Materials
All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) unless otherwise noted. Polyclonal antibodies against HMGB1 were raised in rabbits (Cocalico Biologicals, Reamstown, PA, USA), and titers were determined by immunoblotting as previously described [13]. Anti–HMGB1 antibodies were affinity-purified by using cyanogen bromide–activated Sepharose beads following standard procedures. Neutralizing activity of anti-HMGB1 was confirmed in HMGB1-stimulated macrophage cultures by assay of TNF-α release. In the presence of anti-HMGB1 antibody, neutralizing antibody was defined as inhibiting > 80% of HMGB1-induced TNF release. Sham IgG antibodies were purified from non-immunized rabbit IgG.
Ethical considerations
This research protocol complied with the regulations regarding the care and use of experimental animals published by the National Institutes of Health and was approved by the Institutional Animal Use and Care Committee of the University of Tampere Medical School. Male C57BL/6 mice weighing 20–25 g (University of Kuopio animal care center, Kuopio, Finland) were used in this study. The animals were maintained at the University of Kuopio Animal Research Center with a 12-hour light–dark cycle and free access to standard laboratory food and water. The animals were fasted over night prior to the experiments.
Animal experiments
In the first experiment, 10 mice were randomized into the APAP group and the control group (n = 5 for each group). 5 mice in the APAP group were i.p. injected with a single sub lethal dose of APAP (300 mg/kg dissolved in 1 mL sterile saline) and 5 mice in the control group were injected with same volume of saline not containing APAP. 24 hrs after APAP injection, the animals in each group were anesthetized with sodium pentobarbital (90 mg/kg i.p.) and blood was aspirated from the heart to measure serum HMGB1 by western blot.
In the second experiment, ALI was induced by a single dose of APAP (350 mg/kg dissolved in 1 mL sterile saline) administered by i.p. injection. 14 APAP challenged mice were then randomized into the anti-HMGB1 group (n = 6) and the sham IgG group (n = 8). 6 mice injected with saline not containing APAP served as the control group. The animals in the anti-HMGB1 group were given 1 dose of anti-HMGB1 antibody (400 μg per dose dissolved in 0.5 mL sterile saline) 2 hrs after APAP injection. The same amount of sham IgG and saline were given to the sham IgG group or the control group at equivalent time points. 24 hrs after APAP injection, all surviving mice in each group were anesthetized with sodium pentobarbital (90 mg/kg i.p.), serum was collected to measure ALT, AST and HMGB1 and the left lobe of the liver was stored in 10% formalin for pathology (HE staining).
In the third experiment, ALI was induced the same as above described. APAP injected mice were then randomized into the anti-HMGB1 group (n = 10) and the sham IgG group (n = 11). 6 mice injected with saline not containing APAP served as the control group. The animals in the anti-HMGB1 group were given 2 doses of anti-HMGB1 antibody (400 μg per dose dissolved in 0.5 mL sterile saline): the first dose was given 2 hrs after APAP injection, the second dose were given 24 hrs after the first dose of anti-HMGB1 antibody.
The same amount of sham IgG and saline were given to the sham IgG group or the control group at equivalent time points. 48 hrs after APAP injection, all surviving mice in each group were anesthetized with sodium pentobarbital (90 mg/kg i.p.), and the following procedures were performed: 1) blood was aspirated from the heart to measure serum ALT and AST; 2) the left lobe of the liver was stored in 10% formalin for pathology (HE staining); 3) the rest of the liver tissue was harvested and frozen for subsequent protein extractions.
In the fourth experiment, 3 separate groups of mice were used. ALI was induced the same as above described. The treatments remained the same as described in the second experiment except one more dose of anti-HMGB1 or sham IgG was given 48 h after the first dose and animals were sacrificed at 72 h time point. Except relatively smaller necrosis (8%) in the sham IgG group at 72 time point, the other parameters such as NF-κB and cyclin D1 at 72 h were comparable to the 48 h time point, therefore, this study focused on 24 h and 48 h time points.
Serum HMGB1 measurements
HMGB1 levels were measured by western immunoblotting analysis as described [7]. In brief, serum samples (100 μL) were ultrafiltered with Centricon 100 (Millpore). The elute was fractionated by SDS/PAGE, transferred to nylon membranes (Bio-Rad), and probed with purified IgG from anti-HMGB1 antiserum (5 μg/ ml) for western blot analysis. Polyclonal anti-HMGB1 IgG was purified by using protein A agarose according to the manufacturer’s instructions (Pierce). Membranes were visualized with an Enhanced Chemiluminescence substrate (ECL, Amersham Pharmacia Biotech) and exposed to X-ray film according to the manufacturer’s instructions.
Serum aminotransferase measurements
Serum levels of AST and ALT were measured at 37°C with a commercially available kit (Sigma Diagnostic).
Myeloperoxidase assay for hepatic neutrophils infiltration
Neutrophils infiltration was measured at 48 hours by determining MPO activity in liver tissue homogenates as previously described [14] and was used as an index of neutrophils infiltration in all groups. The MPO levels were expressed as units per gram of tissue (U/g).
Light histology studies
Formalin-fixed hepatic tissue was sectioned, stained with hematoxylin and eosin (H&E) and examined using light microscopy. Blind analysis was performed on all samples to determine the degree of lesion as previously described [15]. The percentage of necrosis was estimated by evaluating the number of microscopic fields with necrosis compared with the entire cross section. Inflammatory cell infiltration results were scored semi-quantitatively by averaging the number of inflammatory cells per microscopic field at a magnification of 200×. Five fields were evaluated per tissue sample; six animals in each group were examined at 24 h time point; six animals in the control group, nine animals in the sham IgG group and 10 animals in the anti-HMGB1 group were examined at 48 h.
Western blot for cyclin D1 measurement
Liver protein was extracted as previously described [13]. Equivalent amounts of protein were boiled in sample buffer and subjected to 7.5% precast SDS-polyacrylamide gels (Bio-Rad) and transferred to nylon membranes. Membranes were then probed with a specific antibody against cyclin D1 (Cell signaling Technology, Lexington, KY) protein, visualized with an Enhanced Chemiluminescence substrate (ECL, Amersham Pharmacia Biotech) and exposed to X-ray film according to the manufacturer’s instructions.
Assessment of NF-кB activation
NF-κB activation was determined by EMSA, as previously described [16]. The gels were dried and exposed to Biomax film (Kodak, Rochester, NY) at −70°C overnight with use of an intensifying screen.
Hepatocyte proliferation (DNA synthesis)
Since at 24 h time point, the APAP challenged C57/BL6 mice only showed occasional hepatocyte nuclei labeled for 5-bromo-2-deoxyuridine (BrdU) [17], therefore, BrdU test was performed only at 48 h time point in this study. To evaluate hepatocyte regeneration, mice from the 48 h groups were administered BrdU (50-mg/ kg i.p. injection at 46 h time point) 2 hours before they were killed. Parraffin-embedded liver sections were prepared and processed for immunohistochemistry using BrdU in-situ staining kits from BD Pharmingen (San Jose, CA, USA) according to the manufacturer’s instructions. Digital images of 5 low-power fields from each liver were obtained in a random and blinded fashion, and the number of BrdU-labeled hepatocyte nuclei was counted. The average number of BrdU-positive hepatocytes in each animal was used for subsequent analysis.
Statistics
Data are presented as means ± SEM. Continuous data were analyzed using student’s t-test or analysis of variance followed by Fisher’s LSD test. Probability levels less than 0.05 were considered significant (p < 0.05).