Acetaminophen apap

Mouse studies were approved by the Italian Ministry of Health. Mice were group-housed (2–5 mice per cage) in standard mouse cages and maintained under a 12 h light/dark cycle with free access to water and standard mouse chow. Wild-type six-week-old male C57BL/6N mice (weight: 20–22 g) purchased from Charles River Laboratories were injected intraperitoneally (i.p.) with 0.5 μg/g of body weight of CD95-activating antibody (CD95-Ab; BD Biosciences), 0.6 μg/g of body weight of α-amanitin (Sigma-Aldrich), 0.4 mg/g of body weight of acetaminophen (APAP) (Sigma-Aldrich), or vehicle (saline). 20 mg/kg/day of garcinol (Enzo Life Sciences) or vehicle (saline) was injected i.p. for five days prior to the injection of CD95-Ab. Galloflavin at the doses of 10, 20, 40 or 80 mg/kg diluted in 40% dimethyl sulfoxide (DMSO) or vehicle (40% DMSO) was injected i.p. at the same time as CD95-Ab, α-amanitin, or APAP injections and was continued daily until mouse sacrifice by cervical dislocation at 72 h after CD95-Ab, α-amanitin, or APAP injections. Blood samples were collected by retro-orbital bleedings and were analyzed for alanine aminotransferase levels by colorimetric/fluorometric assay kit (Biovision) according to manufacturer’s instructions.

Acetaminophen (APAP) was brought from Sigma-Aldrich (St. Louis, MO, USA), Phps was obtained from Baoyifeng Biology of Xi’an (Shaanxi, China). Glutathione (GSH), myeloperoxidase (MPO), aspartate aminotransferase (AST) and glutamic-pyruvic transaminase (ALT) detection kits were acquired through the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). Primary antibodies against HO-1, AMPKα, phosphorylation-AMPKα (p-AMPKα), AMPKβ, phosphorylation-AMPKβ (p-AMPKβ), AKT and phosphorylation-AKT (p-AKT), were procured from Cell signal Technology (Boston, MA, USA). Antibodies against GCLC, GCLM, NQO1 were acquired by Abcam (Cambridgeshire, CA, UK). Anti-Nrf2 was gained from Affinity Biosciences. Antibodies against Lamin B, β-actin were acquired from Proteintech Group Inc. (Boston, MA, USA). HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were afforded by Boster Biological Technology (Wuhan, Hubei, China). All other chemicals were of reagent grade.

Human embryonic kidney 293 (HEK293) cells were purchased from American Type Culture Collection (Manassas, VA, USA). HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 10 units/ml penicillin and 10 mg/ml streptomycin (Thermo Fisher Scientific). The HEK293 cells were routinely tested for mycoplasma contamination by PCR. Mouse primary hepatocytes were incubated in M199 medium (Sigma-Aldrich) with 10% FBS, 10 units/ml penicillin, 10 mg/ml streptomycin, 23 mM HEPES, and 10 nM dexamethasone (Sigma-Aldrich). Acetaminophen (APAP) was purchased from Sigma-Aldrich. Plasmids were transiently transfected into HEK293 cells using polyethylenimine (PEI). Lipofectamine 2000 (Thermo Fisher Scientific) was used for transfection into mouse primary hepatocyte cells.

Amikacin sulfate (AMK; AMIKACIN Sulfate Injection 100 mg “Nichiiko”) was purchased from Nichi-Iko Pharmaceutical Co., Ltd. (Toyama, Japan). Aprindine hydrochloride (AP; Aspenon^® for iv inj. 100) was purchased from Bayer Yakuhin, Ltd. (Osaka, Japan). Vancomycin hydrochloride (VCM; Vancomycin) and Doripenem Hydrate (DRPM; FINIBAX^®) were purchased from SHIONOGI & Co., Ltd. (Osaka, Japan). Sodium 2-propylvalerate (VPA) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Acetaminophen (APAP) was purchased from Sigma-Aldrich (St. Louis, MO., USA). Bicarbonate buffer (Sublood-BSG) and physiological saline were purchased from Fuso Pharmaceutical Industries, Ltd. (Osaka, Japan) and Heparin sodium (Heparin sodium 5,000 units/5 mL for Inj. MOCHIDA) was purchased from MOCHIDA PHARMACEUTICAL Co., Ltd. (Tokyo, Japan). Cilastatin sodium salt was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). I-STAT^® cartridge EC8+ was purchased from Abbot Japan Co., Ltd. (Chiba, Japan). The miniaturized polyethersulfone (PES) dialyzer was provided by the Artificial Organ Development Center of the Nipro Research and Development Laboratory (Shiga, Japan). The dialyzer was shown in Fig 1 A. All other chemicals were of the highest grade and obtained from commercial sources.

IMP (98.8% purity) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Guangzhou, China). Acetaminophen (APAP) and obeticholic acid (OCA) were purchased from Sigma Chemical Co. (St. Louis, MO).

For exposures to compounds, PCLS were cultured in 2.5 mM VPA until day 3 of culture. From day 3 to day 5 of culture, VPA concentration was reduced to 1 mM and PCLS were simultaneously exposed to compounds or corresponding solvent control. Compounds concentrations used: 10 ng/mL TGF-β1 (PreproTech), 1 mM acetaminophen (APAP) (Sigma-Aldrich), 10 μM SB-525334 (Alk5 inhibitor) (Sigma-Aldrich), 20 mM N-acetyl-L-cysteine (NAC) (Sigma-Aldrich). On day 5 of culture (after a 48 h exposure), PCLS and culture medium were collected for analysis.