2.1 Materials
Anti-NLRP3, anti-N-terminal gasdermin D (GSDMD-N), anti-cleaved caspase-1, anti-Caspase-1, anti-caspase-11, anti-p-PI3K, anti-p-Akt, anti-PI3K, anti-Akt and anti-GAPDH antibodies were purchased from Abcam (Cambridge, MA, USA). The anti-villin antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). ELISA kits were purchased from Boster Biological Technology (Wuhan, China). Opti-MEM™ medium was purchased from Gibco (Grand Island, NK, USA). Trinitrobenzene sulfonic acid (TNBS) was purchased from Sigma (St. Louis, MO, USA). Nodakenin was purchased from Topscience (Shanghai, China).
2.2 Animals
Wild-type (WT) mice (C57/BL6, 6-8 weeks old) were purchased from the Model Animal Research Center of Nanjing University and maintained in a specific pathogen-free (SPF) environment. All mice were fed at 25 °C with light/dark cycles for 12 h. Our animal experiments in vivo were approved by the Animal Ethics Committee of Bengbu Medical College (Bengbu, China).
2.3 Animal intervention
C57BL/6 mice were randomly divided into three groups with 10 mice in each group: WT, TNBS and Nod. For the WT group, WT mice were used as negative controls (WT). Experimental colitis models, were induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in the mouse colon, as previously described[24]. Briefly, mice were anesthetized by intraperitoneal injection of 0.7% pentobarbital. Next, 5% TNBS (Sigma‒Aldrich) solution was mixed with an equal volume of 50% ethanol solution, dropped into the mouse colon, and the mice were held inverted for 30 min to allow the drug to fully contact the intestine. TNBS-induced mice were divided into two groups, with mice in the TNBS group given saline therapy, and mice in the Nod group given Nod treatment (20 mg/kg, p.o.)[18, 25]. After 8 days of intervention, mouse colonic tissue was dissected, and its length was measured and divided into two parts, one fixed in 10% formalin for H&E analysis and immunofluorescence detection, and the other stored at -80 °C for western blotting and ELISA detection.
2.4 Organoid culture and intervention
The dissociated mouse small intestine was cut into tissue fragments and the crypts were isolated by vigorous suspension and centrifugation as described[26, 27]. Essentially pure crypts were expanded and cultured in IntestiCult organoid growth medium (Stem Cell Technologies). A model of intestinal organoid pyroptosis was established by stimulation with 5 mM adenosine 5-triphosphate (ATP) alone for 2 h followed by 200 ng/mL lipopolysaccharide (LPS) alone for 8 h [13].
Colonic organoids were divided into three groups: Con, LPS+ATP and LPS+ATP+Nod. After 4 days of normal culture, the Con group was not treated with LPS, ATP or Nod; the LPS+ATP group consisted of ATP-/LPS-induced organoids without Nod treatment; the LPS+ATP+Nod group containing ATP-/LPS-induced organoids was treated with Nod (10 μM) for 24 h. Finally, organoids were collected for subsequent assays.
2.5 Assessment of colitis symptoms
As described by Spencer et al., a numerical system was used to score the inflammatory bowel disease activity index (DAI) in mice with TNBS-induced colitis[28]. DAI was determined by assessing concealed blood in faeces, fur folds, soft stools, and rectal prolapse <1 mm, and each feature was scored as 1 point. An additional point was added when severe rectal prolapse exceeded 1 mm or diarrhoea was present. The DAI was obtained on a scale of 6 points (0-5).
2.6 Histological analysis
Mouse intestinal tissue was stained with haematoxylin and eosin (H&E) and subjected to pathological examination. According to Schultz et al.[29], intestinal tissues are graded on a scale of 0-4 based on intestinal inflammation grading. All specimens were graded by 2 independent pathologists who were blinded to the treatments.
2.7 Intestinal permeability
Mice were fasted for 4 h and then administered fluorescein isothiocyanate (FITC)-dextran (600 mg/kg) (Sigma‒Aldrich, St. Louis, MO). Blood was obtained by cardiac puncture at 4 h, serum was separated by centrifugation, and serum FITC levels were assessed by fluorometry.
2.8 Bacteriological analysis
As we previously described,[30] the mesenteric lymph nodes (MLNs), spleen, liver, and blood were collected using aseptic techniques, and the tissues were weighed and homogenized to a concentration of 1 g/mL. A total of 100 μL of homogenate or blood was plated onto MacConkey agar for 24 h at 37 °C and the resulting colonies were counted. Bacterial numbers were expressed as colony forming units of tissue/g or blood/mL. Culture results were considered positive when more than 102 colony-forming units/g of tissue were found.
2.9 Measurement of Colonic TEER
Fresh colon tissue from mice was gently rinsed with PBS to clean the luminal contents, and then the intestinal segments were placed in Krebs buffer (NaCl: 115 mM, KCl: 8 mM, CaCl2: 1.25 mM, MgCl2: 1.2 mM, KHPO4: 2.0 mM, NaCO3: 25 mM, pH: 7.33–7.37). Bowel tubes were dissected along the mesenteric axis, cut to 2.8 mm × 11 mm, and loaded into an Ussing chamber system (Chamber Systems CSYS-4HA, Warner Instruments, USA). The voltage and current were adjusted by the system and analysis software, and transepithelial electric resistance (TEER) was measured.
2.10 Immunofluorescence analysis
Mouse colon tissue sections were deparaffinized, antigen-exposed, blocked, and incubated with anti-GSDMD-N (1:200) and anti-villin (1:100) antibodies and then FITC-conjugated goat anti-rabbit IgG (H+L) (1:500) and/or Alexa Fluor® 594-conjugated goat anti-mouse IgG (H+L) (1:500). Mouse intestinal organoids were blocked and incubated with anti-GSDMD-N (1:200), anti-NLRP3 (1:400), anti-caspase-11 (1:400) and FITC-conjugated goat anti-rabbit IgG (H+L) (1:500). Finally, the nuclei were stained with DAPI.
2.11 ELISA
Mouse colon tissue was homogenized with 1 mL of saline containing protease inhibitors or blood or mouse intestinal organoid medium supernatant and centrifuged at 1000 × g for 30 min, and interleukin (IL)-1β, IL-18 and LPS levels were measured by ELISA according to the manufacturer's instructions. Mouse peripheral blood was obtained via the eyeball, and the supernatant (400g, 10 min) was collected to detect intestinal fatty acid binding protein (I-FABP) by ELISA.
2.12 qRT–PCR
Total RNA was isolated from mouse colon tissue or mouse intestinal organoids by a TRIzol total RNA isolation system. One microgram of total RNA was synthesized with a cDNA synthesis system kit (TaKaRa, Japan). According to the instructions, the TB Green® Premix Ex Taq™ II detection system (TaKaRa) was used for real-time PCR. GAPDH was used as the internal control, and the mouse-specific primers are shown in Table 1.
2.13 Western blotting
Mouse colon tissue or mouse intestinal organoids were fully lysed, the supernatant was collected by centrifugation and denatured by boiling, and the proteins were separated and transferred to a PVDF membrane (Millipore, Massachusetts, USA). The membrane was incubated with anti-NLRP3 (1:1000), anti-GSDMD-N (1:1000), anti-caspase-1 (1:1000), anti-p-PI3K (1:1000), anti-p-Akt (1:1000), anti-GAPDH (1:1000), and HRP-conjugated goat anti-mouse/rabbit IgG. Finally, an imager (ChemiDoc MP, Bio-rad, USA) was used for visualization.
2.14 Protein‒protein interaction (PPI) networks
Intersecting targets were obtained by combining the Nod target library with the IBD target library using an online Venn analysis tool (http://bioinformatics.psb.ugent.be/webtools/Venn/). All intersecting targets were imported into the string online database (https://string-db.org/) to obtain protein‒protein interaction network (PPI) data. The obtained protein data network was imported into Cytoscape 3.9.1(http://www.cytoscape.org), the PPI relationship network was established and topological analysis was performed.
2.15 Pathway Enrichment Analysis
Targets obtained from MCODE plugin screening in Cytoscape 3.9.1 were imported into the DAVID (https://david.ncifcrf.gov/) database for GO enrichment analysis and KEGG pathway enrichment analysis. The first 20 enrichment results were visualized using p-value, and outputs with P<0.05 were retained the filtered results.
2.16 Statistical analysis
SPSS version 23.0 was used for data analysis. Continuous, normally distributed data are shown as the mean ± SD. The unpaired t test or Mann‒Whitney test was used to compare data between two groups (parameter or non‒parameter). The chi-square test was used to compare binary and categorical data to generate a contingency table. P value<0.05 was considered statistically significant.