Mice

Male C57BL/6 mice (6–8 weeks old) were purchased from Chengdu Yaokang Biotechnology Co., Ltd. (Chengdu, China). All mice were specific pathogen-free, maintained under 12 h of light/dark conditions at 22–24 °C and 40–60% relative humidity, with unrestricted access to food and water for the duration of the experiment. All animal experiment protocols in this study were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Research Ethics Society of Chengdu University of Traditional Chinese Medicine (No. 2,024,024).

Cell culture

Mouse bone marrow-derived macrophages (BMDMs) were prepared as follows. The femurs of adult C57BL/6 mice were removed, and the bone marrow contents were repeatedly rinsed with Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 11,965,092) supplemented with 10% fetal bovine serum (Gibco, 10,100), and 1% penicillin/streptomycin (Solarbio, P1400). At the same time, 50 ng/mL recombinant murine macrophage colony-stimulating factor (MedChemExpress, HY-P7085) was added to the medium, and the cells were placed in a 5% CO2 atmosphere in a humidified incubator. On Day 5 of cell culture, BMDMs were resuspended and inoculated into culture plates at a density of 1 × 106 cells/mL for subsequent experiments.

Inflammasomes activation

The methods used to induce inflammasomes activation have been described previously [23]. Briefly, BMDMs were seeded at a density of 1 × 106 cells/mL in 12-well plates overnight, followed by stimulation with 50 ng/mL ultrapure LPS (Invivogen, tlrl-3pelps) for 4 h. Butein (MedChemExpress, HY-16,558, purity 99.95%) was dissolved in dimethyl sulfoxide (DMSO). The medium was then changed to serum-free Opti-MEM (Gibco, 31,985,070) containing butein (2.5, 5, or 10 µM) for 1 h, followed by treatment with ATP (5 mM, Sigma-Aldrich, A2383), or nigericin (7.5 µM, Invivogen, tlrl-nig) for 1 h; or transfection with poly(I: C) (2 µg/mL, Invivogen, tlrl-picw), or monosodium urate (MSU) crystals (250 µg/mL, Invivogen, tlrl-msu) for 6 h. Poly(dA: dT) (2 µg/mL, Invivogen, tlrl-patn) and ultrapure flagellin (100 ng/mL, Invivogen, tlrl-epstfla) were transfected with Lipo8000™ (Beyotime, C0533) to induce AIM2 and NLRC4 inflammasomes activation, respectively. To induce non-canonical NLRP3 inflammasome activation, BMDMs were primed with Pam3CSK4 (1 µg/mL, Invivogen, tlrl-pms) for 4 h, and then transfected with ultrapure LPS (1 µg/mL) with Lipo8000™ for 6 h. To investigate fatty acid [(sodium palmitate/sodium oleate (PO), Sigma, P9767/O7501)]-induced NLRP3 inflammasome activation, BMDMs were primed with LPS, followed by stimulation with PO (300 µM) for 6 h.

Cell viability assay

BMDMs (1 × 105 cells/well) were seeded in 96-well plates overnight and then exposed to butein (10, 20, 30, 40, 50, 60, 70, 80, or 90 µM) for 24 h. Cell Counting Kit-8 (CCK-8) reagent (MedChemExpress, HY-K0301) was added to the cell culture medium and incubated with the cells for 30 min. Optical density values at a wavelength of 450 nm were analyzed using a multifunctional microplate reader (Tecan, Infinite 200Pro).

Antibodies

Anti-mouse caspase-1 (AG-20B-0042) and anti-NLRP3 (AG-20B-0014) were purchased from Adipogen. An anti-mouse IL-1β antibody (AF-401-NA) was purchased from R&D System. An anti-ASC (SC-22,514-R) was purchased from Santa Cruz Biotechnology. Anti-GSDMD (ab209845) was purchased from Abcam. Lamin B1 polyclonal antibody (12987-1-AP), NRF2 polyclonal antibody (16396-1-AP), heme oxygenase-1 (HO-1/HMOX1) polyclonal antibody (10701-1-AP), goat anti-rabbit IgG (SA00001-2), and goat anti-mouse IgG (SA00001-1) were purchased from Proteintech.

Lactate dehydrogenase (LDH) assay

LDH secretion in culture supernatants was determined using the LDH Cytotoxicity Assay Kit (Beyotime, C0016) according to the manufacturer’s instructions. Briefly, 60 µL of each supernatant sample was collected in a 96-well plate. Then, 60 µL of LDH Cytotoxicity Assay Kit reagent was added to each well and incubated for 30 min. The absorbance signal was measured at 490 nm using a microplate reader (Tecan, Infinite 200Pro).

Caspase-1 activity assay

Caspase-1 activity in the cell culture supernatant was assessed using the Caspase-1-Glo® 1 Inflammasome Assay according to the manufacturer’s instructions (Promega, G9951). Caspase-Glo® 1 buffer was transferred to a bottle containing the Z-WEHD substrate to reconstitute the substrate. MG-132 inhibitor was added to the reconstituted Z-WEHD substrate to prepare the Caspase-Glo® 1 reagent. An equal volume of Caspase-Glo® 1 Reagent and cell culture supernatants were then used to measure luminescence (Tecan, Infinite 200Pro).

Enzyme-linked immunosorbent assay (ELISA)

Determination of IL-1β (Elabscience, E-MSEL-M0003), mouse TNF-α (Elabscience, E-EL-M3063), and mouse IL-6 (Dakewe, 1,210,602) in cell culture supernatants and mouse serum was performed according to the manufacturer’s instructions.

Hoechst 33,342/propidium iodide (PI) fluorescence staining

BMDMs were stained with 5 µg/mL Hoechst 33,342 (Beyotime, C1025) for 10 min at 37 °C in the dark, followed by incubation with 10 µg/mL propidium iodide (PI, Beyotime, ST511) for 15 min at 25 °C in the dark. The cells were observed by fluorescence microscopy (Olympus, U-RFL-T).

ASC oligomerization

After activation of inflammasome as described above, the cells were lysed with Triton buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, and protease inhibitor cocktail] for 30 min. The lysates were subsequently centrifuged at 6000 g for 15 min at 4 °C. The pellets were washed in 500 µL of ice-cold PBS and resuspended in 200 µL of PBS. A 2 mM disuccinimidyl suberate crosslinker (MedChemExpress, HY-W019543) was added to the resuspended pellets and incubated at 30 °C for 30 min. The samples were then centrifuged at 6000 g for 15 min at 4 °C. The cross-linked pellets were resuspended in 30 µL of 1×SDS loading buffer and then boiled for 15 min and analyzed by immunoblotting with an anti-ASC antibody.

Intracellular potassium (K+) assay

BMDMs were seeded in 12-well plates overnight and stimulated as described above. The cells were then washed 3 times with saline, followed by the addition of HNO3 to lyse the BMDMs, and the samples were collected and boiled at 100 °C for 30 min. Intracellular K+ was detected by inductively coupled plasma-mass spectrometry (ICP-MS).

Reactive oxygen species (ROS) assay

BMDMs were seeded overnight in 12-well plates and then stimulated as described above. Cells were washed twice with Hank’s balanced salt solution (HBSS) and then stained with 10 µM DCFH-DA (MedChemExpress, HY-D0940) for 20 min at 37 °C, immediately followed by two washes with HBSS. Cells were resuspended in HBSS and ROS were detected using a Navios flow cytometer (Beckman Coulter, Brea, USA).

LPS-induced peritonitis mouse model

Butein was dissolved in saline containing 10% DMSO, 40% PEG 400 and 5% Tween-80. C57BL/6 male mice were injected intraperitoneally with butein (n = 10, 10 or 20 mg/kg body weight) for 1 h followed by 20 mg/kg body weight LPS [from Escherichia coli (O55:B5), Sigma-Aldrich, L2880] [24]. Mice were sacrificed 2 h after LPS injection, and serum was collected. The peritoneal cavity was washed with ice-cold 1× PBS, and peritoneal lavage fluid was collected. ELISAs were performed to measure the levels of IL-1β and TNF-α production in serum and peritoneal lavage fluid. A Navios flow cytometer (Beckman Coulter, Brea, USA) was used to analyze polymorphonuclear neutrophils (Ly6G+CD11b+, BioLegend, 127,613, 101,205) in the peritoneal lavage fluid.

DSS-induced colitis mouse model

DSS (2.5% (wt/vol), MP Biomedicals, 9011-18-1) was dissolved in daily drinking water for 9 days to induce colitis [25]. Mice received butein (n = 10, 10 or 20 mg/kg body weight) by intragastric gavage during the modeling period. The weight, fecal consistency, and presence of blood in the feces of the mice were recorded daily. The disease activity index (DAI) score, which is used to assess and quantify the severity of intestinal damage during DSS administration, was calculated based on the composite score of diarrhea (0 points = normal, 1–2 points = soft stools, and 3–4 points = watery diarrhea), bloody stools (0 points = normal stools, 1–2 points = slight bleeding, and 3–4 points = excessive bleeding), and body weight loss (0 points = no weight loss, 1 point = 1-5% weight loss, 2 points = 6-10% weight loss, 3 points = 11-20% weight loss, and 4 points = > 20% weight loss). On Day 9 after colitis induction, the mice were necropsied, and the serum was collected to determine the IL-1β levels. The colon was removed, washed with saline, and measured for length. A portion of the colon tissue was placed in neutral formalin, and hematoxylin-eosin (HE) staining was performed to assess pathological injury. The expression of IL-1β and caspase-1 in colon tissues was analyzed by immunohistochemical staining, while the expression of inflammasome-associated proteins in colon tissues was detected by immunoblotting.

HFD-induced NASH mouse model

After 1 week of acclimatization, 10 male C57BL/6 mice fed a regular diet (RD, XTCON50J, containing 4.3% fat, 19.2% protein, and 67.3% carbohydrate) were randomly selected as the control group. Twenty mice fed a HFD (XTHF60, containing 10% fat, 20% protein, and 70% carbohydrate) were randomized into the HFD group (n = 10), butein low-dose group (n = 5), or butein high-dose group (n = 5). Eight weeks later, butein (10, or 20 mg/kg body weight) was administered intragastrically for 4 weeks. Control and model mice received saline containing 10% DMSO, 40% PEG 400 and 5% Tween-80 once daily continuously for 4 weeks. Mouse body weights were recorded once a week throughout the experiment. After 12 weeks of dietary intervention, all the mice were fasted overnight.

Blood samples were collected after anesthesia, and serum IL-1β secretion was measured by ELISA. Serum triglyceride (TG, A110-1-1), total cholesterol (TC, A111-1-1), alanine aminotransferase (ALT, C009-2-1), and aspartate aminotransferase (AST, C010-2-1) levels were measured using assay kits from Nanjing Jiancheng Institute of Bioengineering according to the manufacturer’s instructions. Livers were harvested and weighed. Protein samples were extracted from liver tissues for immunoblot analysis, and liver histopathological changes were evaluated by HE staining, oil red O staining, and Masson’s staining.

Statistical analyses

Statistical analysis was performed using Prism 8 software (GraphPad Software). All experimental data are expressed as the mean ± standard error of the mean (SEM). Significant differences were statistically evaluated using unpaired Student’s t test for two groups or one-way ANOVA followed by Dunnett’s post hoc test for multiple groups. Difference was considered statistically significant when P < 0.05.