Mice
Female wild-type C57BL/6 mice (age: 6–8 weeks) were obtained from SPF Biotechnology Co., Ltd. (Beijing, China). Nlrp3 knockout (Nlrp3-/-) mice were obtained from the National Center of Biomedical Analysis (NCBA, Beijing, China) supplied by Dr. Tao Li. All mice were maintained under a 12-h light/dark cycle at 22–24 °C with ad libitum access to food and water. All animal protocols in this study were performed according to the guidelines outlined for care and use of laboratory animals and approved by the Animal Ethics Committee of the Fifth Medical Centre, Chinese People’s Liberation Army General Hospital (animal ethics committee approval No. IACUC-2017-003).
Cell culture
Bone marrow-derived macrophages (BMDMs) were isolated from the femoral bone marrow of 10-week-old female WT or Nlrp3-/- female C57BL/6 mice and were cultured in the Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 50 ng/mL murine macrophage colony-stimulating factor (M-CSF). THP-1 cells were cultured in the RPMI 1640 medium supplemented with 10% FBS and 1% P/S. Cells were maintained in a humidified 5% (v/v) CO2 incubator at 37 °C.
Antibodies and reagents
Adenosine triphosphate (ATP), nigericin, SiO2, poly (deoxyadenylic-thymidylic) acid sodium salt (poly (dA:dT)), polyinosinic: polycytidylic acid [poly (I:C)], Pam3CSK4, dimethyl sulfoxide (DMSO), and LPS (Escherichia coli, 055: B5) were purchased from Sigma-Aldrich (Munich, Germany). Epimedin A (110623–72-8, purity 99.0%), epimedin A1 (140147–77-9, purity 99.92%), epimedin B (110623–73-9, purity 99.39%), epimedin C (110642–44-9, purity 99.1%), icariin (489–32-7, purity 97.64%), caritin (118525–40-9, purity 99%), icariside I (56725–99-6, purity 99.47%), and anhydroicaritin (38226–86-7, purity 99.51%) were purchased from TargetMol. Salmonella strains were kindly provided by Dr. Tao Li from the National Center of Biomedical Analysis. MCC950 was obtained from TargetMol (Boston, MA, USA). Anti-mouse caspase-1(1:1000, AG-20B-0042) was purchased from Adipogen (San Diego, CA, USA). Anti-mouse IL-1β (1:1000, 12,507) and anti-NLRP3 (1:2000, 15101S) antibodies were obtained from Cell Signaling Technology (Boston, MA, USA). Anti-ASC (1:1000, sc-22,514-R) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GAPDH (1:2000, 60,004–1-1 g) was purchased from Proteintech (Chicago, IL, USA). The color prestained protein marker (20AB01) was purchased from GenStar (Beijing, China).
Inflammasome activation
To induce inflammasome activation, BMDMs were seeded at a density of 5×105 cells/well in 0.5 mL of the medium in 24-well plates and were incubated overnight. Next, the medium was replaced with the fresh medium the following day, and BMDMs were subjected to stimulation with 50 ng/mL LPS or 1 μg/mL Pam3CSK4 for a duration of 4 h. The cells were subjected to treatment with Epimedin B for 1 h and were then stimulated as follows: 5 mM ATP for 1 h, 7.5 μmol/L nigericin for 30 min, or 250 μg/mL silicon dioxide (SiO2) for 6 h. Cells were transfected with poly (I:C) (2 μg/mL), poly(dA:dT) (2 μg/mL), or LPS (1 μg/mL) for 6 h using Lipofectamine 2000 according to the manufacturer’s instructions.
Western blotting
Cell extracts and precipitated supernatants were subjected to lysis using 1×loading buffer containing radioimmunoprecipitation assay buffer. The samples were subjected to denaturation at 105 °C for 15 min. Equal amounts of protein samples were separated by performing 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and protein bands were transferred onto 0.2-mm polyvinylidene fluoride membranes. The membrane was blocked with 5% non-fat milk for 1 h at room temperature. Next, the indicated primary antibodies were added and the bands were incubated at 4 °C overnight and then subjected to treatment with anti-goat IgG (1:3000), anti-mouse IgG (1:5000), or anti-rabbit IgG (1:5000) for 1 h at room temperature. The signals generated thereafter were analyzed using an enhanced chemiluminescence reagent.
Caspase-1 activity assay
The Caspase-Glo® 1 Inflammasome Assay (Promega, Madison, WI, USA) was used to assess caspase-1 activity in cell culture supernatants according to the manufacturer’s instructions.
Enzyme-linked immunosorbent assay (ELISA)
Cell culture supernatants and mouse serum were analyzed for mouse IL-1β (R&D Systems, Minneapolis, MN, USA), IL-1β, TNF-α, and IL-6 (Dakewe, Beijing, China), respectively, according to the manufacturer’s instructions.
Alanine aminotransferase (ALT) and aspartate transaminase (AST)
Serum ALT and AST levels were determined according to the Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and the Nanjing Jiancheng Bioengineering Institute (GOT) assay kit instructions [23].
Lactate dehydrogenase (LDH) assay
The release of LDH into the culture supernatant was assessed using the CytoTox 96® 1 Non-radioactive Cytotoxicity Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
ASC oligomerization
Cells were subjected to lysis using Triton buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% Triton X-100, and EDTA-free protease inhibitor cocktail). The samples were then centrifuged at 6,000 × g at 4°C for 15 min. The supernatant was referred to as Triton X-soluble, and the pellet fractions were referred to as Triton X-insoluble fractions. To enable ASC oligomer cross-linking, the Triton X-100-insoluble fractions were subjected to washing steps and were resuspended in 200 μL of PBS, followed by the establishment of cross-linking at 37°C with 2 mM disuccinimidyl suberate (DSS) for 30 min. The pellets were centrifuged at 6,000 × g for 15 min, after which they were collected and dissolved in 1×SDS loading buffer for immunoblot analysis.
Intracellular K+ measurement
BMDMs were seeded in 12-well plates overnight and were primed with 50 ng/mL LPS for 4 h. The cells were subjected to treatment with epimedin B and were then stimulated with nigericin for 30 min. The culture medium was thoroughly aspirated and subjected to washing steps thrice using potassium-free buffer. Ultrapure HNO3 was added to perform lysis of the cells. Samples were collected in glass bottles and boiled for 30 min at 100 °C. Intracellular K+ measurements were performed via inductively coupled plasma mass spectrometry.
Measurement of intracellular Ca2+ levels
BMDMs were seeded in a 384-well plate at a density of 2.5×104 cells/mL overnight. Then, the cells were primed with LPS for 4 h, followed by stimulation with ATP for 45 min with or without epimedin B. A trace showing ATP-induced Ca2+ flux was analyzed using the FLIPRT Tetra system (Molecular Devices, San Jose, CA, USA).
Mitochondrial reactive oxygen species assay
BMDMs were seeded at a density of 1×106 cells/mL in culture dishes with a diameter of 100 mm and primed with LPS (50 ng/mL) for 4 h. The cells were added in a test tube, subjected to washing steps with Opti-MEM, and were stimulated as per methods described previously. For mitochondrial reactive oxygen species (ROS) measurements, BMDMs were subjected to staining procedures using 4 μM MitoSOX for 20 min at 37 °C and then washing steps were conducted twice with HBSS, followed by assessments using flow cytometry. After the completion of staining and washing procedures, flow cytometry was performed to measure mtROS levels.
Hepatic mRNA expression
RNA extraction from the liver tissue was performed using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Subsequently, total RNA samples were reverse-transcribed into first-strand cDNA using the RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific). A total of 5 μL of the PowerUp SYBR Green Master Mix (A25742, Invitrogen), 3 μL DEPC water, 0.5 μL F primer, 0.5 μL R primer, and 1 μL cDNA were introduced into each well of a 384-well plate, and each sample was analyzed in triplicate via reverse-transcriptase quantitative PCR (RTqPCR).
Assessment of the effects of LPS/epimedin B cotreatment-induced DILI in vivo
C57BL/6 mice (6-8-week-old female) were subjected to starvation for 24 h and were administered with LPS (2 mg/kg) or saline vehicle via tail vein (i.v.). Following an observation period of 2 h, Epimedin B (20 mg/kg, 40 mg/kg, and 80 mg/kg) or its vehicle was administered via intraperitoneal injection. Mouse serum and a fraction of liver tissues were collected 6 h after epimedin B treatment. Serum ALT, AST, IL-1β, and TNF-α levels were measured. Histopathological analysis was performed via hematoxylin and eosin (H&E) staining. F4/80-positive macrophages in the liver were also estimated.
In the second experiment, female C57BL/6 mice (age: 6-8 weeks) were administered with MCC950 (40 mg/kg) or saline vehicle through intraperitoneal injection. After 1 h, LPS (2 mg/kg) or saline vehicle was administered intravenously via tail vein. Epimedin B (40 mg/kg) was administered via an intraperitoneal injection after 2 h. Mouse serum and a fraction of liver tissues were collected after 6 h. H&E staining was performed, and the serum IL-1β, TNF-α, ALT, and AST levels were determined.
Statistical analyses
Prism 6 and SPSS statistics (version 21.0) were used for statistical analysis. All experimental data are expressed as mean ±SD. A two-tailed unpaired Student’s t-test was conducted to evaluate significant differences between the two groups. Statistical significance was set at P < 0.05.