D galn

Male wild-type (C57BL/6) mice (aged 8–12 weeks) were purchased from Capital Medical University (Beijing, China) and housed in the Capital Medical University animal facility under specific pathogen-free condition and received humane care according to Capital Medical University Animal Care Committee guidelines.
The mice were intraperitoneally injected with D-GalN (700 mg/kg; Sigma, St. Louis, MO, USA) and LPS (10 μg/kg; InvivoGen, San Diego, CA, USA) to induce ALF or with saline in the control animals. The PPARα activator Wy-14 643 (6 mg/kg; Sigma) was administered via injection into the tail vein 2 h before D-GalN/LPS exposure. Suppression of autophagy was achieved by tail vein injection of 3-MA (10 mg/kg; Sigma) or siRNA for Atg7 (50 μM/kg). CQ (C6628; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in normal saline solution. Mice were pre-treated with CQ (50 mg/kg, intraperitoneally) 2 h before D-GalN/LPS exposure. The mice were killed at various time points after D-GalN/LPS treatment, and liver and serum samples were collected for future analysis.

Animals were randomly assigned to one of the three following groups with 20 rats per group: i) Control; ii) LPS/D-GalN; or iii) crocetin+LPS/D-GalN. Each group was then further divided into four subgroups according to the time of assessment: 0, 6, 12 or 48 h. Animals in the LPS/D-GalN group received an intraperitoneal injection of 300 mg of D-GalN per kg of body weight (Sigma-Aldrich; Merck KGaA) and then were injected intradermally with 50 mg of LPS per kg of body weight (Sigma-Aldrich; Merck KGaA) (15 (link)). Rats were sacrificed after 0, 6, 12 and 48 h after LPS/D-GalN injection. Animals in the crocetin+LPS/D-GalN group were pre-treated with 200 µl of 5 µmol/l crocetin once, (Sigma-Aldrich; Merck KGaA) (16 ,17 (link)) 1 day before the injection of LPS/D-GalN. Furthermore, no treatment was performed on the rats in the Control group. All the rats were anaesthetized with avertin (250 mg/kg) and sacrificed by exsanguination from the femoral artery. Liver and 100 µl blood samples were collected for subsequent analysis. All rats received humane care according to the Guidelines for the Care and Use of Research Animals established by Southern Medical University and the experimental protocol was approved by the Ethics Committee of the Fifth Affiliated Hospital of Southern Medical University.

Conditioned media (CMs) from human and mouse macrophages, i.e., M0 CM, M(IFN-γ)
CM, M(IFN-γ→IL-4) CM, M(IL-4) CM, and M(IL-4→IFN-γ) CM were collected and
centrifuged to remove cell debris. Then, CMs from above-mentioned groups were
incubated with primary mouse hepatocytes or human liver cell lines (HL-7702 and
HepG2) for 6 h, and then cell apoptosis was induced by human and mouse TNF-α (50
μg/ml, Peprotech)/d-GalN (100 mg/ml, Sigma-Aldrich) (GA for short) for
12 h.^27,28 To evaluate hepatocyte apoptosis, primary mouse
hepatocytes and HL-7702/HepG2 cells were stained with rabbit anti-mouse cleaved
caspase-3 (Abcam, Cambridge, MA, USA) and FITC-conjugated goat anti-rabbit IgG
(eBioscience, San Diego, CA, USA). A Nikon Inverted Fluorescence Microscope
ECLIPSE Ti and NIS-Elements F3.0 Software (Nikon Corporation, Tokyo, Japan) was
applied for image capture. Image J software was used to quantify the expression
of cleaved caspase-3.

The normal human liver cell line (L02) was purchased from The Cell Collection Center of Wuhan University. The Cell Counting Kit-8 (CCK-8) assay was purchased from Dojindo Chemical Technology Co., Ltd. GLY was purchased from Selleck Chemicals. The lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH) and ROS kits were purchased from Beyotime Institute of Biotechnology. The primary antibodies against glutathione peroxidase 4 (GPX4, cat. no. sc-166570) was purchased from Santa Cruz Biotechnology, Inc. GAPDH (cat. no. 10494-1-AP) were purchased from ProteinTech Group, Inc. The primary antibodies against high mobility group box 1 (HMGB1, cat. no. 3935), nuclear factor E2-related factor 2 (Nrf2, cat. no. 12721) and homooxygenase-1 (HO-1, cat. no. 43966) were purchased from Cell Signaling Technology, Inc. The goat anti-rabbit IRDye fluorescent secondary antibody IRDye800CW (cat. no. 926-32211) was purchased from LI-COR Biosciences. Cy3 (cat. no. BA1031 and BA1032) and FITC (cat. no. BA1101 and BA1105) fluorescently labeled rabbit anti-goat secondary antibodies were purchased from Wuhan Boster Biological Technology, Ltd. The iron ion detection kit was purchased from Abcam. D-GalN, LPS and TNF-α were purchased from Sigma-Aldrich; Merck KGaA.