Capsular polysaccharide TP2 was provided by Guangzhou Zhiyi Biotechnology Co., Ltd. (Guangzhou, China), with a total polysaccharide content exceeding 98%, protein content below 1%, and nucleic acid content less than 0.5%. Following preparation, it is stored in a freezer at −20°C. 2, 4, 6-trinitro-Benzenesulfonicacid (TNBS) was purchased from Sigma-Aldrich (St. Louis, United States), and prednisolone was obtained from SinePharm (Shanghai, China). Antibodies for Claudin-1 and Muc-2 were purchased from Abcam Biotechnology (Cambridge, United Kingdom), Claudin-1, PI3K, AKT, and GAPDH from Proteintech (Chicago, IL, United States), p-PI3K(Tyr458) and p-AKT (Ser473) from CST (Danvers, MA, United States), ZO-1 and Claudin-2 from Affinity Biosciences (Jiangsu, China), and Cleaved-Caspase-3, Bcl-2, BAX from Biodragon (Beijing, China). TNF-α, INF-γ, IL6, IL10, IL-17A, IL22 enzyme-linked immunosorbent assay (ELISA) kits were purchased from MultiSciences (Lianke) Biotechnology (Hangzhou, China), and the IL23 kit was obtained from JianglaiBiology (Shanghai, China). RIPA Lysis Buffer and Enhanced BCA Protein Assay Kit were purchased from Beyotime (Shanghai, China). All other reagents used in the experiments were of analytical grade.

Male Wistar rats (120 g ± 10 g) were procured from Guangzhou Ruige Biological Technology Co. Ltd (Guangzhou, China) with license number SCXK (Yue) 2023–0,059. The rats were accommodated in the animal facility of Guangzhou Ruige Biological Technology Co. Ltd., at a temperature of 21°C–24°C with a 12 h light/dark cycle. The animals were housed in IVC cages, with five rats per cage, and provided with unrestricted access to water from water bottles, along with ad libitum access to food. All animals were adapted to the new surroundings for at least 5 days before use in experiments. The animal use protocol listed below has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ruige Biotechnology (Number: 20,230,701–001).

A total of 60 male Wistar rats, six-week-old, were used in this study and were randomly allocated into the following groups: normal control group (NC), model control group (MC), 9 mg/kg prednisolone group (PD), 1 mg/kg TP2 group (LTP2), 2 mg/kg TP2 group (MTP2), and 4 mg/kg TP2 group (HTP2), each comprising 10 rats. To establish a colitis rat model, TNBS powder dissolved in 30% ethanol was used at a concentration of 60 mg/mL. This concentration has been shown to effectively induce colitis in previous studies (Motavallian-Naeini et al., 2012). Rats were fasted for 40 h before the experiment, during which they received subcutaneous injections of 5% glucose saline (10 mL/kg, once a day). On the first day of the experiment, fasting rats were anesthetized with intraperitoneal injection of 1% pentobarbital sodium at a dose of 40 mg/kg. The model control group, the prednisolone group, and the TP2 administration group used a disposable rectal administration tube inserted into the left colic flexure (approximately 8 cm from the anus) to induce colitis with TNBS enema (0.5 mL/piece). The normal control group received 0.5 mL of normal saline. To prevent enema fluid reflux, the animals’ heads were lowered for 15 min after enema, and they were kept in the Trendelenburg position until they woke up. On the first day post-modeling, the TP2 administration group and the prednisolone group received their respective drugs, while the blank group and the model group were instilled with the same volume of purified water for 7 days. Daily records were maintained for animal weights, and fecal status was scored. On day 8, animals were euthanized by cervical dislocation, and the colon was swiftly removed and washed with 0.9% saline. Measurements included the ulceration area, weight, and colon length (from the ileocecal junction to the anal margin) to calculate the weight/length (W/L) ratio.

DAI score is the important indicator to assess the pathological degree of colitis. According to Ho Pan Sham et al.’ report (Sham et al., 2018), the rat DAI scores were evaluated though the following formula: DAI = (weight loss percentage score + stool score + rectal bleed score)/3 (Table 1).

To evaluate histopathological changes in the colon, both hematoxylin and eosin (HE) staining and Periodic Acid-Schiff (PAS) staining were conducted. Rat small intestinal tissue was fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into 4 μm-thick slices for HE staining. Colonic mucosal damage was observed under a light microscope, and histopathological evaluation was based on the severity of inflammation, crypt loss, and mucosal damage, as outlined in Table 2 (Rahmani et al., 2020). PAS staining was performed following the manufacturer’s instructions to assess the structure of colonic goblet cells.

The colon tissues were cut into small pieces and lysed in ice-cold RIPA lysis buffer with a protease and phosphatase inhibitor cocktail. Subsequently, The tissue was lysed by an ultrasonic cell disruption apparatus. The protein concentration was determined using a BCA kit and then heated with loading buffer. Protein samples were separated by 10% gel electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk for 1 h at room temperature, the membranes were washed with Tris-Buffered Saline with Tween 20 (TBST) three times and then incubated with primary antibodies (1:1,000) overnight at 4°C refrigerator. After incubation with secondary antibodies (1:2000) for 1 h at room temperature, the membranes were washed with TBST three times, and the proteins were detected by ECL using ChemiDoc Imaging System (Biorad). GAPDH was used as an internal reference.

Cytokine concentrations in colon tissue were quantified using ELISA. Colon tissue was homogenized in a PBS solution, centrifuged at 3,500 rpm for 20 min at 4°C, and the supernatant was collected and stored at −80°C for further analysis. Levels of cytokines (IL-1β, IL-6, IL-10, IL-17A, IL-22, IL-23, TNF-α, IFN-γ) were determined using ELISA kits following the manufacturer’s protocol.

The Rat feces were collected, and total DNA was extracted using the QIAmp DNA microbiome kit (Qiagen). The 16S rRNA gene (V3-V4 region) was sequenced using the Illumina MiSeq system. The effective tags were clustered into operational taxonomic units (OTUs) with a similarity of ≥97% using Usearch software (version 11.0.667). Chimeric sequences and singleton OTUs (with only one read) were removed, after which the remaining sequences were sorted into each sample based on the OTUs. The tag sequence with the highest abundance was selected as a representative sequence within each cluster. Bacterial and fungal OTU representative sequences were classified taxonomically by blasting against the RDP Database and UNITE fungal ITS Database, respectively. All alpha diversity indices (including Chao1,Simpson, and Shannon indices) was calculated with Mothur software (version 1.30) and beta diversity were analyzed by R vegan package (version 2.5–6). Difference comparison was used to identify features with significantly different abundances between groups using LefSe (version 1.1.0). PICRUSt (v1.1.4) was utilized to predict functional profiles based on Kyoto Encyclopedia of Genes and Genomes (KEGG).

All data are presented as mean ± SEM. Statistical data analysis and graph creation were performed using GraphPad Prism10 software (Ver. 8.2.0). For each figure, one-way ANOVA was used to evaluate the statistical significance of differences among group means. If significant differences were detected by ANOVA, Tukey’s post hoc test was applied to perform pairwise comparisons between each group’s mean and the means of all other groups. Significance levels were defined as follows: p-value < 0.05 is considered as significant, p < 0.01 as very significant, p < 0.001 as highly significant, p < 0.0001 as extremely significant and p > 0.05 as not significant (NS), respectively.

UC symptoms were assessed based on factors such as body weight, DAI, and colon-related characteristics. In comparison to the NC group, the final body weight of rats in all modeled groups significantly decreased, while the DAI scores in the MC group rats significantly increased. Rats in the LTP2, MTP2, HTP2 and PD groups exhibited a significant decrease in DAI scores compared to the MC group (p < 0.001) (Figures 1B, C). Additionally, we evaluated colon tissue ulcer area, colon length, and colon weight/colon length (CW/CL). The ulcer area in the MC group was significantly larger than in the NC group (p < 0.0001), whereas the LTP2, MTP2, HTP2 and PD groups showed a significant reduction in ulcer area compared to the MC group (p < 0.01, p < 0.01, p < 0.05 and p < 0.01, respectively) (Figures 1D, F). Colon length in the MC group was significantly shorter than in the NC group (Fig. F), while the LTP2 and HTP2 groups exhibited significantly longer colon lengths than the MC group. The CW/CL in the LTP2, MTP2, HTP2 and PD groups was significantly lower than in the MC group (p < 0.0001, p < 0.0001, p < 0.01 and p < 0.001, respectively) (Figure 1E). Thus, TP2 alleviates the symptoms of TNBS-induced colitis, indicating its potential as a therapeutic agent for UC.

We histologically assessed the function of TP2 inrestoring intestinal epithelial integrity by HE staining. In the NC group, the intestinal wall exhibited normal tissue architecture with intact mucosal structure, clear crypt architecture, and evenly distributed glands. The MC group showed the disappearance of normal colonic wall structure, accompanied by partial cell necrosis. Crypt cells, goblet cells, and the majority of glands were largely absent, with a noticeable infiltration of inflammatory cells. In comparison to the MC group, the LTP2, MTP2 and PD groups displayed a mostly intact colonic structure with fewer inflammatory lesions. There was a slight loss of crypt cells and goblet cells (Figure 2A). These results were corroborated by histological score analysis. The MC group had significantly higher scores than the NC group, indicating severe colonic damage induced by TNBS-induced ulcerative colitis. Both the LTP2, MTP2 HTP2 and PD groups had significantly lower scores than the MC group (p < 0.01, p < 0.001, p < 0.05 and p < 0.01, respectively), indicating TP2 significantly reduced histopathological damage (Figure 2B).

The intestinal mucosal barrier serves as the host’s first line of defense, protecting the intestinal epithelium from invasion (Pelaseyed et al., 2014). Structural weakening of the colonic mucosal barrier promotes the development of ulcerative colitis (van der Post et al., 2019). We utilized PAS staining and Western blot analysis to evaluate the protective effects of TP2 on the intestinal mucosal barrier. The results indicate severe damage to goblet cells in the MC group, with a significant decrease in Muc-2 expression. Treatment with TP2 and PD significantly alleviated the TNBS-induced reduction in goblet cells and Muc-2 secretion (Figures 3A, B). Considering that the MTP2 group demonstrated optimal protective efficacy in clinical symptoms, HE staining, and PAS staining in colitis rats, we selected the MTP2 group for further analysis in subsequent mechanistic investigations (In the following discussions, TP2 will be used to represent this group).

Furthermore, intestinal tight junction proteins are crucial components of the intestinal barrier, preventing the leakage of harmful substances from the intestinal lumen (Buckley and Turner, 2018; Chelakkot et al., 2018). To investigate the protective effect of TP2 on TNBS-induced tight junction disruption, we measured the expression of ZO-1, Occludin, Claudin-1, and Claudin-2. The results showed a significant decrease in the expression of ZO-1, Occludin, Claudin-1, and Claudin-2 in the MC group compared to the NC group. TP2 and PD treatment, however, upregulated the expression levels of these proteins (Figures 3C–H). These findings suggest that TP2 restores the damaged intestinal barrier function by protecting goblet cells, upregulating Muc-2 expression, and enhancing the expression of tight junction proteins.

To assess apoptosis in colonic tissues, we investigated several proteins associated with apoptosis. Firstly, the proto-oncoproteins Bcl-2 and Bax were detected. As shown in Figure 4A, the expression of the anti-apoptotic protein Bcl-2 was downregulated, while the pro-apoptotic protein Bax was upregulated, resulting in a marked reduction in the Bcl-2/Bax ratio in the colonic tissues of TNBS-induced UC rats (p < 0.05). Additionally, Cleaved-Caspase-3 was selected to evaluate the effect of TP2 on TNBS-induced apoptosis. Under the challenge of TNBS, the expression of Cleaved-Caspase-3 significantly increased (p < 0.0001). By contrast, TP2 and PD treatment remarkably abrogated the downregulation of the Bcl-2/Bax ratio (p < 0.01, p < 0.05) (Figure 4B) and reduced Cleaved-Caspase-3 expression (p < 0.001, p < 0.001) (Figure 4C). In summary, TP2 suppressed apoptosis in colon tissues in TNBS-induced UC rats.

To gain a deeper understanding of the mechanism by which TP2 alleviates colitis, we utilized ELISA technology to assess typical pro-inflammatory and anti-inflammatory cytokines in rats’ colonic tissues, including TNF-α, IFN-γ, IL-1β, IL-6, IL-10, IL-17A, IL-22, and IL-23. As depicted in Figure 5, the MC group showed significantly increased levels of TNF-α (p < 0.05), IFN-γ (p < 0.05), IL-1β (p < 0.01), IL-6 (p < 0.01), and IL-23 (p < 0.01), indicating serious inflammation in UC rats. In comparison to the MC group, TP2 significantly enhanced the expression of IL-10 (p < 0.0001)and IL-22 (p < 0.05) in the colonic tissues of colitis-induced rats. Simultaneously, it reduced the levels of TNF-α (p < 0.055), IL-1β (p < 0.05), IL-6 (p < 0.05) and IL-23 (p < 0.01), while TP2 did not exhibit a significant impact on IFN-γ and IL-17 A. These research findings suggest that TP2 can promote the expression of anti-inflammatory cytokines and inhibit the secretion of pro-inflammatory cytokines, thereby mitigating colitis.

Immunological dysregulation stands as a pivotal pathogenic factor in UC, manifested through an aberrant balance in the expression of pro-inflammatory and anti-inflammatory cytokines. The classical PI3K/AKT signaling pathway plays a crucial role in modulating diverse cellular processes, encompassing proliferation, differentiation, apoptosis, and inflammation-related responses. Numerous investigations underscore the interconnectedness of cytokine expression and closely associated proteins with the PI3K/AKT signaling pathway in the progression of UC (Huang et al., 2011; Chen et al., 2017; Li et al., 2022a). Consequently, we conducted Western blot analysis to scrutinize the influence of TP2 on the PI3K/AKT signaling pathway. The data revealed no discernible disparities in the total expression levels of PI3K and AKT across the experimental groups. Nevertheless, a noteworthy elevation in the expression levels of p-PI3K and p-AKT was observed in the MC group, signifying the induction of PI3K/AKT signaling pathway activation by TNBS (Figure 6A). Remarkably, treatment with TP2 and prednisolone effectively reversed this observed trend (Figures 6B, C). This aligns with the established regulatory effects of TP2 on inflammation, tight junction proteins, and cellular apoptosis. In summary, our research findings substantiate the proposition that TP2 mitigates TNBS-induced colonic injury by impeding the activation of the PI3K/AKT pathway.

To investigate the changes in the structure and function of rat intestinal flora after drug administration, we performed 16S rRNA gene amplicon sequencing. Alpha diversity analysis at the OTU level showed that multiple indices (ACE, Chao, Shannon, Simpson) differed significantly in different groups (Figures 7A–E), the results of Principal Co-ordinates Analysis (PCoA) at the OTU level showed the same trend (Figure 7G), in which the MC group showed a significant separation compared to the NC group, and the TP2 group showed clustering with the PD and NC groups. The mean relative abundance at the genus level showed that TNBS reduced the abundance of major genera such as Lactobacillus, Limosilactobacillus, Ligilactobacillus and Akkermansia compared to the NC group, with the opposite trend in the TP2 and PD groups relative to the MC group (Figures 7E, F). These results suggest that TP2 treatment restored TNBS-induced disruption of UC intestinal microbiota. In addition, Functional prediction analysis of KEGG for bacteria and archaea was performed using PICRUSt (Figure 7G), Functional abundance heatmap showed that the abundance of probable phosphoglycerate mutase [EC:5.4.2.12], protein-tyrosine phosphatase [EC:3.1.3.48], probable phosphoglycerate mutase [EC:5.4.2.12] and L-lactate dehydrogenase [EC:1.1.1.27] was different in MC group and NC group, however, the abundance was similar in TP2 group and PD group and NC group. This may predict an important role for these KEGG ontologys (KOs) in the pathogenesis of inflammatory bowel disease.