Acetic acid (H1808008), propionic acid (A1903124), n-butyric acid (E1814125), and isobutyric acid (K1822187) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Tryptophan (T0254), 5-hydroxytryptamine (H952), kynurenine (K8625), and 3-hydroxykynurenine (H1771) were purchased from Sigma. 5-hydroxyindoleacetic acid (121282500) was purchased from ACROS. Quinolinic acid (P63204) was purchased from ALDRICH.

A. muciniphila (ATCC BAA835) was purchased from BeNa Culture Collection (Beijing, China). BHI broth (CM917) was purchased from Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Chen et al. in our research group had previously prepared Wuji Pills with stable quality, and Guo et al. had carried out sufficient qualitative and quantitative analyses (Chen et al., 2017; Guo et al., 2022). The chemical composition analysis of Wuji Pill from previous studies were provided in the Supplementary Figures S1, S2 and Supplementary Table S1.

Sprague–Dawley (SD) pregnant rats were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (Certificate of Qualification: SCXK (Beijing, China) 2016-0011). All animals were housed fed with food and water ad libitum (temperature 22 ± 2 °C, 12 h light/dark cycle). All experiments were provided by the Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences (SYXK (Beijing, China) 2020-0042, SYXK (Beijing, China) 2022B191). All animal experiments were strictly approved by the Animal Experiment Welfare and Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences and followed the international guidelines for animal research.

The IBS combined with depression model was induced by exposure to multiple stressors during the juvenile and adult stages of female SD rats, as previously described (Gong et al., 2022). During the juvenile stage (from day 7 to day 21 of birth in rats), percutaneous transluminal coronary angioplasty (PTCA) balloon dilation catheter (specification: 3.0 mm × 20 mm, Cordis) stimulation, maternal separation (MS), and chronic restraint stress (CRS) were used for modeling. Firstly, a PTCA balloon (Paraffin lubricated) was inserted into the anus of neonatal rats for 2 cm, and the intestinal tract was stimulated for 1 min after the balloon was fully inflated. Secondly, after the balloon was removed, the forelimbs of neonatal rats were bound, and the MS stimulation was performed for 3 h (Varghese et al., 2006). Finally, the neonatal rats were stimulated again with PTCA balloon, and then put back into the cage. In the adult stage (from day 49 to day 70 of birth in rats), the rats were stimulated by CRS for 9 h every day (Reagan et al., 2004).

Abdominal wall withdrawal reflex test (AWR) was used to evaluate visceral sensitivity in rats. The catheter (Paraffin lubricated) was inserted into the rat anus for 4 cm and fixed at the root of the rat tail. After the rats were immobile, 0.4 or 0.6 mL of normal saline was injected into the catheter balloon. The visceral sensitivity of rats was scored according to the scoring principle of AWR experiment, as shown in Figure 1B (Al-Chaer et al., 2000).

The distal colonic motility was recorded to evaluate the intestinal motility of rats. After fasting overnight, the rats were fixed in the bonder. The catheter (Paraffin lubricated) was inserted into the rat anus for 4 cm and 0.4 mL of normal saline was injected into the catheter balloon. The area under the colonic motility curve (motility index, MI) was recorded after 30 min of adaptation (BIOPAC, MP150, physiograph; BIOPAC, Goleta, CA, United States), and the colonic motility was quantitatively described by MI, as shown in Figure 1C (Iwa et al., 2006).

To minimize animal suffering and distress, euthanasia was performed under anesthesia induced by isoflurane. Following the completion of the behavioral test, the rats were anesthetized using an experimental bench animal anesthesia ventilation system (Midmark) with a 3% concentration of inhaled isoflurane. Subsequently, euthanasia was performed under anesthesia by collecting blood via the abdominal aorta. The feces of rats were collected aseptically and stored at −80 °C for further analysis. Subsequently, the hippocampus and colon tissues were carefully cleaned in cold normal saline. Excess water was gently absorbed using filter paper, after which the tissues were promptly immersed in liquid nitrogen for cryopreservation. The samples were then transferred and stored at −80 °C for subsequent analysis. Cardiac perfusion was performed after anesthesia in rats, and the colon and brain tissues were fixed in 4% paraformaldehyde for histological analysis.

The colon samples fixed in paraformaldehyde were embedded in paraffin and sliced. The colonic mucus secretion was detected by alcian blue periodic acid Schiff (AB-PAS) staining according to the protocol (Yamabayashi, 1987). The paraffin sections were dewaxed to distilled water and 3% acetic acid solution for 3 min. Then, the colon was stained according to the instructions of AB-PAS (CAS12040-44-7, Shanghai Tensus Biotech Co., Ltd.). Finally, the secretion of colonic mucus was observed under a microscope. ImageJ software was used to measure the percentage of acidic mucin area relative to the total tissue area.

The morphology of microglia was detected by immunofluorescence staining according to the protocol (Im et al., 2019). Briefly, euthanasia was performed under anesthesia induced by isoflurane. The rats were anesthetized using an experimental bench animal anesthesia ventilation system (Midmark) and inhaled isoflurane at a concentration of 3% and subjected to cardiac perfusion. For the cardiac perfusion process, first perfuse with pre-cooled 0.9% normal saline to wash out the blood in the blood vessels. Subsequently, immediately perform perfusion fixation with pre-cooled 4% paraformaldehyde phosphate buffer solution (0.01 M PBS) (SparkJade). The brain sample was fixed in paraformaldehyde, then embedded in paraffin and sliced. Brain sections were dewaxed to water, antigen repair, hydrogen peroxide and serum blocking in turn. After washing off the sealing solution, the primary antibody (1:500, GB113502, Servicebio) was added and incubated at 4 °C overnight. After eluting, the secondary antibody (1:500, GB23301, Servicebio) was added and incubated for 50 min. After incubation with iF555-TSA (1:500, Servicebio, G1233), the sections were cleaned, microwave treated and sealed. DAPI (Servicebio, G1012) was used to stain the nuclei, and then the sections were rinsed and sealed.

Total RNA from colon and hippocampus was extracted according to RNA Easy Fast Tissue/Cell Kit (DP451, Tiangen Biotech). The quantity and quality of RNA were performed by instrument (Biochrom SimpliNano, America). The total RNA was transcribed into cDNA according to EasyScript^® First-Strand cDNA Synthesis SuperMix (AE301, TransGen Biotech). Further, amplification was performed by PerfectStart^®Green qPCR SuperMix procedure (AQ601, TransGen Biotech) on a real-time fluorescent quantitative PCR (LightCycler 480II, Roche). The primer sequence was shown in the Table 1. Calculate according to the average Ct of multiple holes. Using GAPDH as the internal reference gene.

The genomic DNA of A. muciniphila was extracted by TIANamp Bacteria DNA Kit (DP302-02, Tiangen Biotech). TIANamp Stool DNA Kit (DP328-02, Tiangen Biotech) was used to extract genomic DNA from stool samples of rats. The above operations are carried out in strict accordance with the instructions. Further, qPCR was performed on a real-time fluorescent quantitative PCR instrument (lightcycle 480 II, Roche) using SYBR green reagent (AQ601, TransGen Biotech). The gene specific primers of A. muciniphila were synthesized according to the literature (Qin et al., 2018). The average Ct of multiple holes is substituted into the standard curve for calculation.

The 16S rRNA sequencing was used to detect the composition and relative abundance of gut microbiota. Briefly, DNA was extracted from fecal samples in strict accordance with the protocol in the instructions (Wang et al., 2025). The V3–V4 hypervariable region of the 16S rRNA was amplified using universal primers 341F (5’-CCTACGGGNGGCWGCAG-3′) and 805R (5’-GACTACHVGGGTATCTAATCC-3′). QIIME2 were used for sequences processing. Amplicon sequence variants (ASVs) were obtained after using DADA2 for dereplication and denoising. The raw data obtained after sequencing on the machine were subjected to subsequent bioinformatics analysis. This part of the sequencing experiment was assisted by Shanghai Zhongke New Life Biotechnology Co., Ltd. The Bray-Curtis distance was used for Principal coordinates analysis (PCoA). Alpha diversity analysis (including Chao1 index and Shannon index) was used to analyze the microbial differences. Linear discriminant analysis Effect Size (LEfSe) was used to identify differentially abundant taxa between groups. The raw sequence data were deposited in the Genome Sequence Archive in National Genomics Data Center (CNCB-NGDC Members and Partners, 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA023334).

The metabolites in the hippocampus, colon and feces were analyzed using the AB SCIEX triple Quad ™ 6,500 LC–MS/MS system (AB Sciex, Canada). The hippocampus (about 30 mg) was added with 150 μL methanol: acetonitrile (v/v = 1:1) to prepare homogenate. Add 10 μL of the internal standard working solution to 100 μL of the homogenate, and then perform a centrifugation operation (13,000 rpm, 4 °C, 20 min). The supernatant was used for LC–MS/MS analysis. The colon (about 200 mg) was added with 1 mL methanol: acetonitrile (v/v = 1:1) to prepare homogenate. Add 20 μL internal standard working solution to 200 μL homogenate and centrifuge (13,000 rpm, 4 °C, 20 min). The supernatant (160 μL) was concentrated by vacuum centrifugation (37 °C, 45 min), then added 80 μL methanol: acetonitrile (v/v = 1:1) for centrifugation (13,000 rpm, 4 °C, 15 min). The supernatant was used for LC–MS/MS analysis. The fecal samples were prepared according to the method described previously (Wang et al., 2025).

For hippocampus and colon samples, Waters Acquity UPLC HSS PFP column (2.1 × 100 mm, 1.8 μm) was used for chromatographic separation. The mobile phase consisted of solvent A (0.1% formic acid water) and solvent B (0.1% formic acid acetonitrile) at 35 °C with a typical flow rate (0.3 mL·min-1). The elution gradient of mobile phase was as follows: 0–1.5 min, 2% B; 1.5–5 min, 2% B -98% B; 5–7 min, 98% B; 7.1–10 min,2% B. Multiple reaction monitoring mode (MRM) and cationic electrospray ion source (ESI+) were used. Other parameters were as follows: ion source spray voltage, 4,500 V; inlet voltage, 10 V; impact chamber outlet voltage, 11 V; air curtain pressure, 35 psi; collision pressure, 9 psi; ion source temperature: 450 °C. For fecal samples, Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) was used for chromatographic separation. The mobile phase consisted of solvent A (0.1% formic acid water) and solvent B (isopropanol) at 50 °C with a typical flow rate (0.2 mL·min⁻¹). The elution gradient of mobile phase was as follows: 0–1 min 15% B; 1–8 min 15% B-36% B; 8–8.5 min 36% B-50% B; 8.5–10.5 min 50% B; 10.5–10.51 min 50% B-15% B; 10.51–15 min 15% B. Other parameters were set as mentioned above. Analytical parameters for target ion pairs were summarized in Tables 2, 3.

Statistical analyses were performed with GraphPad Prism 9 (La Jolla, CA, United States). All data of the experimental results were expressed by mean ± SEM. The data of each group were processed by one-way ANOVA or Kruskal-Wallis test. Fisher’s LSD test or Dunn’s post-hoc test was used for multiple comparison analysis. P-value < 0.05 was considered statistically significant.

In order to simulate the symptoms of gastrointestinal motility disorders accompanied by depression in IBS patients, we successfully established an animal model of IBS by PTCA balloon stimulation, MS and CRS. The procedure was shown in Figure 1A. The experimental results demonstrated that IBS model rats exhibited significantly higher AWR scores compared to the control group, indicating that two stages stimulation protocols effectively induced visceral hypersensitivity in the intestinal tract (Figure 1B; Supplementary Figure S3A), and the colonic motility index was significantly increased (Figure 1C). Furthermore, the exercise time of IBS rats did not change (Supplementary Figure S3B), and the sucrose consumption significantly decreased in both the juvenile and adult stages (Figure 1D). Meanwhile, the immobility time in the FST of IBS rats significantly increased (Figure 1E).

At the same time, pinaverium bromide (antispasmodic) and sertraline (antidepressant) were employed as positive drugs, which are commonly used to improve IBS in clinic. Pinaverium bromide can significantly reduce the AWR score and colonic motility index in IBS rats, the sucrose consumption was significantly increased and the immobility time in the FST was decreased, which plays a certain role in improving depressive-like behaviors. Sertraline can significantly increase the sucrose consumption and reduce the immobility time in the FST. Wuji Pill can significantly improve the symptoms of abdominal pain and colonic motility in rats (Figures 1B,C). Furthermore, the treatment with Wuji Pill remarkably ameliorated the depression-like behaviors in IBS rats. The IBS rats administered with Wuji Pill exhibited an augmented sucrose consumption (Figure 1D), and alleviated the desperate state of rats in a stressful environment (Figure 1E). These results indicate that Wuji Pill has the potential to improve the intestinal symptoms and depression-like behaviors of IBS rats.

In this study, 16S rRNA gene sequencing technology was used to evaluate changes in the gut microbiota of rats. Based on the principal coordinate analysis (PCoA) of Bray-Curtis distance, we revealed that the gut microbiota composition of the model group was significantly different from that of the control group and Wuji Pill group (Figure 2A). Chao1 index and Shannon index belong to alpha diversity analysis, representing the abundance and diversity of taxon composition, respectively (Shi et al., 2023). The Chao1 index of IBS rats increased significantly (Figure 2B), and the Chao1 index and Shannon index decreased significantly after oral administration of Wuji Pill (Figures 2B,C). In addition, the taxon composition results showed that Bacteroidota and Firmicute were the dominant bacteria at the phylum level (Supplementary Figure S3C). Further, Prevotella was the main difference between the control group and the model group (Supplementary Figure S3D). Prevotellaceae, Muribaculaceae, Firmicutes and A. muciniphila were the main difference bacteria between the model group and the Wuji Pill group (Figure 2D). The relative abundance of Prevotellaceae_UCG-001 in the model group increased significantly (Figures 2E,G). Interestingly, the currently highly regarded next-generation probiotic A. muciniphila significantly increased in the Wuji Pill group (Figures 2F,H). Muribaculaceae showed an increasing trend in the Wuji Pill group (Figure 2I). In short, the composition of the gut microbiota was significantly changed after treatment with Wuji Pill.

A germ-free animal model is often used to explore the relationship between gut microbiota and the host. We established a pseudo germ-free model by using mixed antibiotics to explore the improvement effect of Wuji Pill on IBS (Figure 3A). Wuji Pill also improved intestinal visceral sensitivity (Figure 3B) and reduced intestinal motility index (Figure 3C) in ABX group. Intestinal mucus constitutes a crucial component of the intestinal barrier (Aburto and Cryan, 2024). Goblet cells and other secretory cells play an important role in the secretion of intestinal mucus (Birchenough et al., 2015). The results showed that the secretion of mucus in the colon of IBS rats was significantly reduced (Figure 3D). Notably, oral administration of Wuji Pill significantly ameliorated this colonic mucus depletion. Furthermore, antibiotic intervention induced a marked reduction in intestinal mucus secretion (Figure 3D). These results indicate that gut microbiota plays an essential role in maintaining mucosal secretory homeostasis.

We further investigated the improvement effect of Wuji Pill on depressive-like behaviors in pseudo germ-free IBS rats. Similarly, the ABX group also has the effect of improving the depressive-like behavior of IBS rats. In the ABX group, the distance of movement in the central area of the OFT increased (Figure 3E). Moreover, both the time of NSFT and the immobility time of FST were shortened (Figures 3F,G). In addition, IBA-1, as a marker of microglia, reflects health and activation status of microglia (Lituma et al., 2021). Compared with the control group, the expression of IBA-1 in the cortex of the model group was significantly up-regulated. Notably, Wuji Pill was able to downregulate the expression of IBA-1 (Figure 3H).

16S rRNA gene sequencing technology and real-time fluorescent quantitative PCR technology were used to compare the changes of gut microbiota. The results demonstrated that antibiotic intervention significantly reduced the diversity of gut microbiota (Figures 4A,B). The absolute abundance of A. muciniphila in Wuji Pill group was significantly greater than that in model group, and A. muciniphila in feces was significantly removed by antibiotics (Figure 4C). The results of LC–MS/MS indicated that the levels of acetic acid, propionic acid and total short-chain fatty acids (SCFAs) in the feces of IBS rats were decreased (Figure 4D). Wuji Pill could significantly reverse the level of acetic acid (Figure 4D). After the administration of antibiotics, the levels of SCFAs in feces decreased significantly (Figure 4D).

In the subsequent experiment, intragastric administration was used for microbiota transplantation and the improvement of gut microbiota on IBS was evaluated. Microbiota transplantation includes fecal microbiota transplantation (the gut microbiota following the intervention of Wuji Pill) and A. muciniphila (the probiotics significantly increased after Wuji Pill intervention) (Figure 5A). Compared with the model group, the visceral sensitivity of IBS rats was significantly reduced after the intervention of FMT and A. muciniphila (Figure 5B). In addition, abnormal colonic motility was relieved in IBS rats (Figure 5C). Meanwhile, the secretion of colonic mucus was significantly restored by the fecal microbiota and A. muciniphila (Figure 5D).

After the intervention of FMT and A. muciniphila, the ratio of distance in the central area (Figure 5E) and the exploration interest (Figure 5F) were significantly increased of IBS rats. The immobility time of FST was reduced (Figure 5G). It is further verified that FMT and A. muciniphila intervention can improve the depression-like behavior of IBS rats. In addition, the microglia in cortex were significantly inhibited after the intervention of FMT and A. muciniphila (Figure 5H).

The changes of SCFAs in feces after intervention of microbiota were verified. The levels of acetic acid, n-butyric acid and isobutyric acid were significantly increased in FMT group (Figure 6A). A. muciniphila intervention significantly increased the levels of acetic acid and isobutyric acid (Figure 6A). These findings demonstrate that microbiota intervention directly influences the levels of SCFAs.

The levels of tryptophan pathway metabolites in the hippocampus and colon of IBS rats after the intervention of gut microbiota were summarized in Table 4. In the hippocampus of IBS rats, the level of 5-hydroxytryptamine (5-HT) was significantly decreased and the levels of 3-hydroxykynurenine (3-HK) and quinolinic acid (QA) were significantly increased. After the intervention of FMT, 5-HT was significantly increased, and the levels of 3-HK were significantly reversed. 5-hydroxyindoleacetic acid (5-HIAA) was significantly decreased after A. muciniphila intervention. In the colon, 5-HT levels were increased in the model group. After FMT, the levels of tryptophan and 5-HIAA were significantly increased. 5-HT was significantly decreased after A. muciniphila intervention.

Tryptophan metabolizing enzymes are the key points for regulating the tryptophan metabolic pathway. Further, we detected the levels of tryptophan metabolizing enzymes in the hippocampus and colon of IBS rats after the intervention of FMT and A. muciniphila (Figure 6B; Supplementary Figure S4). In the hippocampus of IBS rats (Figure 6B), the mRNA expression levels of MAOA, MAOB, and QPRT were significantly elevated. Conversely, in the FMT group, the mRNA expression levels of MAOB and QPRT were notably reduced. In the AKK group, the mRNA expression levels of MAOA, MAOB, and QPRT exhibited a reversal trend. In the colon (Supplementary Figure S4), the mRNA expression levels of THP1, MAOB and IDO1 increased in the model group. In the FMT group, the mRNA expression levels of THP1 and IDO1 decreased. Moreover, the level of IDO1 mRNA was decreased after A. muciniphila intervention.