Effect of hepatocyte apoptosis induced by TNF-alpha on acute severe hepatitis in mouse models

Abstract

AIM:To study the effect of hepatocyte apoptosis and necrosis induced by TNF-alpha on the pathogenesis of acute severe hepatitis (ASH).METHODS:The model of ASH was prepared in D-galactosamine (GalN) sensitized BALB/c mice by injection of either endotoxin (ET) or tumor necrosis factor-alpha(TNF-alpha. Morphological changes of apoptotic hepatocytes were studied by both light and electron microscope and in site end labeling method (ISEL). Molecular biological changes of DNA ladder were observed by electrophoresis of extract from liver tissues. Biochemical changes were measured by alanine aminotransferase (ALT), aspartic aminotransferase (AST) and TNF-alpha. The relation between apoptosis and necrosis was evaluated simultaneously.RESULTS:The sequence of hepatocyte apoptosis, necrosis, and final death from ASH was observed both in GalN/ET and GalN/TNF-alpha group. Apoptosis was prominent at 3.5h and 6h after injection of inducer, while necrosis became dominant at 9h after challenge. The appearance of apoptosis was earlier in GalN/TNF-alpha group than that in GalN/ET group. Pretreatment of mice with antiTNF IgG1 may completely prevent the liver injury induced by GalN/ET.CONCLUSION:TNF-alpha can cause liver amage by inducing hepatic apoptosis and necrosis in mice with endotoxemia.

Figure 1

Liver cells apoptosis in mice induced by GalN/ET.A 3.5 h after GalN/ET, individual apoptotic cells are visible, apoptotic positive signal mainly locates in nucleus. ISEL × 400 B 9 h after GalN/ET, further increase of apoptotic liver cells and pieces of liver cell necrosis and bleeding with leukocytes infiltration appear. Apoptotic cells (a), apoptotic bodies (b), leukocytes (1) and necrosis (n). HE × 400

Figure 2

Liver cells apoptosis in mice induced by GalN/TNF-α. A. 6 h after GalN/TNF-α, a lot of apoptotic liver cells can be seen. Apototic bodies appear on the section, but liver necrosis is not obvious. HE × 200 B. 6 h after GalN/TNF-α, a great number of apoptotic liver cells are found. ISEL × 400

Figure 3

Analysis of DNA ladder from liver cell extract. A. (1) Marker (λ DNA/Hind III); (2) GalN/ET 9 h; (3) GalN/ET 6 h; (4) GalN/ET 3.5 h; (5) GalN 6 h; (6) ET 6 h; (7) Normal liver cells; (8) Dexamethasone induced apoptosis of thymus cells. B. (1) Marker (λ DNA/Hind-III); (2) GalN/TNF 9 h; (3) GalN/TNF 6 h; (4) GalN/TNF 3.5 h; (5) GalN 6 h; (6) TNF 6 h; (7) Normal liver cells; (8) Anti-TNF-α/GalN/ET 9 h.

Figure 4

Apoptitic liver cell under electron microscope. Chromatin condensation in apoptotic cell to form cresent moon body locating near the nucleus lining. × 6000