Ethics statement. Animal care and experiments were performed in accordance with the standards in the ARRIVE 47 and PREPARE 48 guidelines. In addition to these, our study was in accordance with the relevant guidelines and regulations, which was approved by the Animal Care Committee of Kansai Medical University (19-009 and 20-059). Animal models were created in compliance with the criteria of the ARRIVE 47 and PREPARE 48 guidelines. All methods proposed in these studies were also carried out according to the standards of relevant institutional guidelines and regulations.
Drugs. OMZ (20 mg) and recombinant human IL-1β (2 × 107 U/mg protein) were purchased from Nichi-iko Co., Ltd. (Toyama, Japan) and MyBioSource (San Diego, CA, USA). Isoflurane, pentobarbital sodium, collagenase, Transaminase CII-test kit, GalN, 10% formalin, and PicaGene Luminescence kit were from Wako Pure Chemical Industries (Osaka, Japan). LPS (Escherichia coli; O111:B4) and mouse anti-β-tubulin were from Sigma–Aldrich Japan (Tokyo, Japan). Enzyme-linked immunosorbent assay (ELISA) kits were from Life Technologies Japan (Tokyo, Japan). TRIzol™ Reagent was from Thermo Scientific (Waltham, MA, USA). T4 polynucleotide kinase, Oligo (dT) Primer (25 ng), dNTPs Mixture, RNase Inhibitor, and Rever Tra Ace® were from Toyobo (Osaka, Japan). Beta-Glo kits and mouse immunoglobulin κ light chain were from Promega (Fitchburg, WI, USA).
Animals. Male Wistar and Sprague–Dawley rats were purchased from Charles River Laboratories Japan (Yokohama, Japan) and maintained in a room at 22°C under a 12-h light/dark cycle were conducted with a diet of γ-irradiated CRF-1 (Oriental Bioservice, Kyoto, Japan) and water ad libitum.
Rat GalN/LPS and PH/LPS models. To examine effect of LPS on survival, acute liver injury was induced in the in vivo model. Male Sprague–Dawley rats (8 weeks old, 300 g ± 20) were anesthetized with isoflurane (Abbott Laboratories, Abbott Park, IL, USA) before receiving an i.v. injection of GalN/LPS (500 mg/kg GalN and 0.5–50 µg/kg LPS) via the penile vein 49. Survival was monitored for 3 days after GalN/LPS injection. To examine the effects of OMZ on survival in the liver injury model with GalN/LPS (2.5 µg/kg LPS), rats that were randomly assigned to receive OMZ were injected (i.p.) with various doses of OMZ (40–240 mg/kg) 1 h before GalN/LPS treatment. Survival was monitored for 5 days.
To investigate the effects of LPS on survival, the PH/LPS model was induced. Rats were anesthetized with pentobarbital and isoflurane prior to undergoing 70% hepatectomy, as reported previously 29,50. Forty-eight hours after surgery, LPS (6.25–250 µg/kg) was injected into the penile vein. Survival was monitored for 3 days after LPS injection. To determine the effects of OMZ levels on survival in the liver injury model with PH/LPS (25 µg/kg), rats were randomly divided into control, OMZ, PH/LPS, and PH/LPS + OMZ groups. Forty-eight hours after surgery, LPS (25 µg/kg) was injected into the penile vein. The rats that were randomly assigned to receive OMZ were injected (i.p.) with various doses of OMZ (40–100 mg/kg) 1 h before LPS treatment. Survival was monitored for 5 days. The rats were killed when they appeared weak and moribund because of the progression of liver failure, congestion, and multi-organ failure. We used the NIH Office of Animal Care and Use 51 score and severity assessment to assess the animals following liver resection 52. Liver and blood samples were collected from the rats 1 and 6 h after GaIN/LPS treatment, and 1 and 4 h after LPS treatment in PH/LPS.
EMSA. EMSA was performed as described previously 53,54 with a minor modification, as described elsewhere 6, 55. Nuclear extracts were prepared from frozen liver at −80°C or cultured hepatocytes. Binding reactions were undertaken by incubating the nuclear extracts in reaction buffer (20 mM HEPES-KOH, pH 7.9, containing 1 mM EDTA, 60 mM KCl, 10% glycerol, and 1 µg poly[dI-dC]) with a probe (40,000 dpm) for 20 min at room temperature. Products were electrophoresed on a 4.8% polyacrylamide gel in high-ionic-strength buffer, and dried gels were analyzed by autoradiography. An NF-κB consensus oligonucleotide (5′-AGTTGAG GGGA-CTTTCCCAGGC) from the mouse immunoglobulin κ light chain was purchased and labelled with [γ-32P]-ATP (PerkinElmer, Tokyo, Japan) and T4 polynucleotide kinase. Protein was measured using the Bradford method 56. Bands corresponding to NF-κB were quantified by densitometry using ImageJ 57.
Reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was extracted from the frozen liver samples or cultured hepatocytes using TRIzol® reagent (the guanidinium thiocyanate-phenol-chloroform mixture) 58. cDNA was synthesized from 1 µg total RNA from each sample with Oligo(dT)20 Primer (25 ng/µL), 5× RT Buffer (5 µL), 10 mM dNTPs Mixture (2.5 µL), RNase Inhibitor (20 units/0.5 µL), Rever Tra Ace (100 units/µL), and UltraPure™ DNase/RNase-free distilled water (total volume, 25 µL). The conditions of thermal cycling using iCycler (Bio-Rad Laboratories, Hercules, CA, USA) were 42°C for 60 min and 95°C for 5 min. Real-time PCR was performed using SYBR Green and primers for each gene. Primer sequences were synthesized by Eurofins Genomics (Tokyo, Japan) (Table 1). The conditions of thermal cycling using a Rotor-Gene Q (Qiagen, Stanford, VA, USA) were 95°C for 5 min followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. Collection and analyses of data were undertaken using the system software. mRNA expression levels of each gene were measured as CT threshold levels and normalized to those of eukaryotic elongation factor-1α. The cDNA sequence for rat NOS2 mRNA was deposited in the DNA Data Bank of Japan/European Bioinformatics Institute/GenBank under accession number AB250951.
Serum biochemical analyses. Serum ALT and AST levels were quantified using commercial kits. The serum levels of nitrite and nitrate (stable metabolites of NO) were measured using a commercial kit (Roche, Mannheim, Germany) according to the Griess method 59.
Histopathological analyses. Excised liver specimens from the Sprague–Dawley rats were collected and fixed in 10% formalin and embedded in paraffin. Sections of 3–5 µm in size were cut and stained with HE. Neutrophil infiltration was evaluated by staining with MPO using anti-MPO antibodies (A0398; DAKO, Glostrup, Denmark) before HE staining. Apoptotic bodies in the hepatocyte nuclei were detected by TUNEL staining using an in-situ Apoptosis Detection Kit (MK500; Takara Bio Inc., Kusatsu, Shiga, Japan). The number of MPO- and TUNEL-positive cells per square millimeter was counted by analysts who were blinded to the treatment arm.
Preparation of rat primary cultured hepatocytes. Collagenase perfusion was used to isolate hepatocytes from male Wistar rats (200–250 g, 6–7 weeks old) 60,61. The isolated hepatocytes were cultured with Williams’ medium E (supplemented with 10% fetal calf serum, HEPES (5 mmol/L), penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin B (0.25 µg/mL), aprotinin (0.1 µg/mL; Roche, Basel, Switzerland), dexamethasone (10 nmol/L), and insulin (10 nmol/L). After 7 h, the medium was changed with fresh hormone-free medium and the cells were cultured overnight. The number of cells attached to the dishes was estimated by counting the number of nuclei 62 and applying a ratio of 1.37 ± 0.04 nuclei/cell (mean ± standard error; n = 7 experiments).
Treatment of the cultured hepatocytes with OMZ. OMZ was dissolved in Williams’ medium E under sterile conditions. On day 1 after cell culture, the hepatocytes were washed with fresh serum- and hormone-free Williams’ medium E and incubated with IL-1β (1 nmol/L) in the same medium, either in the presence or absence of OMZ (dose range, 0.1–0.5 mmol/L).
Determination of NO production and LDH activity in the cultured hepatocytes. The amount of nitrite (a stable metabolite of NO) in the cell culture medium of the hepatocytes was measured using the Griess method 59. Cell viability was measured on the basis of LDH activity using a commercial kit (Cytotoxicity LDH Assay Kit-WST; Dojindo Inc., Tokyo, Japan).
Western blotting in the cultured hepatocytes. Total cell lysates were obtained from the cultured hepatocytes using a previously described method with minor modifications 4,46. Immunostaining was performed with primary antibodies against mouse iNOS (Affinity BioReagents, Golden, CO, USA), human inhibitor of κB alpha (IκBα; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rat β-tubulin. Immunoreactive proteins were visualized by an enhanced chemiluminescence detection kit (GE Healthcare Biosciences, Piscataway, NJ, USA).
Transfection and luciferase assay in the cultured hepatocytes. Transfection of the cultured hepatocytes was performed using a previously described method 63. Hepatocytes were cultured at 3 × 105 cells/dish (35 × 10 mm) in Williams’ medium E with serum, dexamethasone, and insulin for 7 h before undergoing magnet-assisted transfection. Reporter constructs pRiNOS-Luc-SVpA (for detecting the transactivation of the NOS2 promoter) or pRiNOS-Luc-3′UTR (for detecting the stability of mRNA) (1 µg) and the cytomegalovirus promoter-driven β-galactosidase plasmid pCMV-LacZ (1 ng; internal control) were mixed with a magnet-assisted transfection reagent (1 µL; IBA Lifesciences, Göttingen, Germany) in fresh serum- and hormone-free Williams’ medium E (1.5 mL), followed by incubation with cultured cells. After a 15-min incubation period on a magnetic plate at room temperature, the medium was replaced with fresh Williams’ medium E with serum. The cells were then cultured overnight and treated with IL-1β in the presence or absence of OMZ.
Statistical analyses. Quantitative results were obtained from three to four independent experiments for each of the various analyses, and the mean values and their standard deviations were calculated. Differences between groups and survival rates were identified using the Student’s t-test and log-rank test, respectively (JMP® 14, SAS Institute Inc., Cary, NC, USA). P <0.05 was considered significant.