B6.129P2-Nos2tm1Lau/J (iNOS−/−), B6.129P2-Lgr5tm1(cre/ERT2)Cle/J (Lgr5-EGFP-IRES-creERT2), and B6;129S6-Gt(ROSA)26Sortm14(CAG−tdTomato)Hze/J (tdTomato) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The genotypes of these mice were determined by polymerase chain reaction (PCR) and DNA agarose gel electrophoresis. When the progeny of iNOS−/−, Lgr5-tdTomato and Lgr5-tdTomato; iNOS−/− mice reached 6 to 8 weeks of age, male mice were selected for subsequent studies along with their wild-type littermate controls. All mice were maintained in a specific pathogen-free (SPF) facility with a 12 h light/dark cycle and were allowed standard food and water ad libitum. All experimental procedures were carried out in accordance with the guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Sichuan Cancer Hospital & Institute (SCCHEC-04-2023-009).

Intestine samples were collected, fixed with 4% cold paraformaldehyde (BL539A, Biosharp, China) for 72 h, dehydrated and paraffin embedded using the standard histological protocol of our laboratory. Sections of 4 μm thickness were used for hematoxylin–eosin (H&E) (E607318, BBI, China), immunohistochemistry (IHC) (PV-6001/6002, ZSGB-BIO, China) and immunofluorescent (IF) staining. Antigen retrieval was performed using boiled TRIS–EDTA Antigen Retrieval Solution (BL618A, Biosharp) for 20 min, and tissues were blocked with phosphate buffered solution (PBS, SH30256.01, HyClone, USA) containing 1% bovine serum albumin (BSA, A7906, Sigma‒Aldrich, USA) and subsequently incubated with specific primary antibodies against Olfm4 (39141S, CST, USA), BrdU (ab152095, Abcam, UK), Ki67 (ab1667, Abcam), pHH3 (9718 T, CST), FABP1 (13368 T, CST), lysozyme (ab108508, Abcam), chromogranin A (60,135–1-1 g, Proteintech, USA), DCLK1 (21,699–1-AP, Proteintech), β-catenin (610,153, BD Biosciences, USA), and p27 Kip1 (3686 T, CST, USA) at a 1:200 dilution ratio according to the manufacturer’s instructions. Goblet cells were stained with an Alcian Blue and Nuclear Fast Red staining kit (E670107, BBI). Measurements for each quantitative outcome were collected from 30 crypts or villi per mouse analyzed and from more than 3 independent fields of the ileum.

Fresh intestinal tissues were flushed with ice-cold PBS and opened longitudinally on ice. Intestines were cut into 3 to 5 mm pieces, washed briefly with PBS, and placed into PBS supplemented with 5 mM EDTA (25,300,096, Invitrogen, USA). The tissue was incubated for 30 min at 4 °C and then washed with PBS. Villi and crypts were isolated by vigorous shaking and were passed through a 70-μm strainer to separate crypts. Individual crypts were collected by centrifugation at 4 °C and 800 ×g for 3 min. Crypts, mixed with Matrigel (354,230, Corning, USA), were seeded into 96-well flat-bottom plates (100 crypts/well). The plates were incubated at 37 °C for 10 min. Then, IntestiCult™ Organoid Growth Medium (06005, STEMCELL Technologies, Canada) supplemented with 100 μg/mL streptomycin and 100 units/mL penicillin (15,140-122, Invitrogen) was added to the wells. 1400W (HY-18731, MCE, China), the specific inhibitor of iNOS, was used to treat intestinal organoids in order to mimic the deficiency of iNOS.

Images of H&E and IHC staining on slides and of organoids in culture plates were captured by M5000 (Thermo Fisher, USA), Cytation 5 (BioTek, USA) or BX53 (Olympus, Japan) microscope. Image parameters were generated by ImageJ (NIH, USA) for each image. IF staining images were captured by a A1R confocal microscope (Nikon, Japan). Each value was calculated based on at least three independent replicates.

Total RNA extraction was performed using RNAiso Plus (9109, TaKaRa, Japan) following the manufacturer’s recommendations, and then the total RNA concentration was determined using a NanoDrop2000 spectrophotometer (Thermo Scientific). Hifair II 1st Strand cDNA Synthesis SuperMix (11137ES60, YEASEN, China) was used to synthesize cDNA, and qPCR was performed using Hieff qPCR SYBR Green Master Mix (11203ES08, YEASEN). The expression levels of Nos2, Lgr5, MKi67, Lyz2, Wnt3a, etc., were measured by qPCR using a C1000 instrument (Bio-Rad, USA). Primer sequences used in this study were listed in Table 1. Gene expression results were normalized to that of β-actin, and relative expression was determined by the 2−ΔΔCt method.

Ileum tissue samples (from three wild-type mice and three iNOS−/− mouse littermates) were isolated, gently washed with DEPC-treated water (10601ES76, YEASEN), frozen and then used for transcriptome RNA-Seq. Total RNA extraction, RNA integrity evaluation, library construction, and sequencing were performed according to the manufacturer’s standard protocol. RNA-seq and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Differentially expressed genes (DEGs) were identified using the absolute value of log2 (ratio) ≥ 1 as the threshold. The t test threshold (P values < 0.05) and fold-change threshold (> 1.5 or < 0.5) were set as the thresholds for significantly DEGs. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database analyses were performed. MetaboAnalyst of DEGs was performed using R based on the hypergeometric distribution. Gene set enrichment analysis (GSEA) was performed to determine pathways that were significantly enriched in DEGs for each group compared with those in the GSEA molecular signature database. RNA-Seq results were further validated by qPCR.

All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, USA). The results are expressed as the mean ± standard deviation (SD) values. Statistical significance was determined using the Student’s t test for two-group comparisons and one-way ANOVA for three or more groups. P values of less than 0.05 were considered statistically significant.