Recombinant murine TNF-α and immunoglobulin (Ig) G1 fraction of anti-murine TNF-α were purchased from Pepro Tech EC LTD. Terminal-deoxynucleotidyl transferase (TdT) and Bio-11-dUTP Na salt were purchased from Dako LTD. Salmonella abortus equi endotoxin (ET) was purchased from Sigma Chemical Co. GalN was purchased from Chong Qing Medical University. Endogen mouse TNF-α ELISA Kit was provided by Endogen Inc. Unless otherwise specified, all other reagents were analytical reagents.

One hundred and thirty specific pathogen-free male BALB/c mice (from Shanghai Second Medical university animal breeding house) were divided into 7 groups: ① Control; ② GalN; ③ ET; ④ TNF-α; ⑤ GalN/ET; ⑥ GalN/TNF-α; and ⑦ anti-TNF-α IgG1/GalN/ET.

Each group contained 20 mice except Group 7 which contained 10 mice. Both GalN (800 mg/kg) and ET (2.4 μg/kg) were injected intraperitoneally; Both TNF-α (1.0 μg/kg) and anti-TNF-α IgG1 (100 μg/mouse) were injected in tail vein. The control group received the same volume of normal saline.

Five mice were killed at 1.5 h, 3.5 h, 6 h, and 9 h respectively after injection. TNF-α, ALT, and AST were measured in the blood samples taken from the mouse heart. Tissue samples taken from liver were also prepared for morphological and molecular biological examinations.

The sections of liver tissue were stained with hematoxylin and eosin (HE), and ISEL[12] for detection of apoptotic liver cells were performed, DNA was extracted from fresh liver tissue for further analysis of DNA ladder.

Hepatic lobular architecture was clear and intact without any abnomalities in the liver section of control group. Only mild swelling of hepatic cell was presented in GalN, ET and TNF-α groups. With the time prolonging from 1.5 h, 3.5 h, 6 h, to 9 h, the mild swelling become moderate degree. Neither apoptosis nor necrosis was present in HE and ISEL staining. No DNA ladder was found in any group mentioned above.

No obvious liver cell alterations were present at 1.5 h on the section of HE staining and ISEL staining failed to detect positive signals of apoptosis.

Mild swelling of liver cells on HE section and a few apoptotic cells on ISEL section were present at 3.5 h (Figure 1A).

Open in New Tab   Full Size Figure   Download Figure

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

Obvious swelling of liver cells and lots of apoptotic cells were found at 6 h and mild dotted necrosis was also observed. Strong apoptotic positive signal s could be detected in the cell nuclei.

Nine hours after GalN/ET injection, enormous apoptotic liver cells and pieces of hepatic necrosis with leukocytes infiltration can be seen (Figure 1B).

These data demonstrate that apoptosis occurred as an early event during the development of hepatic failure. Compared with the ET model, the majority of changes occurred earlier in the TNF model, which agrees with the phenomena that ET act on hepatocytes by inducing TNF-α.

Morphological changes of the liver in this group were basically the same as those in GalN/ET group but severer at 1.5 h, 3.5 h, 6 h and 9 h ( Figure 2A, B), 1.5 h, 3 h, 6 h and 9 h after adiministrating inducer, apoptosis positive rates of GalN/ET group were 0.0%, 0.2%, 1.21% and 3.14% respectively, while for the GalN/TNF-α group, were 0.2%, 0.5%, 2.57% and 3.19% respectively.

Open in New Tab   Full Size Figure   Download Figure

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

DNA ladder was found at 6 h and 9 h after GalN/ET, GalN/TNF-α adiministration, but was not found at 1.5 h and 3.5 h after GalN/ET, GalN/TNF-α injection, and DNA ladder was not found in other groups (Figure 3A, B).

Open in New Tab   Full Size Figure   Download Figure

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.

Electron microscopic study showed the chromatin condensation of apoptotic cells and near the nuclear lining (Figure 4).

Open in New Tab   Full Size Figure   Download Figure

Figure 4 Apoptitic liver cell under electron microscope. Chromatin condensation in apoptotic cell to form cresent moon body locating near the nucleus lining. × 6000

In the GalN/ET group pretreated with anti-TNF-α IgG1, only mild swelling of liver cells could be seen on the HE staining. Neither apoptosis nor necrosis could be found on ISEL staining. No DNA ladder was present on electrophoresis of agarose gel (Figure 3A).

We compared the time course of typical morphology of apoptosis (chromatin condensation, apoptotic bodies) with ALT and AST (as a parameter for necrosis) in each group (Table 1). ALT and AST remained normal at 3.5 h after GalN/TNF-α challenge, at this time point, a small amount of apoptotic liver cells could be found. Apoptotic liver cells become obvious at 6 h, ALT and AST were elevated mildly at the same time. This finding indicated that apoptosis was already developed while the liver cell membrane still remained intact 6 h after challenge, suggesting that apoptosis occurred earlier than necrosis.

The prominent increase of ALT and AST occurred at 9 h, when a great number of necrotic liver cells were observed. Meanwhile, profuse apoptotic liver cells were also present even after the death of mice associated with ASH and eletrophoresis of agarose gel still showed DNA ladder at the final stage.