8
Figure 8
Western blot analysis of Fas, Fas ligand (FasL) and caspase-3 expression in liver tissues. The GAPDH antibody was used as internal control. Sample 1 from the untreated control mice; Samples 2–8 from the mice at 8 h after the d-galactosamine (d-GalN)/lipopolysaccharide (LPS) injection.
Figure 9
Figure 9
Flow cytometric analysis of apoptotic liver cells. (A) Untreated control mouse; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse. Live cells show only a low level of fluorescence; apoptotic cells show a higher level of blue fluorescence, and dead cells show low-blue and high-red fluorescence. Q1 district showing injury cells; Q2 district showing necrotic cells and apoptotic cells at late stage; Q3 district showing normal live cells; Q4 district showing apoptotic cells at early stage.
Figure 9
Figure 9
Flow cytometric analysis of apoptotic liver cells. (A) Untreated control mouse; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse. Live cells show only a low level of fluorescence; apoptotic cells show a higher level of blue fluorescence, and dead cells show low-blue and high-red fluorescence. Q1 district showing injury cells; Q2 district showing necrotic cells and apoptotic cells at late stage; Q3 district showing normal live cells; Q4 district showing apoptotic cells at early stage.
Figure 10
Figure 10
Transmission electron microscopy analysis of apoptotic liver cells (magnification, ×15,000). (A) Untreated control mouse showing normal hepatocyte structure; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse showing chromatin condensation in apoptotic cells to form crescent moon apoptotic body (arrowhead) located near the nucleus membrane.
Figure 10
Figure 10
Transmission electron microscopy analysis of apoptotic liver cells (magnification, ×15,000). (A) Untreated control mouse showing normal hepatocyte structure; (B) d-galactosamine (d-GalN)/lipopolysaccharide (LPS)-treated mouse showing chromatin condensation in apoptotic cells to form crescent moon apoptotic body (arrowhead) located near the nucleus membrane.
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