Thirty-six 6-week-old specific pathogen-free (SPF) female BALB/c mice were purchased from Vital River Laboratory Animal Technology Co., Ltd (Being, China). The mice were acclimatized for one week under SPF conditions at the China Agricultural University’s facility, with a 12 h light-dark cycle, at 25 ± 2°C and 50% ± 5% humidity. The experimental design was approved by the China Agricultural University’s Ethical Committee (Approval No. Aw61103202-5-1), in accordance with the EU Directive 2010/63/EU.

BL-99 was sourced from the China General Microbiological Culture Collection Center (CGMCC 15650).

Mice were randomly divided into six groups (n = 6/group): control (Ctrl), constipation model (LOP), positive control (POS), BL-99, FOS, and a synbiotic combination of BL-99 and FOS (BL-99+FOS). Except for the Ctrl group, all groups received intragastric administration of loperamide hydrochloride (10 mg/kg) from days 8 to 17 following a 7-day acclimatization period to induce constipation. Upon successful modeling, treatments were administered from days 18 to 31. The control and model groups received saline, the FOS group received FOS solution (700 mg/kg), the BL-99 group received BL-99 bacterial suspension (5×10⁹ CFU/day), and the synbiotic group received a mixture of BL-99 and FOS (700 mg/kg FOS + 5×10⁹ CFU/day BL-99). The positive drug control group received polyethylene glycol 4000 solution (0.17 g/kg).

With reference to the research of Lim YJ et al., the fecal parameters were carried out, which is briefly described as follows (21). After fasting for 16 h, mice were gavage with 0.2 mL activated carbon powder solution to record the first black stool discharge time, and the number of mice defecation granules within 2 h. Fresh feces were dried at 105 °C for 16 h, and the fecal moisture content is calculated according to the following formula: Fecal moisture content = (W_wet - W_dry)/W_wet × 100%.

After fasting for 16 h, 0.1 mL carmine solution was administered orally. Mice were euthanized 30 min later, and the entire intestine was dissected. The lengths of the intestine (S₁) and the distance traveled by the carmine marker (S₂) were measured to calculate the propulsion rate with the formula D = S₂/S₁×100%.

Tissues were dehydrated, embedded, and sectioned for hematoxylin and eosin (H&E) staining to assess colon histological changes.

Serum levels of neurotransmitters and cytokines, including serotonin (5-HT), acetylcholine (ACh), substance P (SP), vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), Interleukin-10 (IL-10), Interleukin-4 (IL-4), Interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were quantified using ELISA (Shanghai Enzyme-linked Biotechnology Co., Ltd., China), following the manufacturer’s instructions.

Data were expressed as mean (M) ± standard deviation (SD). Differences between groups were analyzed using one-way ANOVA followed by LSD post-hoc test for multiple comparisons. A P-value < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism version 9.5.0.

To evaluate the efficacy of prebiotics, probiotics, and synbiotics, mice were pretreated with loperamide for two weeks, followed by administration of FOS, BL-99, and BL-99+FOS for another two weeks. Loperamide treatment significantly induced constipation, evidenced by prolonged first black stool defecation time, reduced fecal number, lower fecal weight, decreased fecal moisture content, and decreased gastrointestinal transit rate compared to the Ctrl group (P < 0.05, Figure 1 ). Compared with the LOP group, the FOS, BL-99, FOS+BL-99, and POS groups all alleviated defecation-related parameters. Compared to the FOS and BL-99 groups, the FOS+BL-99 group significantly decreased the first black stool defecation time, and significantly increased the fecal number, fecal weight, and the gastrointestinal transit rate (P < 0.05). Although the BL-99+FOS group did not show significant differences in some defection indexes such as fecal number, fecal weight, and fecal moisture content, there was still a trend of improvement compared with the FOS and BL-99 groups.

Histological staining was employed to assess alterations in colonic histomorphology ( Figure 2 ). An intact colonic structure was observed in the normal control group, while the constipation model exhibited reduced goblet cells and inflammatory infiltrations. Compared to the constipation model group, colonic sections from the FOS group, BL-99 group, and FOS+BL-99 group showed a restoration of villus structure, resembling morphology observed in the Ctrl group.

Through the measurement of the excitatory neurotransmitters 5-HT, ACh, and SP, and the inhibitory neurotransmitters VIP and NOS, we explored whether treatment with FOS or BL-99 alone or combined can ameliorate intestinal function by modulating the levels of intestinal neurotransmitters ( Figure 3 ). In terms of excitatory neurotransmitters, the serum levels of 5-HT, SP, and ACh were significantly lower in the LOP group mice compared to the Ctrl group (P < 0.05). Treatment with FOS or BL-99 alone significantly increased the levels of ACh (P < 0.01) and showed a trend towards modulating 5-HT and SP levels, although these changes were not statistically significant compared to the LOP group. Notably, the combined use of BL-99 and FOS significantly increased the levels of 5-HT and ACh (P < 0.05) compared to the LOP group, more effectively restoring the balance of intestinal neurotransmitters and potentially promoting the recovery of intestinal function. In terms of inhibitory neurotransmitters, the serum levels of VIP and NO were significantly elevated in the LOP group mice (P < 0.05). Treatment with BL-99, FOS, and their combination all effectively reduced the levels of VIP and NO (P < 0.05), with the combined treatment showing the most significant reduction (P < 0.01). These findings indicate that both individual and combined treatments of FOS and BL-99 can modulate intestinal neurotransmitter levels, with the combined treatment showing the most pronounced effects in restoring excitatory neurotransmitter levels and reducing inhibitory neurotransmitter levels, thereby potentially ameliorating intestinal function.

The serum levels of the anti-inflammatory cytokines IL-4 and IL-10, as well as the pro-inflammatory cytokines TNF-α and IL-6, were assessed to quantify the effects of prebiotics, probiotics, and synbiotics on inflammatory factors in constipated mice ( Figure 4 ). In constipated mice induced by loperamide hydrochloride, there was a statistically significant decrease in the levels of the anti-inflammatory cytokines IL-4 and IL-10 in the constipated mice compared to the control group. This indicates an imbalance in inflammatory cytokines, with a dominance of pro-inflammatory cytokines in the serum of constipated mice. The administration of FOS or BL-99 independently, or in combination, significantly increased the serum levels of the anti-inflammatory cytokines IL-4 and IL-10 and reduced the pro-inflammatory cytokine TNF-α and IL-6 (P < 0.05). The combination of BL-99 and FOS showed a synergistic effect, with a significantly greater increase in IL-10 levels compared to BL-99 or FOS used alone. This suggests that the combination of BL-99 and FOS can effectively improve inflammation of constipated mice.

The cecal microbial diversity in mice was significantly enhanced by the combined treatment with BL-99 and FOS compared to the LOP group. This increase in microbial diversity and richness was further corroborated by notable improvements in alpha diversity indices like Sobs, Shannon, and Chao1 ( Figures 5A–D ). beta diversity was effectively depicted through Principal Coordinates Analysis (PCoA) using Bray-Curtis distances ( Figure 5E ). This analysis distinctly separated the microbial compositions between the Ctrl and LOP groups, particularly in mice treated with BL-99 and FOS. The Adonis test (P = 0.001) confirmed significant differences in community composition, highlighting the substantial impact of the treatments on the gut microbiome’s structure.

Further analysis of the predominant bacterial phyla in the intestinal microbiota across various intervention groups revealed the primary phyla as Bacteroidetes, Firmicutes, Desulfobacterota, Deferribacterota, and Patescibacteria ( Figure 5G ). Results showed an increase in Bacteroidetes and a decrease in Firmicutes within the LOP group. Additionally, the Bacteroidetes-to-Firmicutes ratio was significantly higher in the LOP group compared to the Ctrl group ( Figure 5F ). Post-intervention assessments revealed a significant reduction in this ratio following treatment with BL-99, FOS, and BL-99+FOS, indicating a potential restoration of microbial balance. At the genus level, the top ten in terms of abundance included Alistipes, Lachnospiraceae NK4A136 group, Bacteroides, Oscillibacter, Prevotellaceae UCG-001, Rikenellaceae RC9 gut group, Colidextribacter, Alloprevotella, Lactobacillus, and Intestinimonas ( Figure 5H ). Using the LEfSe method with an LDA score threshold greater than 3, significant microbial variations within the cecum contents were observed, with each group showing distinct microbial compositions ( Figure 5I ). In the LOP group, Prevotellaceae UCG-001 and Parabacteroides were more abundant compared to the Ctrl group, while Odoribacter, Rikenellaceae RC9 gut group, Alistipes, Mucispirillum, and Peptococcus showed lower relative abundance. Notably, significant differences were observed for Alistipes, Prevotellaceae, Rikenellaceae, Mucispirillum, and Peptococcus (P < 0.05). In the BL-99+FOS group, the Lachnospiraceae NK4A136 group, Blautia, Candida, Peptococcus, Lachnospiraceae UCG-006, Lachnoclostridium, Turicibacter, and Clostridium sensu stricto 1 were significantly enriched (P < 0.05), whereas Alistipes, Bacteroides, Eubacterium ventriosum group, and Akkermansia were significantly less abundant compared to the LOP group (P < 0.05). Moreover, compared to individual use of FOS and BL-99, their combination significantly enriched Blautia and Clostridium sensu stricto 1 (P < 0.05, Figure 5J ). The influence of synbiotic treatment on the cecal microbiota was further evidenced by alterations in community structure, impacting their metabolic pathways. To elucidate these effects, PICRUSt2 software was utilized for functional prediction analysis based on 16S rRNA gene sequences ( Figure 5K ). The KEGG Pathway enrichment bar charts indicated that primary metabolic pathways involved metabolism, genetic information processing, cellular processes, environmental information processing, organismal systems, and human diseases.

To explore the differences in cecal metabolites among different mouse groups, Principal Component Analysis (PCA) was initially employed to analyze the metabolic profiles of the cecal contents in mice, but PCA results not revealed good separation among groups ( Figure 6A ). From the secondary identification, all 2863 metabolites identified were indexed in the HMDB database and classified into 17 HMDB super-classes. Lipids and lipid-like molecules contained 839 metabolites, accounting for the largest category (29.30%), followed by organic acids and derivatives, which constituted 26.02% ( Figure 6B ). Our findings reveal that the combined intervention of BL-99 and FOS significantly modulated various metabolites ( Figure 6C ). The nature of these metabolic changes was significantly distinct across the different intervention approaches, with the combined treatment of BL-99 and FOS ameliorating these metabolic shifts. In the BL-99+FOS treated groups, pathways related to the metabolism of fructose and mannose, as well as the biosynthesis of phenylalanine, tyrosine, and tryptophan, tryptophan metabolism, and fatty acid biosynthesis and degradation, were significantly enhanced compared to the LOP group (P < 0.05, Figure 6D ). Conversely, pathways involved in cell apoptosis and galactose metabolism were significantly reduced (P < 0.05).

The biosynthesis of 5-HT requires its precursor, tryptophan, which is ingested through dietary proteins and subsequently metabolized by the liver before entering the bloodstream. Once in the human body, tryptophan is initially converted into 5-HT by the enzyme tryptophan hydroxylase through an oxidative process, and then rapidly transformed into 5-HT by the enzyme 5-HT decarboxylase. Studies have identified a significant negative correlation between the levels of tryptophan and its metabolic precursors, such as 5-HT, and delayed colonic transit times. As illustrated in Figure 6E–G , in a constipation model using mice, the levels of tryptophan and its metabolites in the cecal contents are significantly reduced. The intervention of BL-99+FOS notably increases the levels of N-hydroxy-L-tryptophan, indole, and indole-3-aldehyde, demonstrating a statistically significant elevation (P < 0.05).

Dietary fibers in the intestinal tract are fermented by bacteria, predominantly producing SCFAs, which play a central role in the microbiota-gut-brain axis communication. As illustrated in Figure 6H–J , the levels of the three SCFAs were significantly reduced in the constipation model group compared to the control group (P < 0.05). All intervention groups exhibited an increase in SCFAs levels, with the combined use of BL-99 and FOS proving most effective in elevating SCFAs levels in the cecal contents of constipated mice, surpassing those of the groups using either agent alone. Overall, the joint application of BL-99 and FOS effectively promoted gastrointestinal microbial fermentation producing SCFAs.

To investigate the interrelationships and variability among different bacterial genera and metabolites, we employed a heatmap to visually represent the clustering patterns of diverse metabolites and bacterial genera within the cecum. In Figure 7 , we observed distinct patterns of correlation between various bacterial genera and metabolite levels: Genera such as Bacteroides, Clostridium sensu stricto 1, Lachnospiraceae_UCG-006, Akkermansia, Lachnospira, and Candida displayed a negative correlation with glycocholic acid, taurocholic acid, malic acid, glycosides, butyrate, and aspirin levels. Conversely, they exhibited a positive correlation with acetate, propionate, butyrate, N-hydroxy-L-tryptophan, deoxycholate, taurodeoxycholate, α-muricholic acid, indole, and indole-3-carboxaldehyde levels. In contrast, genera such as Prevotella and Ruminococcus demonstrated an opposite trend in relation to the aforementioned metabolite levels.

Pearson correlation analysis was further conducted to elucidate the relationship between differentially enriched microorganisms and serum neurotransmitters or inflammation parameters. Bacteroides enriched in the LOP group showed a strong inverse correlation with cecal IL-4 levels (P < 0.05) ( Figure 8 ). Notably, oral administration of FOS+BL-99 resulted in significant alterations in the intestinal microbiota of mice with constipation, suggesting a pivotal role of the gut microbiota in mitigating loperamide-induced constipation. Lachnospiraceae_UCG-006, enriched in the FOS+BL-99 group, exhibited a significant positive correlation with serum IL-10 levels (P < 0.05) and a significant negative correlation with VIP, NOS, and IL-6 levels in the cecum (P < 0.05). These findings underscore the critical influence of FOS+BL-99 on gut microbiota modulation, highlighting its potential to alleviate constipation symptoms induced by loperamide.

The transcription levels of key genes in the 5-HT pathway within colonic tissues are crucial for maintaining intestinal health. As illustrated in Figure 9 , the transcription levels of Fxr, Vdr, TGR5, and Tph1 genes in the colonic tissues of mice in the LOP group were significantly reduced under constipated conditions compared to the Ctrl group. This reduction indicates a weakened defense against pathogens in the colon, thereby increasing the risk of colitis, a finding corroborated by the inflammation indicators shown in Figure 4 . In contrast, the transcription levels of TGR5 and Tph1 genes were significantly elevated in both the BL-99 and FOS groups compared to the LOP group. Furthermore, the BL-99+FOS group exhibited a significant increase in the transcription levels of Fxr, Vdr, TGR5, and Tph1 genes compared to the LOP, FOS, and BL-99 groups. This enhancement in gene transcription indicates a strengthened pathogen defense capability, reduced inflammation, and effective alleviation of constipation symptoms in mice.