D galactosamine d galn

BML-111 was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). LPS (Escherichia coli, O55:B5) and d-galactosamine (d-GalN) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Rat tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) interleukin-10 (IL-10) enzyme-linked immunosorbent assay (ELISA) kits were from BD Biosciences (Bedford, OH, USA). Transforming growth factor-β1 (TGF-β1) and cyclooxygenase-2 (COX-2) polyclonal antibodies were purchased from Anbo Biotechnology Company (San Francisco, CA, USA). Assay kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), myeloperoxidase (MPO), malondialdehyde (MDA), nitric oxide (NO), the activation of inducible nitric oxide synthase (iNOS), catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) and hydroxyl radical-scavenging ability were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Caspase 3 activity assay kits were obtained from Beyotime Institute (Shanghai, China).

Male rats were selected to generate the animal model of ALF as previously described [7 (link)]. In the protocol, rats received simultaneous intraperitoneal (i.p.) injections of d-galactosamine (d-GalN) at 950 mg/kg body weight (Sigma, St. Louis, MO, USA) and lipopolysaccharide (LPS) at 10 μg/kg body weight (Sigma, St. Louis, MO, USA).

Recombinant rat interleukin-22 (rIL-22) was purchased from R&D Systems (Minneapolis, MN, USA). Commercial enzyme-linked immunosorbent assay (ELISA) kits of rat COX-2 rat TNF-α were purchased from R&D Systems and IBL International (GmbH, Hamburg, Germany), respectively. D-Galactosamine (D-GalN) and phenol extracted lipopolysaccharide from Escherichia coli (LPS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other used chemicals and reagents are stated under the sections of their applications.

D-Galactosamine (D-GalN) was purchased from Sigma Aldrich Corporation (St. Louis, MO, USA). S. boulardii was purchased from Biocodex (France). After ruling out baseline differences in blood and fecal samples, the experimental mice were randomly divided into three groups (n = 5 per group): (1) mice that served as vehicle control (CTRL group), (2) mice that were treated with D-GalN (D-GalN group), and (3) mice that were treated with D-GalN and probiotic S. boulardii (D-GalN + SB group). The D-GalN + SB group were gavaged with 1 ml of S. boulardii (1 × 10⁹ CFU/ml) for 4 weeks prior to exposure to D-GalN. And the CTRL group and D-GalN group received the same volume of saline solution. D-The GalN group and the D-GalN + SB group were then intraperitoneally (i.p.) injected with 200 mg/kg D-GalN, while the CTRL group were injected with saline solution. All the mice were sacrificed 24 h after D-GalN challenge. Serum samples, liver tissue specimens and gut microbiota were compared between groups.

C57BL/6 female mice, 10-week old with body weight of 18–20 g, were injected intraperitoneally with 10 μg/kg of LPS (Sigma-Aldrich, St. Louis, MO, USA) and 700 mg/kg D-galactosamine (D-GalN) (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 200 μl PBS for induction of liver failure. For the indicated experiments, these mice were pre-treated with intraperitoneal injection of vehicle (5% DMSO) or 10 mg/kg GSK126 (Selleckchem, Houston, TX, USA) dissolved in 200 μl 5% DMSO for 2 h, and then subjected for liver failure induction. The harvested serum and liver tissues were analysed at the indicated time points. Kupffer cells isolation were also perform as described in supplementary materials. All animal studies were approved by the Institutional Animal Care and Use Committees of Ruijin Hospital.

Lipopolysaccharide (LPS, #L2630), D-galactosamine (D-GalN, #12,662) and Dimethyl sulfoxide (DMSO, #41,640) were procured from Sigma, St Louis, USA to be used as reagents. Dexamethasone (Dex, #14,648) was purchased from MedChemExpress. The source of 8 standard sample was as follows: Myrcene (CAS: 123-35-3, LOT: X30A11S122905, Shanghai Yuanye Biotech); D-limonene (CAS: 5989-27-5, LOT: 1,012,026,665, Sigma-Aldrich); γ-terpinene (CAS: 99-85-4, LOT: A29J11L119842, Shanghai Yuanye Biotech); 4-Carvomenthenol (CAS: 562-74-3, LOT: J13A9S58322, Shanghai Yuanye Biotech); α-Terpineol (CAS: 98-55-5, LOT: J28J7S9465, Shanghai Yuanye Biotech); β-elemene (CAS: 515-13-9, LOT: J14GB154783, Shanghai Yuanye Biotech); β-Caryophyllene (CAS: 87-44-5, LOT: A05GB156905, Shanghai Yuanye Biotech); Germacrene D (CAS: 23986-74-5, LOT: G367765, Toronto Research Chemicals). Furthermore, a range of antibodies against markers like β-actin (#4967), TBK1 (#3013), phospho-TBK1 (#5483), TAK1 (#5206), phospho-TAK1 (#9339), IRF3 (#4302), phospho-IRF3 (#29,047), IKK (#2370), phospho-IKK (#2697), IκB (#4812), phospho-IκB (#2859), NF-κB p65 (#4764), phospho-NF-κB p65 (#3033), p38 (#9212), phospho-p38 (#9211), JNK (#3708), phospho-JNK (#4668), ERK (#9102), phospho-ERK (#9101), F4/80 (#2370), CD68 (ab125212), TLR4 (ab9105), MyD88 (ab219413), and TRIF (ab13810) were obtained from Cell Signaling Technology or Abcam.

All animal procedures were approved by the Animal Experiment Committee of the 302 Military Hospital of the People’s Liberation Army. Male C57BL/6 mice weighing 20 ± 2 g were obtained from the laboratory animal center of the Sibei Fu (Beijing) Laboratory Animal Science and Technology Corporation. Food and water were available ad libitum. All studies were performed in accordance with the guidelines of the Council on Animal Care of the Academia Sinica. The animals were randomly divided into four groups with twenty mice in each group. Sophocarpine (Us150218) was dissolved in normal saline and intragastrically administered to mice (60 mg/kg for the low dose and 120 mg/kg for the high dose) for 12 days. Finally, all animals in each group except the control group were intravenously injected with Polyinosinic-polycytidylic acid sodium (Poly I: C, Sigma) dissolved in pyrogen-fee saline at a concentration of 75 μg/kg (Huang et al., 2013 (link)). The mice were simultaneously intraperitoneally injected with D-galactosamine (D-GalN, Sigma) at a dose of 500 mg/kg (Huang et al., 2013 (link)). The mice were sacrificed 12 h after injection. The blood and liver were collected. The blood was centrifuged at 3000 g for 10 min to separate the serum without hemolysis. All of the serum and the rest of the liver tissue after hematoxylin-eosin staining were stored at -80°C.