Animals
Male thromboxane receptor knockout (TP−/−) mice (8 weeks old) were generated as described previously [19]. Eight-week-old male C57BL/6 wild-type (WT) mice were purchased from CLEA Japan (Tokyo, Japan). TP-floxed mice were generated as previously described [16]. Mice with Tp deletions in myeloid cells (C57BL/6 background) were generated by crossing TP-floxed homozygous mice with LysM-Cre mice. In this study, LysMCre-Tp-floxed mice were referred to as TP△mac mice, whereas littermate control mice were referred to as control mice. Mice ubiquitously expressing green fluorescent protein (GFP) were kindly provided by Dr. Okabe (Genome Information Research Center, Osaka University, Osaka, Japan). All mice were maintained under controlled humidity (50% ± 5%) and temperature (25°C ± 1°C) with a standard light/dark cycle of 12/12 h and were given ad libitum access to food and water.
The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Kitasato University School of Medicine (Approval no. 2023-062). All experimental studies were performed in accordance with the institutional guidelines for animal experimentation, based on the Guidelines for Proper Conduct of Animal Experiments published by the Science Council of Japan.
Animal procedures
The animals were fasted overnight and injected intraperitoneally (i.p.) with 300 mg/kg APAP (Sigma-Aldrich, St. Louis, MO, USA) dissolved in warm pyrogen-free saline (final concentration, 20 mg/mL). At the indicated times, the mice were anesthetized with isoflurane (Pfizer, Manhattan, NY, USA), and blood was drawn from the heart. Liver tissues were collected, a small section of each liver was placed in 10% formaldehyde, and the remainder was immersed in RNAiso Plus reagent (Takara Bio, Shiga, Japan) for further analysis. Following this procedure, the animals were euthanized by cervical dislocation, and death was verified by the lack of heartbeat, respiration, and corneal reflex. Liver injury was determined by measuring serum alanine aminotransferase (ALT) using a Dri-Chem 7000 Chemistry Analyzer System (Fujifilm, Tokyo, Japan).
Treatments
Ozagrel sodium (Takata Pharmaceutical Co., Saitama, Japan), a TXS inhibitor, or vehicle (phosphate-buffered saline [PBS]) was administered (150 mg/kg, i.p.) [13] 2 h after APAP treatment. ANG-3777 and HGF mimetics (Selleck Houston, TX, USA) (50 mg/kg) [20] were dissolved in 0.2 mL PBS containing 4% dimethyl sulfoxide and intraperitoneally administered 2 h and 24 h after APAP administration.
Clodronate
Clodronate and control liposomes were purchased from FormuMax Scientific (Sunnyvale, CA, USA). Clodronate or control liposomes (100 µL/mouse) were injected intraperitoneally 48 h before APAP administration.
Histology and immunohistochemistry
Excised liver tissues were fixed immediately with buffered 10% formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Sections (3.5 µm thick) were prepared from paraffin-embedded tissues and subjected to either hematoxylin and eosin (H&E) staining or immunostaining. Images of the H&E-stained sections were captured using a microscope (Biozero BZ-700 Series; Keyence, Osaka, Japan). The level of necrosis (as a percentage of the total area) was determined in five fields (100×) from each animal by measuring the necrotic area relative to the entire histological section using the ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA). The results are expressed as percentages.
Proliferating cell nuclear antigen (PCNA)
With regard to PCNA immunohistochemistry, liver sections were stained with a rabbit monoclonal anti-PCNA antibody (Thermo Fisher Scientific, Waltham, MA, USA) at a dilution of 1:200. Immune complexes were detected using Histofine Simple Stain MAX PO (MULTI) (Nichirei, Tokyo, Japan). Images of stained liver sections were captured using a microscope (Biozero BZ-700 Series). The number of PCNA+ hepatocytes was counted in five fields (200×) per animal using the ImageJ software. The percentage of PCNA+ hepatocytes was then calculated, and the results were expressed as percentages.
Immunofluorescence staining
Liver tissues from APAP-treated mice were fixed in 4% periodate-lysine-paraformaldehyde overnight at 4°C, transferred to 30% sucrose prepared in 0.1 M phosphate buffer (pH 7.2), and stored at 4°C for 3 days, followed by mounting the liver tissue in Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and stored at − 20°C. Liver tissue sections (8-µm-thick) were cut and blocked with 1% bovine serum albumin in 0.5% Triton X-100 in PBS. The sections were incubated with a rabbit anti-mouse TP (1:100; Cayman Chemical, Ann Arbor, MI, USA), a rabbit anti-mouse TXS(1:100; Bioss, Woburn, MA, USA), a rat anti-mouse CD68 antibody (1:100; Bio-Rad Laboratories, Hercules, CA, USA), a rat anti-mouse CD41 (BioRad Laboratories), and a rabbit anti-mouse HGF (1:100; Proteintech Group, Rosemont, Il, US,) at 4°C overnight. The sections were then incubated with the following secondary antibodies at 4°C for 1 h: Alexa Fluor 488-conjugated donkey anti-rabbit IgG, Alexa Fluor 594-conjugated donkey anti-rabbit IgG, or Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR, USA). Nuclei were stained with 4'-6-diamidino-2-phenylindole (DAPI). Images were obtained using a fluorescence microscope (Biozero BZ-700 Series; Keyence, Osaka, Japan). After labeling, five optical fields (×200) per animal were randomly selected, and the number of positive cells was counted.
Bone marrow (BM) transplantation
BM cells were obtained by flushing the cavities of freshly dissected femurs and tibiae from donor mice (7 weeks old) using PBS. The flushed BM cells from each donor were dispersed by pipetting and resuspended in PBS at a density of 1 × 107 cells/mL. Mice were lethally irradiated (9.0 Gy) using an MBR-1505R X-ray irradiator (Hitachi Medico Co., Tokyo, Japan) with a filter (copper, 0.5 mm; aluminum, 2 mm) to monitor the cumulative radiation dose. Donor BM mononuclear cells (1 × 106) in 200 µL of PBS were transplanted into the tail veins of irradiated mice. Bone marrow transplantations were performed to generate the chimeric mice as follows: WT mice reconstituted with WT BMMs (WT→WT) or TP −/− BMMs (TP −/−→WT) and TP −/− mice reconstituted with WT BMMs (WT→TP −/−) or TP−/− BMMs (TP −/−→TP −/−). In a separate experiment, donor BM cells were harvested from GFP+-WT mice, and GFP+-WT BM cells were transplanted into WT mice. The chimeras were treated with APAP for eight weeks after BM transplantation.
Measurement of glutathione (GSH) and glutathione disulfide (GSSG)
The frozen tissues were homogenized and centrifuged to separate the supernatants. Hepatic GSH and GSSG levels were measured using a spectrophotometric/microplate reader with a GSSG/GSH Quantification Kit (Dojindo Molecular Technologies, Kumamoto, Japan).
Measurement of TXB2
The concentrations of TXB2, a stable metabolite of TXA2, in liver tissues were measured using a Thromboxane B2 ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA).
Isolation of intrahepatic leukocytes
Animals were anesthetized by i.p. injection of mixed anesthetic agents containing 4.0 mg/kg midazolam (Sandoz, a Novartis division, Basel, Switzerland), 0.75 mg/kg medetomidine hydrochloride (Nippon Zenyaku Kogyo, Fukushima, Japan), and 5.0 mg/kg butorphanol (Meiji Seika Pharma, Tokyo, Japan). The liver was perfused with Hank’s balanced salt solution through the portal vein. Excised livers were incubated in Roswell Park Memorial Institute (RPMI) medium containing 0.05% collagenase (Type IV; Sigma Chemical Co., St. Louis, MO, USA) for 20 min at 37°C. The liver homogenates were filtered through a 70 µm cell strainer. Non-parenchymal cells were purified by density-gradient centrifugation on 33% Percoll (GE Healthcare Life Sciences, Piscataway, NJ, USA) as previously reported [21].
Flow cytometric analyses
Isolated non-parenchymal cells were incubated with an anti-mouse CD16/32 antibody (TruStain FcX; BioLegend, San Diego, CA, USA) to block nonspecific binding of the primary mAb. Cells were stained with a combination of the following reagents: PE-conjugated anti-CD45 (30-F11; BioLegend, San Diego, CA, USA), APC/CY7-conjugated anti-Ly6G (1A8; BioLegend), PE/Cy7-conjugated anti-CD11b (M1/70; BioLegend), Brilliant Violet 510-conjugated anti-Ly6C (HK1.4; BioLegend), and anti-F4/80 (BM8; BioLegend). Cells positive for 7-aminoactinomycin D (BioLegend) were excluded from the analysis. Samples were analyzed using a FACSVerse cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using the Kaluza software v2.1 (Beckman Coulter, Brea, CA, USA). We quantified and presented the number of cells normalized to liver tissue weight (cells/g).
RT-qPCR analysis
Total RNA was extracted from the liver tissues using RNAiso Plus (Takara Bio). cDNA was constructed with 1 µg of total RNA using the ReverTra Ace qPCR RT Kit (TOYOBO Co., Ltd., Osaka, Japan). Quantitative PCR amplification was performed using the TB Green Premix Ex Taq II (Tli RNase H Plus; Takara Bio, Inc. Shiga, Japan). PCR amplification was performed with the following conditions: 95°C for 10 s, followed by 40 cycles at 95°C for 3 s and 60°C for 20 s. The mRNA expression levels were calculated based on the comparative threshold cycle and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in each sample. The primer sequences are listed in Supplementary Table S1.
Cell preparation and culture
To generate BM-derived macrophages, BM cells were isolated from the femurs and tibiae of 8-week-old male mice. BM cells were cultured in 6-well plates (1.0 × 106 cells/well) and maintained in RPMI 1640 medium (Gibco, Thermo Scientific, Waltham, MA, USA) containing 10% fetal calf serum and 20 ng/mL macrophage colony-stimulating factor (BioLegend, San Diego, CA, USA), as previously described [21]. On day 7, BM-derived macrophages were stimulated with lipopolysaccharide (LPS) (10 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) and recombinant murine interferon-gamma (IFN-γ) (20 ng/mL; BioLegend) to polarize toward a pro-inflammatory macrophage phenotype or recombinant murine interleukin (IL)-4 (20 ng/mL; BioLegend) and to polarize toward a reparative phenotype in RPMI 1640 medium for 18 h. Cultured BM-derived macrophages were harvested and homogenized in RNAiso Plus (Takara Bio), and mRNA levels were measured using RT-qPCR.
Statistical analysis
All results are expressed as the mean ± standard deviation. All statistical analyses were performed using GraphPad Prism, version 8 (GraphPad Software, La Jolla, CA, USA). Data were compared between two groups using unpaired two-tailed Student’s t-tests and between multiple groups using one-way analysis of variance followed by Tukey’s post hoc tests. Statistical significance was set at P < 0.05.