Eight-month-old female BALB/c retired breeder mice (body weight 24 ± 2 g) were purchased from Guangzhou Chase Reward Co. Ltd, China. The mice were bred in the animal facility of the Fifth Affiliated Hospital of Sun Yat-sen University under controlled conditions (12-h light/12-h dark; 22 ± 2°C). All mice were maintained in a specific pathogen–free environment and received sterile food and filtered water ad libitum. All experimental protocols were performed following approval from the Institutional Animal Care and Use Committee of the Fifth Affiliated Hospital of Sun Yat-sen University.

The proteoglycan (PG)-induced AS model has been described previously (29). The mice were immunized on days 0, 21, and 42 by intraperitoneal injections with 200 µl of an emulsifier containing 100 µg of cartilage PGs (Sigma-Aldrich, St. Louis, MO, USA) in 100 µl PBS and 100 µl complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA).

The mice were randomly and evenly divided into three groups (n = 10 per group): 1) control group containing 10 healthy mice; 2) AS model group (PG group), in which AS model mice received daily intraperitoneal injections of vehicle control (phosphate-buffered saline, PBS); and (3) IAA group, in which AS model mice received daily intraperitoneal injections of IAA (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 50 mg/kg body weight. The IAA dosage used in the present study was based on previously published data (30, 31) and our preliminary experiment. This dosage was identified as safe for mice. IAA was completely dissolved in PBS (stock solution 10 mg/mL) by adding NaOH (1 N), followed adjusting the pH to 7.4 with 25% (v/v) HCl. IAA or vehicle treatment began in week 10, after PG induction, and continued for 4 weeks. The entire experiment lasted 14 weeks ( Figure 1A ).

Arthritis severity was evaluated by two investigators blinded to the animal treatments by examining changes in the polyarthritis index and the volume of hind paw swelling. The polyarthritis index was scored weekly, starting after the third immunization (week 7) until euthanasia (week 14), using a standard visual scoring system (32): 0, no symptoms; 1, redness and swelling in one toe; 2, redness and swelling in more than one toe; 3, toe stiffness; and 4, deformity or ankle involvement. The accumulated maximum score, including all four paws for each mouse, was 16 points. Hind paw volumes were measured with a PV-200 volume meter (Chengdu Technology Market Co. Ltd., Sichuan, China) before and after immunization. Paw swelling degree was calculated as paw swelling at each time point (weekly during weeks 7–14) − paw swelling (baseline, week 0).

At the end of week 14, the mice were sacrificed. The hind paws and spine were dissected and fixed in buffered 10% formalin, followed by further decalcification in 10% Titriplex EDTA for 1 month and embedding in paraffin. Serial 5-µm sections were stained with hematoxylin and eosin (H&E), and images were acquired using a Pannoramic 250 (3DHISTECH, Budapest, Hungary). Stained sections were semi-quantitatively scored by two independent, blinded observers.

For arthritis score assessments, entire digital images were analyzed according to synovial inflammation, cartilage degradation, and bone erosion, as previously described (33). Synovial inflammation was scored using the following scale: 0, normal; 1, slight thickening of the lining layer, with some infiltrating cells in the sublining layer; 2, moderate thickening of the lining layer, with a moderate number of infiltrating cells in the sublining layer; and 3, severe inflammation with a massive immune cell infiltrate into the synovium. Cartilage degradation was scored using the following scale: 0, normal; 1, mild cartilage destruction; 2, evidence of cartilage destruction with synovial cell invasion; and 3, severe loss of cartilage. Bone erosion was scored using the following scale: 0, normal; 1, mild loss of cortical bone at a few sites; 2, moderate loss of cortical and trabecular bone; and 3, marked loss of bone at many sites. The scores for each criterion were added together to obtain an overall inflammation score for each sample, ranging from 0 to 9 points.

For the vertebral joint score assessment, entire digital images were analyzed according to the level of inflammation, as previously described (34). Inflammation was scored using the following scale: 1, enthesitis, inflammatory cell accumulation around the intervertebral disks (IVD) or infiltration of the annulus fibrosus; 2, <50% absorption/erosion of the IVD; 3, essentially complete resorption (> 50%) of the IVD; and 4, cartilaginous/bony ankylosis.

At the end of week 14, the mice were anesthetized by exposure to 3% isoflurane. Approximately 1 mL of blood was obtained from each mouse via eyeball enucleation and allowed to clot at room temperature for 1 hour. Serum samples were separated by centrifuging at 2000 × g for 10 min at 4°C and stored at −80°C until analysis. IL-6, TNF-α, IL-17A, IL-23, and IL-10 levels were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Beyotime Biotechnology, Shanghai, China), following the manufacturer’s instructions.

At the end of week 14, intestinal tissues were quickly removed following sacrifice. After irrigation with PBS, some intestinal tissue samples of each individual mouse were immediately stored at −80°C for future western blot analysis, whereas others were immediately fixed in buffered 10% formalin. Paraffin-embedded sections 4µm-thick were stained with H&E, and images were acquired using Pannoramic 250. Inflammation was evaluated in a blinded manner by two investigators using a previously described scoring system (35, 36). Entire digital images were analyzed based on three criteria: inflammatory cell infiltration, epithelial changes, and mucosal architecture. Inflammatory cell infiltration was scored as follows: 1, mild mucosal infiltration; 2, moderate mucosal and submucosal infiltration; and 3, marked mucosal, submucosal, and transmural infiltration. Epithelial changes were scored as follows: 1, mild hyperplasia (<25%); 2, moderate hyperplasia (25%–50%); and 3, marked hyperplasia (>50%). The mucosal architecture was scored as follows: 1, mild villous blunting (villous-to-crypt-length ratio of 2:1 to 3:1); 2, moderate villous blunting (villous-to-crypt-length ratio of 1:1 to 2:1); and 3, marked, villous atrophy, branched crypts. The scores for each criterion were combined to obtain an overall inflammation score for each sample, ranging from 0 to 9 points.

Immunohistochemistry staining for zonula occludens-1 (ZO-1) and Occludin, types of tight junction proteins in the intestine, in 4µm-thick paraffin-embedded intestinal sections were performed as previously described (37). Briefly, tissue sections on slides were deparaffinized in xylene, rehydrated in graded alcohol and washed in PBS, immersed in 3% hydrogen peroxide in PBS for 15 min at room temperature to block endogenous peroxidase. We perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 (ab 93684, Abcam, Cambridge, UK). After washing with PBS buffer, the sections were treated with goat serum for 20 min at room temperature to block nonspecific binding. Subsequently, the sections were incubated overnight at 4°C with primary antibodies: rabbit monoclonal anti-ZO-1 (1:1000; ab221546, Abcam, Cambridge, UK) or rabbit monoclonal anti-occludin (1:200; ab216327, Abcam, Cambridge, UK), and then, incubated with horseradish peroxidase (HRP)-conjugated goat against rabbit IgG secondary antibody (1:1000; ab6721, Abcam, Cambridge, UK) for 30 min at room temperature. The immunocomplexes were visualized using diaminobenzidine (DAB), and the nuclei were counterstained with hematoxylin. The images were acquired using Pannoramic 250 and analyzed using ImageJ v1.46r software by measuring the optical density of ZO-1 and occludin proteins. The arithmetic mean of the quantification in five fields per section was determined.

Total proteins were extracted from intestinal tissue by homogenization in radioimmunoprecipitation assay (RIPA) buffer. The bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China) was used to determine protein concentrations. Equal amounts of protein (40 µg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a polyvinylidene difluoride (PVDF) membrane (Millipore, Boston, MA, USA) using a semidry transfer system (Bio-Rad, Hercules, CA, USA). The membrane was blocked for 1 h at room temperature with 5% bovine serum albumin (BSA) and incubated overnight at 4°C with the following primary antibodies: anti-zonula occludens-1 (ZO-1) (1:1000; ab96587, Abcam, Cambridge, UK), anti-occludin (1:1000; ab167161, Abcam, Cambridge, UK), anti-AhR (1:1000; ab28698, Abcam, Cambridge, UK), anti-FoxP3 (1:1000; ab75763, Abcam, Cambridge, UK), anti-STAT3 (1:1000; ab68153, Abcam, Cambridge, UK), and anti-RORγt (1:1000; 14-6988-82, Thermo Fisher Scientific, Carlsbad, MA, USA). Appropriate HRP-conjugated secondary antibodies (1:3000; ab6721 and 31470) were obtained from Abcam and Thermo Fisher Scientific and incubated with the membrane for 1 h at room temperature. Bands were visualized with an enhanced chemiluminescence (ECL) reagent (Beyotime Biotechnology, Shanghai, China) using the ChemiDoc™ XRS+ imaging system (Bio-Rad, Hercules, CA, USA). The ImageJ v1.46r software was used to measure the optical density of protein bands.

Lamina propria (LP) immune cells from the small intestine were isolated using mouse lamina propria dissociation kit (130-097-410, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instruction. For intracellular analysis of cytokine expression, cells were stimulated in culture medium with cell stimulation cocktail plus protein transport inhibitors (00-4975-93, eBioscience, San Diego, CA, USA) in a cell incubator with 5% CO2 at 37°C for 6h to promote IL-17 and FoxP3 expression and prevent secretion. To discriminate live from dead cells, single-cell suspensions were incubated with Zombie Aqua™ Fixable Viability Kit (1:100; 423101, Biolegend, San Diego, CA, USA) for 30 minutes in the dark at room temperature. For T-helper 17 (Th17) cells, cells were surface stained on ice for 20 min in the dark with surface markers APC/Cyanine7 anti-mouse CD3ϵ (145-2C11, Biolegend, San Diego, CA, USA) and FITC anti-mouse CD4 (RM4-5, Biolegend), then fixed, permeabilized and intracellular stained for 25 min at room temperature with intracellular markers PE anti-mouse IL-17A (TC11-18H10.1, Biolegend). For T regulatory (Treg) cells, cells were surface stained on ice for 20 min in the dark with surface markers FITC anti-mouse CD4 (RM4-5, Biolegend) and APC anti-mouse CD25 (PC61, Biolegend), then fixed, permeabilized and intracellular stained for 30 min at room temperature with intracellular markers PE anti-mouse FoxP3 (MF-14, Biolegend). All appropriate isotype controls were used as negative controls. Cell samples were acquired using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA), and data were analyzed using FCS Express v7.12 (De Novo Software, Pasadena, CA, USA).

Fresh stool samples were collected from mice at week 14, placed into sterile containers, and immediately frozen at −80°C until processing. Microbial DNA was extracted from the fecal material of each sample using the TIANamp Genomic DNA Kit DP328 (TIANGEN Co., Beijing, China), according to the manufacturer’s instructions. The DNA concentration was measured using the Qubit^® dsDNA HS Assay Kit in a Qubit^® 2.0 Fluorometer (Thermo Fisher Scientific, Carlsbad, MA, USA). Sequencing libraries were generated using NEBNext^® Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s recommendations. DNA libraries were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Finally, DNA libraries were sequenced on an Illumina NovaSeq 6000 (Illumina Inc., San Diego, USA), and paired-end reads were generated.

The sequencing resulted in approximately 60 Gb raw reads, with an average number of 4,324,296,072 reads per sample, ranging from 4,053,396,509 to 5,110,089,364. The raw sequence reads underwent quality control, de novo assembly, and gene prediction using the MetaPhlAn3 (version 3.0) pipeline (38). The adaptor was detected and trimmed using Trim Galore (version 0.6.4; https://github.com/FelixKrueger/TrimGalore), and Fastp (39) was used to remove reads with more than 10% N, Q20 >20%, or shorter than 60 bp. Mouse genomic reads were filtered by mapping the metagenomic reads against the latest mouse genome reference (mm10) using Bowtie2 (version 2.3.5.1) (40). Taxonomic annotation was generated using MetaPhlAn3 with the v30 version of the database and default parameters. Functional profiling based on metagenomic sequences was performed using the HUMAnN3 pipeline (version 3.0.0) (41) with default parameters to obtain gene families (UniRef90, 201901) (42).

Sequences were subjected to alpha and beta diversity analyses using the Vegan package, and visualization was performed using the ggplot2 package in R statistics software (version, R 3.6.3). Differences in alpha diversity were calculated according to the richness indicated by observed species and the diversity indicated by the Shannon index. Beta diversity was evaluated using principal coordinate analysis (PCoA) based on Bray–Curtis distance matrices. Permutational multivariate analysis of variance (PERMANOVA) was used to evaluate the effects of grouping variables on sample differences using the Vegan package in R statistics software. Heat map and clustering analyses were performed using the Vegan package in R statistical software. Significant differences in the relative abundance of gut microbiota between the control, PG, and IAA groups were determined using linear discriminant analysis (LDA) effect size (LEfSe) analysis (43), based on the Kruskal–Wallis rank-sum test. Only LDA scores > 3 and p-values < 0.05 (Kruskal–Wallis test) were considered significantly enriched.

For metagenomic analysis, significant differentially abundant genes identified in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between the three groups were identified using the Kruskal–Wallis rank-sum test with Benjamini–Hochberg false discovery rate (FDR) correction.

All data are presented as the mean ± standard deviation (SD) and were analyzed using GraphPad Prism 8.3 (GraphPad Software, California, USA). Significant differences between the three groups were determined by one-way analysis of variance (ANOVA), followed by Bonferroni’s multiple comparisons test. A p-value < 0.05 was considered significant.

To evaluate the effectiveness of IAA treatment on arthritis, we examined arthritis incidence and arthritis severity in mice from the three treatment groups. The experimental design is summarized in Figure 1A . Compared with control mice, mice from the PG group began to develop redness and swelling in the foot and toes starting at week 7, which gradually aggravated over time and showed significant redness and swelling at week 14. This effect was alleviated by IAA treatment, as the incidence of arthritis was reduced and the redness and swelling were relieved to varying degrees during weeks 10–14 among the mice in the IAA group ( Figure 1B ). The arthritis scores and hind paw swelling in the IAA-treated mice were significantly reduced compared with those in the PG group during weeks 13–14 (p < 0.05, Figures 1C, D ). Representative pictures of the hind feet from the different groups are shown in Figure 1E .

The histological sections obtained from the ankle joint of mice in the control group showed normal histological structures, with no signs of inflammatory infiltration or cartilage destruction and a clear synovial space. The ankle joints from mice in the PG group exhibited marked signs of arthritis, characterized by synovial proliferation, inflammatory cell infiltration, cartilage degeneration, and bone erosion ( Figure 1F ). Similarly, we observed inflammatory cell infiltration around the IVD and the destruction of the disk structure in the vertebral joints of mice from the PG group ( Figure 1G ). These histological features of the ankle joint and the vertebral joints were significantly ameliorated in IAA-treated mice. The histological scores for the ankle joint and the vertebral joints in the IAA group were significantly decreased compared with those in the PG group (p < 0.05, Figures 1H, I ). Overall, these findings demonstrated that IAA treatment could inhibit the development and severity of AS in mice.

Inflammatory cytokines have been shown to play important roles in the pathogenesis of chronic inflammatory diseases and represent important therapeutic targets in human and animal models of these diseases (44). To assess the effects of IAA on systemic inflammatory cytokines, the circulating levels of pro-inflammatory and anti-inflammatory cytokines were examined. The serum levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-17A, and IL-23) were significantly increased in the PG group compared with those in the control group, whereas IAA treatment significantly reduced the levels of TNF-α, IL-6, IL-17A, and IL-23 (p < 0.05, Figures 2A–D ). By contrast, the serum level of the anti-inflammatory cytokine IL-10 was significantly decreased in the PG group compared with that in the control group, and IAA treatment significantly increased the level of IL-10 (p < 0.05, Figures 2E ). Meanwhile, the ratios of pro-/anti- inflammatory cytokines (TNF-α/IL-10, IL-6/IL-10, IL-17A/IL-10, IL-23/IL-10) were significantly increased in the PG group compared with those in the control group, whereas IAA treatment significantly reduced the ratios of TNF-α/IL-10, IL-6/IL-10, IL-17A/IL-10, and IL-23/IL-10 (p < 0.05, Figures 2F–I ). These results demonstrated that IAA could reduce the levels of pro-inflammatory cytokines, increase the level of an anti-inflammatory cytokine, and reduce the ratios of pro-/anti- inflammatory cytokines.

Intestinal inflammation is common among patients with AS and is thought to serve as a risk factor for AS development (45, 46). Thus, we investigated whether pathological alterations were observed in the intestinal mucosa of AS mice during AS development. We analyzed pathological sections of ileum tissue stained with H&E ( Figure 3A ). Histological analysis showed normal histological morphology in the control group, with intact epithelium, normal mucosal architecture, and no infiltration of inflammatory cells. Among the AS mice in the PG group, marked levels of epithelial hyperplasia, villous blunting, and inflammatory cell infiltration could be observed. IAA treatment significantly mitigated these pathological alterations, and the ileum samples from the IAA-treated group were characterized by mild levels of epithelial hyperplasia, villous blunting, and inflammatory cell infiltration. The histopathological intestinal inflammation score for the IAA group was significantly decreased compared with that for the PG group (p < 0.05, Figures 3B, C ). Overall, IAA appeared to ameliorate pathological morphology in ileum tissue.

ZO-1 and occludin are important tight junction proteins, which are involved in intestinal leakage (47).. The decreased expression of these proteins increases intestinal permeability, which has been associated with AS pathogenesis (48). To examine whether IAA could protect the intestinal mucosal barrier function in AS mice, immunohistochemistry and western blot analysis were used to detect the expression of tight junction proteins in the ileum. The protein levels of ZO-1 and occludin were significantly decreased in the PG group compared with those in the control group. IAA treatment could partly restore ZO-1 and occludin protein levels (p < 0.05, Figures 3D–H ), indicating that IAA could partially protect against PG-induced intestinal mucosal barrier dysfunction by enhancing intestinal tight junction expression and restoring intestinal integrity.

AhR is a ligand-activated transcription factor that plays critical roles in various autoimmune diseases, including the regulation of Th17/Treg response (49, 50). An imbalance in the Th17/Treg response and the release of associated cytokines are thought to be involved in the pathogenesis of AS. The transcription factor FoxP3 is essential for Treg cell development and function (51). STAT3 and RORγt are important transcription factors involved in Th17 cell differentiation (52, 53). Th17/Treg balance is regulated by these important transcription factors. To further investigate the anti-inflammatory effects of IAA through the regulation of AhR activation and Th17/Treg balance, we first used western blot analysis to detect the expression of AhR and Th17/Treg-related transcription factors, such as RORγt, STAT3, and FoxP3 in the ileum. The protein levels of AhR and FoxP3 were significantly downregulated, and RORγt and STAT3 were significantly upregulated in the PG group compared with the control group. After IAA treatment, the protein levels of AhR and FoxP3 were significantly upregulated, and RORγt and STAT3 were significantly downregulated, in contrast to the PG group (p < 0.05, Figures 4A, B ). These findings indicated that IAA could activate the AhR pathway and regulate Th17/Treg-related transcription factor activity. Due to the increased expression of Foxp3 and decreased expression of RORγt and STAT3, we next investigated whether IAA could regulate Th17/Treg balance by increasing Treg cell numbers and decreases Th17 cell numbers in AS model mice. We used flow cytometry to measure the percentages of Treg cells (CD4⁺CD25⁺Foxp3⁺) and Th17 cells (CD3⁺CD4⁺IL-17⁺) among CD4⁺ T cells in the lamina propria of ileum. The percentage of Th17 cells was increased and that of Treg cells was decreased in the PG group compared with those in the control group. As expected, IAA treatment markedly decreased the percentage of Th17 cells and increased that of Treg cells among CD4⁺ T cells in the ileal lamina propria of AS model mice (p < 0.05, Figures 4C–H ). Taken together, these findings indicated that IAA could activate the AhR pathway and regulate Th17/Treg balance.

We performed metagenomic sequencing on fecal samples obtained from the three groups to investigate changes in the intestinal microbiota compositions. We analyzed the diversity of the intestinal microbiota examined whether diversity was associated with AS. Alpha diversity was measured according to the observed species and the Shannon index, which showed no significant differences between the three groups (p = 0.502 and 0.0545, respectively, Figure 5A ). However, comparative analysis revealed that the Shannon index value increased significantly in the PG group compared with the control group (p < 0.05, Figure 5A ). To identify discrepancies between the intestinal microbiota compositions among the three groups, we assessed overall differences in beta diversity using a PCoA of the Bray–Curtis dissimilarity analysis, paired with PERMANOVA. The results showed that the microbiota community composition of the PG group was distinctly different from that of the control group (PERMANOVA R² = 0.596, p = 0.002, Figure 5B ). These findings showed that the richness and diversity of the intestinal microbiota composition found in AS mice were significantly different from those in control mice, suggesting that AS mice were characterized by disorder among the intestinal microbiota.

Subsequently, we analyzed the composition of the intestinal microbiota and detected whether changes in composition were associated with AS development. Taxon analysis showed differential abundance in the intestinal microbial compositions of the control, IAA, and PG groups. At the phylum level, Bacteroidetes, Proteobacteria, and Firmicutes were the three most dominant taxa (relative abundance >5%), representing greater than 96.5% of the total sequences across all three groups ( Figure 5C ). The relative abundance of Bacteroidetes (61.57%) decreased, and the relative abundances of Proteobacteria (18.97%) and Firmicutes (17.89%) increased in the PG group compared with the control group (84.41%, 8.62%, and 5.95%, respectively). IAA treatment increased the abundance of Bacteroides to 74.04% and decreased the abundances of Proteobacteria to 13.51% and Firmicutes to 9.05%. Moreover, the ratios of Bacteroides/Firmicutes and Bacteroides/Proteobacteria were 3.44 and 3.25 in the PG group, respectively, compared with 14.18 and 9.79 in the control group. IAA treatment increased the ratio of Bacteroides/Firmicutes to 8.19 and the ratio of Bacteroides/Proteobacteria to 5.48.

At the species level, we also found differences in the intestinal microbial community compositions among the three groups ( Figure 5D ). To identify specific individual bacterial taxa, which were differentially abundant across the three groups, we performed LEfSe analysis, using LDA ≥ 3 and p < 0.05 as the criteria to determine whether differences existed in these biomarkers between the three groups. A total of 41 differentially abundant bacterial taxa were identified across the three groups, including 22 bacterial taxa that were significantly more prominent in the PG group, 13 bacterial taxa that were significantly more prominent in the control group, and 6 bacterial taxa that were highly enriched in the IAA group ( Figures 6A, B ). Hierarchical cluster analysis, visualized using a heat map, showed 8 abundant taxa that were significantly different between groups at the species level ( Figure 6C ). The relative abundances of Bacteroides sartorii, Anaerotruncus sp. G3 (2012), Clostridium sp. ASF502, Firmicutes bacterium ASF500, Dorea sp. 5-2, and Clostridium sp. ASF356 were significantly higher in the PG group, and the relative abundance of Bifidobacterium pseudolongum was significantly lower compared with those in the control group. Interestingly, IAA treatment significantly reversed these changes in species abundance associated with AS in the PG group. In addition, the abundance of Mucispirillum schaedleri was significantly higher in the IAA group than in the other two groups ( Figure 6D , Kruskal–Wallis, p < 0.05).

To investigate the functional profiles of the intestinal microbiota compositions among the three groups, we performed a metagenomic analysis of our samples using KEGG pathway analysis in the HUMAnN3 pipeline. In total, we identified 63 KEGG pathways that were differentially abundant in the three groups based on the results of the Kruskal–Wallis test (Benjamini–Hochberg FDR correction). The significantly enriched KEGG pathways identified among the intestinal microbiota of AS mice were primarily distributed in cell growth and death pathways (e.g., oocyte meiosis and cell cycle), drug resistance: antimicrobial (e.g., beta-Lactam resistance), and infectious disease: parasitic (e.g., malaria and toxoplasmosis), indicating that these pathways were significantly disrupted and may potentially be involved in the pathogenesis of AS. After IAA treatment, enriched pathways were primarily distributed in genetic information processing (e.g., sorting and degradation), organismal systems (e.g., ‘development and regeneration’ and ‘excretory system’), environmental information processing (e.g., signal transduction), and metabolism, which included metabolism of cofactors and vitamins (e.g., retinol metabolism), xenobiotic biodegradation and metabolism (e.g., ‘nitrotoluene degradation’, ‘caprolactam degradation’, and ‘ethylbenzene degradation’), metabolism of terpenoids and polyketides (e.g., ‘Geraniol degradation’, ‘Limonene’, and ‘pinene degradation’), and biosynthesis of other secondary metabolites (e.g., penicillin and cephalosporin biosynthesis) ( Figure 6E ). The enrichment pathways in the IAA group were similar to those in the control group, suggesting that IAA treatment was able to recover the function of the intestinal microbiota in AS mice.