Male and female specific pathogen-free (SFP) BALB/c mice aged 6–8 weeks with different microbiota complexity were purchased from Beijing SiPeiFu Biotechnology Co. and kept at 23 ± 2°C under a 12 h light/dark cycle with ad libitum access to food and water. Mice were pretreated with 25 mg streptomycin by oral gavage 24 h prior to infection. S.T ATCC 14028 were grown overnight in lysogeny broth medium at 37°C with shaking at 220 rpm and then cultured (1:33) in fresh lysogeny broth medium for 3–3.5 h. To model S.T infection, mice were administered S.T by oral gavage (1 × 10⁹ CFU/mouse). Six hours after S.T gavage, mice were administered AA (Aladdin, Shanghai, China, CAS: 464-92-6, C₃₀H₄₈O₅, HPLC ≥ 98%) by oral gavage (10 mg/kg) on 3 consecutive days. The infected mice were euthanized 72 h after infection. The sample-size was not predetermined, and mice were randomly assigned to groups. Neutrophils were depleted in mice by daily intraperitoneal injection of anti-mouse Ly6G-Purified (Cat No: L280, Leinco Technologies, USA) (250 μg/mouse) for 3 days.

All animal experiments were approved by the Committee on Animal Care and Use of the Institute of Traditional Chinese Medicine Health Industry (licence numbers: SYXK(Gan)2023-0008).

Intestinal tissue from the anus to the cecum was isolated from mice. Colon tissues were fixed in 4% formaldehyde, embedded in paraffin, and sectioned for staining with hematoxylin-eosin (H&E) (Servicebio, Beijing, China) or Alcian blue/periodic acid-Shiff (AB/PAS) solutions. Sections were observed with an SV200 microscope (Olympus, Japan).

Acidic mucin granules (blue) density was calculated using Jmage J software as follows: Open image → Image → Color → Segmentation Channel → Select Blue → Adjustment → Threshold → Apply → Analyze → Measurement → Acid Mucin Density (%Area).

Based on a previously described method (37), samples were prepared from colon tissues using RIPA buffer (KeyGEN BioTCHE, Nanjing, China). Total proteins were extracted, and protein concentration was measured using a BCA kit. Equal amounts of proteins were separated by 10%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and were then transferred onto nitrocellulose membranes (Cytiva, USA). After blocking with 5% skim milk for 1 h, the membranes were incubated with the indicated primary antibodies at 4°C overnight. The main primary antibodies used are as follows: Occludin (Cat No. 27260, 1:1000, Proteintech, Wuhan, China), Claudin1 (Cat No. 28674, 1:500, Proteintech), β-actin (Cat No. 81115, 1:1000, Proteintech), Hes5 (Cat No. 22666, 1:200,:1000, Proteintech), Hey1 (Cat No. 19929,:1000, Proteintech), Notch2 (Cat No. 5723, 1:1000, CST), Ly6G (Cat No. 14-5931-82, 1:1000, Thermo Fisher Scientific, USA), LGR5 (Cat No. PA5-87974, 1:1000, Thermo Fisher Scientific). The visualization of the protein bands was completed with ECL kit (Advansta, USA).

Three-micron-thick sections were prepared from wax-embedded tissues, deparaffinized in xylene and alcohol, incubated with EDTA at 100°C for 20 min, 3% hydrogen peroxide at room temperature for 25 min, and finally with 5% bovine serum albumin at room temperature for 1 h. Sections were then incubated sequentially with primary and secondary antibodies. The following antibodies and dilutions were used for the staining of different samples: EPCAM/CD326 (Cat No. 21050, 1:200, Proteintech), 1:200 S.T-LPS (Cat No. MA1-83451, 1:200, Thermo Fisher Scientific, USA), Ly6G (Cat No. 14-5931-82, 1:100, Thermo Fisher Scientific, USA), LGR5 (Cat No. PA5-87974, 1:100, Thermo Fisher Scientific) combination with the respective secondary antibodies, anti-Rabblt AlexaFluor™ 488 (Cat No. A11008, 1:500, Thermo Fisher Scientific), anti-Mouse AlexaFluor™ 488 (Cat No. A11011, 1:500, Thermo Fisher Scientific), Cyanine3 (Cat No. A10521, 1:400, Thermo Fisher Scientific), and DAPI (Beyotime Biotechnology, Shanghai, China). Images were observed with an SV200 microscope (Olympus, Japan).

HL-60 cells were stimulated into neutrophils with 1 μM all-trans-retinoic acid, then cultured in 96-well plates. Each well was infected with S.T at a multiplicity of infection of 10. AA (50 and 100 μM) was added to the culture immediately after S.T infection. Following a 3 h infection with S.T, HL-60 cells were lysed with sterile distilled water for 5 min. The cell lysates were then diluted and plated on LB agar and incubated at 37°C for 24 h, permitting an estimation of the CFUs for S.T.

Phagocytosis (%) = (Counts of released bacteria after Triton treatment/Counts of added bacteria) * 100.

Bacteria were grown to early exponential phase in Luria-Bertani broth at 37°C and then diluted to 10⁷ CFU/mL in Luria-Bertani medium supplemented with AA at final a gradient of concentrations ranging from 50 μM and 100 μM. A separate Luria-Bertani culture without the AA was set as the control. Next, 200 μL of the aforementioned cultures with or without AA was transferred to each well of a 96-well, microtiter plates, which were then incubated at 37°C. Growth was again measured by taking OD readings at 600 nm at 24 h.

HERB (High-throughput Experiment-and Reference-guided database of traditional Chinese medicine), HIT (Herbal Ingredients’ Targets Platform), STITCH (Search Tool for Interactions of Chemicals, combined score ≥ 0.8), TargetNet (Probability ≥ 0.8), and NetInfer (Score ≥ 2) were applied to search or predict potential targets. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) platform was used to construct the target interaction network and to perform functional annotation analysis.

Total RNA was extracted from colon tissue using TRIzol Reagent according to the manufacturer’s instructions (Invitrogen, California, USA), and genomic DNA was removed using deoxyribonuclease I (TaKara, Tokyo, Japan). Then, RNA quality was determined by 2100 Bioanalyzer (Agilent, California, USA) and quantified using the ND-2000 (NanoDrop Technologies, Massachusetts, USA). Sequencing was performed by Tianjin Novogene Co., Ltd. (Tianjin, China). Briefly, 1 μg of total RNA was used for reverse transcription into cDNA. Then, A 200-300 bp cDNA target fragment was amplified for 15 RNA cycles, which was used to construct a cDNA library. After quantification by TBS380, the paired-end RNA-seq library was sequenced on the Illumina NovaSeq 6000 (2 × 150 bp read length).

RNA-seq data were initially filtered to obtain clean data. To identify differentially expressed genes (DEGs) between two different samples, the expression level of each transcript was calculated according to the fragments per kilobase of exon per million mapped reads method. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were performed on the genes associated with the core targets using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/). The species was set to “Mus musculus”, the p-value was set to “< 0.05”.

Statistical analyses were conducted using GraphPad Prism software (version 6). Whenever applicable, the one-way analysis of variance (ANOVA) followed by LSD-t test for multiple comparisons was used. All values are presented as mean ± S.D. p < 0.05 is considered statistically significant.

S.T infection can quickly trigger an inflammatory response that modifies the intestinal environment and disrupts the intestinal barrier (38). Mice were infected with wild-type S.T by daily oral administration of 1 × 10⁹ CFU for 3 days ( Figure 1A ). As expected, the length of the colon was reduced by S.T infection ( Figures 1B, C ). To observe the microstructure of the colon, H&E staining was performed, which showed that the crypt depth was significantly reduced in the S.T-infected mice ( Figures 1D, E ). These data confirm that S.T causes significant damage to the colon.

Dextran sulfate sodium-induced colon damage is alleviated by AA (33). Therefore, we hypothesized that a similar response to the S.T-induced colon injury may occur after AA administration. As predicted, AA significantly increased the length of the colon reduced by S.T infection ( Figures 1B, C ) and increased crypt depth ( Figures 1D, E ). Collectively, these results indicate that S.T-induced colon damage is alleviated by AA.

The intestinal mucosal barrier is a crucial line of defense against pathogens. The strength of this barrier is maintained by the integrity and tight connections of IECs. Therefore, we assessed the level of EPCAM, an IEC marker, in the colon of mice by immunohistochemistry. We observed that the level of EPCAM in the colon was not affected by S.T infection ( Supplementary Figure S1 ). Next, IHC and western blotting was used to examine the levels of tight junction proteins. S.T infection significantly decreased the levels of occludin and claudin 1 compared with those in the control group ( Figures 2A–F ). Furthermore, AA significantly increased the levels of occludin and claudin 1 compared to those in the S.T-infected mice ( Figures 2A–F ). IECs are often covered with a layer of mucin secreted by goblet cells, which blocks the penetration of harmful substances and chemicals (39). We therefore performed AB-PAS staining to quantify the levels of acidic mucin granules. Not surprisingly, S.T infection significantly reduced the level of acidic mucin granules in the colon ( Figures 2G, H ). Conversely, AA significantly increased the level of acidic mucin granules ( Figures 2G, H ). These results indicate that AA alleviates S.T-induced damage to the colonic barrier.

S.T infection inactivates ISCs (40); therefore, to observe ISC activation, we performed immunohistochemistry and western blotting for LGR5, a highly specific marker for ISCs located above crypts (41). The results showed significantly reduced levels of LGR5 in the S.T-infected mice ( Figures 3A–D ). AA significantly increased the levels of LGR5 compared with the levels in the S.T-infected mice ( Figures 3A–D ). These data support the reversal of S.T.-induced ISC inactivity by AA.

To explore the mechanism by which AA ameliorates S.T infection, network pharmacology analysis was performed. We obtained 2104 predicted disease targets and 220 drug targets from databases ( Figure 3E ). Among these, 67 AA- and S.T infection-related targets were considered hub targets ( Figure 3E ). A protein-protein interaction (PPI) network containing 20 nodes and 42 edges was constructed that included IL6, TNF, IL1B, IL2, PTGS1, and NFBκ1 ( Figure 3F ). GO ( Figure 3G ) and KEGG ( Figure 3H ) analyses were then performed using the DAVID. In biological processes (BP), hub targets were majorly enriched in response to xenobiotic stimulus, response to oxidative stress, apoptotic process, inflammatory response, cellular response to lipopolysaccharide, and response to lipopolysaccharide. Among 20 KEGG-enriched pathways were the Notch, IL17, and TNF signaling pathways, and Salmonella infection.

Overactivation of the Notch pathway inhibits the differentiation of ISCs (42, 43), and the Notch pathway was significantly enriched in the network pharmacology analysis ( Figure 3H ). To understand the role of the Notch2 pathway in S.T-infected mice, the levels of related proteins were determined using western blotting. The levels of Notch2, Hes5, and Hey1 were significantly increased in the S.T infected colon ( Figures 3I, J ). AA significantly decreased the levels of Notch2, Hes5, and Hey1 compared with those in the S.T-induced mice ( Figures 3I, J ). These results indicate that the S.T-activated Notch2 pathway is inhibited by AA.

To study the targets and signaling pathways involved in the AA-amelioration of Salmonella colitis, we performed transcriptome analysis. This analysis highlighted the involvement of neutrophils. DEGs involved in processes related to neutrophils including Slit2, Thbs4, Nos2, and Cxcl13 were significantly upregulated after the S.T infection ( Figure 4A ). Conversely, these genes were significantly downregulated after the AA treatment ( Figure 4B ). Next, GO and KEGG enrichment analysis were then performed on the DEGs. The results show that the DEGs following the S.T infection and the AA intervention were enriched in multiple neutrophil-related pathways, including neutrophil homeostasis, neutrophil activation involved in immune response, neutrophil mediated immunity, neutrophil activation, regulation of neutrophil chemotaxis, regulation of neutrophil migration, regulation of neutrophil migration, neutrophil activation involved in immune response, neutrophil mediated immunity, and neutrophil degranulation ( Figures 4C–F ). Therefore, we speculate that neutrophils play an important role in AA-induced colonic damage caused by S.T infection.

As an early line of defense against bacterial infections, neutrophils are recruited into the intestinal lumen during pathogen infection (32–34). Immunohistochemistry showed a significantly higher level of S.T around intestinal tissue in the S.T-infected mice than in the control mice ( Figures 5A, B ), which was colocalized with neutrophils ( Supplementary Figure S2 ). Neutrophils were recruited into the intestinal lumen after the S.T infection, which forms a barrier preventing S.T invasion ( Figures 5C, D ). AA significantly reduced the levels of S.T and neutrophils around the colon tissue ( Figures 5A, B ), which may result from the phagocytosis of S.T by neutrophils. We then stained colon tissue for the IEC marker, EPCAM, which showed that the level of IEC efflux was significantly increased in the S.T-infected mice ( Figures 5E, F ). This was significantly attenuated by the AA intervention ( Figures 5E, F ), which may be related to the reduction in S.T counts. These results indicate that IEC efflux is inhibited by AA, which may be related to neutrophils.

To investigate this possibility further, mice were injected intraperitoneally with Ly6G, which significantly reduced the neutrophil numbers ( Supplementary Figure S3 ; Figures 5C, D ). Surprisingly, the large numbers of S.T were found around the colon tissue of the neutrophil-deficient mice ( Figures 5A, B ). AA did not reduce the level of S.T around the colon in the mice with neutrophil depletion ( Figures 5A, B ). After neutrophil depletion, the number of shed IECs increased ( Figures 5E, F ) compared with that in the S.T-infected group. Moreover, the inhibitory effect of AA on IEC shedding was attenuated after neutrophil depletion ( Figures 5E, F ). These results indicate that AA regulation of IEC shedding is associated with neutrophils.

Upon infection, neutrophils rapidly migrate to the intestinal tract and phagocytose pathogens. To demonstrate the relationship between AA and neutrophil phagocytosis of bacteria, HL-60 cells were cultured in vitro and stimulated with all-trans-retinoic acid to induce differentiation into neutrophils (44). The number of S.T outside the neutrophils was significantly reduced after the AA intervention by calculating the phagocytosis% in vitro. ( Supplementary Figure S4A ). Next, we found that AA significantly inhibited the growth of S.T. ( Supplementary Figure S4B ). These results demonstrate that AA enhances the phagocytic function of neutrophils. In summary, neutrophils play an important role in AA-mediated colonic damage caused by S.T stimulation.

To further observe ISC activity in neutrophil-deficient mice, we examined levels of LGR5 by immunohistochemistry and western blotting. LGR5 levels were reduced in the neutrophil-deficient mice compared with those in the S.T-infected mice ( Figures 6A–D ). The effect of AA on ISC activity was significantly reduced after the neutrophil depletion ( Figures 6A–D ). These results demonstrated that the effect of AA on ISCs is dependent on neutrophils during the S.T infection. Next, western blotting was used to detect the levels of key proteins in the Notch2 pathway. As predicted, the levels of Hes5 and Hey1 were significantly increased in the neutrophil-deficient mice than in the S.T-infected mice ( Figures 6E, F ). The inhibitory effect of AA on the Notch2, Hes5 and Hey1 were reduced in the neutrophil-deficient mice ( Figures 6E, F ). These results demonstrate that AA influences the activity of ISCs by neutrophils via Notch2/Hes5/Hey1. In summary, the effect of AA on ISCs is dependent on neutrophils during the S.T infection.

To better define the contribution of neutrophils to the maintenance of the intestinal mucosal barrier in the S.T-infected mice, IHC and western blotting were used to examine tight junction proteins. The levels of occludin and claudin 1 were significantly reduced in the colon tissues of neutrophil-deficient mice compared with those in the S.T group ( Figures 7A–F ). Meanwhile, neutrophil depletion significantly reduced the ability of AA to affect the low levels of claudin 1 and occludin caused by the S.T infection ( Figures 7A–F ). Similarly, the restorative effect of AA on acidic mucin granule levels ( Figures 7G, H ), colon length ( Figures 7I, J ) and crypt depth ( Figures 7K, L ) was reduced in the neutrophil-depleted mice. Moreover, the levels of occludin and claudin 1, acidic mucin granules and crypt length were slightly decreased in mice with isolated neutrophil depletion; however, there was no significant difference compared with the control group ( Supplementary Figure S5 ). These results demonstrate that the effect of AA on S.T colitis is dependent on neutrophils.