A pectic polysaccharide from Lycium ruthenicum Murray alleviates dextran sulfate sodium-induced colitis in mice

Abstract

Inflammatory bowel disorders (IBD) can lead to severe complications like perforation, bleeding, and colon cancer, posing life-threatening risks. Lycium ruthenicum Murray (L. ruthenicum Murr.), rich in polysaccharides, has been utilized in traditional diets for thousands of years. This study explores the protective effects of the polysaccharide of L. ruthenicum on mice with dextran sulfate sodium (DSS)-induced colitis. In the present study, a pectic polysaccharide (LRWP-Ap) containing arabinogalactan (AG) and homogalacturonic acid (HG) structural domains with a Mw of 4.34 kDa was obtained from L. ruthenicum Murr. Fruit. The gavage administration of LRWP-Ap significantly alleviated symptoms of DSS-induced colitis in mice. In this process, LRWP-Ap modulated the balance of Arg-1/iNOS to regulate the metabolism of arginine, and the levels of intestinal tight junction (TJ) (ZO-1, Occludin, and Claudin 1) were increased by LRWP-Ap treatment, which promoted intestinal barrier function. In addition, LRWP-Ap alleviated the inflammatory response while increasing the anti-inflammatory response by reducing the level of proinflammatory factors, enhancing the level of anti-inflammatory factors (IL-10) and improving the balance of Treg/Th17 cells. These effects resulted in the maintenance of intestinal immune homeostasis. Moreover, LRWP-Ap modulated the gut microbiota composition and short-chain fatty acid (SCFA) content, which may maintain relatively favorable intestinal homeostasis. In general, LRWP-Ap has the potential to alleviate IBD, and the use of L. ruthenicum Murr. As a natural functional food to improve gut health in the context of DSS-induced colitis.

Graphical abstract

Highlights

• •A pectic polysaccharide from Lycium ruthenicum Murray shows anti-IBD activity in mice.
• •Lycium ruthenicum Murray pectic polysaccharide fortifies gut barrier in colitis mice.
• •Lycium ruthenicum Murray pectic polysaccharide balances immunity in colitis mice.
• •Lycium ruthenicum Murray pectic polysaccharide alters gut microbiota in colitis mice.
• •Lycium ruthenicum Murray pectic polysaccharide enhances gut health as a functional ingredient.

1. Introduction

Inflammatory bowel disease (IBD), characterized by persistent and recurring inflammation of the digestive system, encompasses conditions such as Crohn's disorder (CD) and colitis ulcerosa (UC)(Tavakoli et al., 2021). The excessive inflammatory response leads to damage in the intestinal tract, consequently raising the risk of colorectal cancer. Despite extensive research, the precise causes of IBD remains unclear; nevertheless, there is a prevalent notion that it arise from a intricate interplay among genetic tendencies, environmental influences, disruptions in the immune system, alongside shifts in the intestinal flora composition(Ananthakrishnan, 2015).

Intestinal damage in IBD is mediated by multiple interconnected mechanisms, including impaired barrier function, dysregulated immune homeostasis, and an imbalanced gut microbiota(Pizarro et al., 2019; Y. Z. Zhang and Li, 2014). Tight junction (TJ) serve as adhesion complexes between intestinal epithelial cells (IECs), creating a protective barrier that separates the external milieu from the organism, preventing detrimental elements within the intestinal lumen to prevent their permeation through the epithelial layer, thus suppressing intestinal inflammation(Capaldo et al., 2017). Once the TJ proteins levels in the colon is reduced, the protective barrier between the intestinal mucosa and the gut is disrupted. This phenomenon allows bacteria or bacterial metabolites to enter the intestinal mucosal layer, where they can then translocate to the bloodstream, stimulating the release of substantial quantities of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α by the organism's defense system(Kaser et al., 2010; Maloy and Powrie, 2011). Excessive release of proinflammatory cytokines triggers an inflammatory cascade that may further exacerbate harm to the intestinal physical barrier, promoting intestinal inflammation and the development of intestinal diseases(Chelakkot et al., 2018). Abnormal activation of specific T-cell subsets, such as Th17 cells, along with dysfunctional Tregs also contributes to IBD progression(A. Luo et al., 2017). Additionally, contrasted with healthy individuals, individuals with IBD exhibit a reduction in the abundance of “beneficial” bacteria, whereas opportunistic pathogens are enriched, thereby altering the levels of metabolic products and disrupting the relationship between the host and its commensal bacteria, ultimately leading to inflammatory responses and tissue damage(Frank et al., 2007; Sartor, 2008; Smith et al., 2013).

The treatment of IBD remains a major challenge. 5-Aminosalicylic acid (5-ASA)-based drugs are widely used for mild-to-moderate colitis, whereas corticosteroids offer rapid control for moderate-to-severe inflammation but are unsuitable for long-term use due to severe side effects(Kornbluth and Sachar, 1997; Lichtenstein et al., 2018). For patients with severe complications (intestinal obstruction, perforation, and malignant tumors) that do not respond to medications, surgical treatment can be used(Frolkis et al., 2013), but even surgery does not provide a complete cure, leaving patients prone to recurrent episodes. In recent years, research on biological agents and fecal microbiota transplantation (FMT) as a therapeutic approach has made some progress(Costello et al., 2019; Moayyedi et al., 2015; Paramsothy et al., 2017; Sands et al., 2019; Sands et al., 2019). However, these treatments are costly, have limited applicability, and are associated with adverse effects. Consequently,it is imperative to discover secure and more effective therapies to improve IBD management.

Polysaccharides are macromolecular compounds widely encountered in nature and exhibit a diverse range of pharmacological activities(Tao et al., 2017; Yin et al., 2019). Lycium ruthenicum (L. ruthenicum) Murr., commonly known as black wolfberry, is a perennial thorny shrub belonging to the Solanaceae family. It has been utilized as a functional and nutritional food in China for thousands of years. Its fruit is rich in anthocyanins, polysaccharides, and other bioactive compounds, making it a valuable food and drink for reducing inflammation by inhibiting inflammation(Q. Peng et al., 2014). The polyphenols extracted from L. ruthenicum Murray exhibit a range of biological activities, including antioxidant, anti-inflammatory, antitumor, and neuroprotective effects(Gao et al., 2022; Lee and Choi, 2023; Pang et al., 2023; M. L. Xu et al., 2024). Additionally, polysaccharides from L. ruthenicum fruits modulate the immune response, as they boost the restoration of the immune organs indices and increase proliferation of both T and B lymphocytes(Gong et al., 2015), providing a theoretical basis for the use of L. ruthenicum Murr. polysaccharides to ameliorate IBD. Hence, in this study, we isolated the pectic polysaccharide LRWP-Ap, explored its protective effect on murine models with dextran sulfate sodium (DSS)-induced colitis and investigated the possible mechanisms involved in the treatment of IBD with LRWP-Ap.

2. Materials and methods

2.1. Preparation and characterization of LRWP-Ap

L. ruthenicum Murr. Fruits were obtained from Dongshangen, Dulan County (N 36.321, E98.111; altitude: 3100 m), Haixi national municipality of Mongol and Tibeta, Qinghai, China, as described in our previous study(Ni et al., 2013). LRWP dissolved in water (5% w/v) was introduced into a DEAE-Cellulose (Hengxin, Shanghai, China) column and then eluted with H₂O and 0.5 M NaCl in turn. The eluents were gathered, concentrated to a reduced volume, dialyzed against tap water and distilled water successively. The solutions were freeze-dried, yielding two distinct polysaccharide extracts: LRWP-N and LRWP-A. Further purification of LRWP-A was achieved by using a DEAE-Sepharose Fast Flow column (Sigma, MO, USA) with a linear gradient elution of 0–0.5 M NaCl to obtain a purified polysaccharide fraction named LRWP-Ap.

The protein contents, uronic acid, alongside total carbohydrate were ascertained via standard colorimetric methods with galacturonic acid and bovine serum albumin as standards. The monosaccharide composition was conducted via the PMP precolumn derivation technique, followed by HPLC employing a Kromasil 100-5C18 column (250 × 4.6 mm i.d., Nouryon B.V., Bohus, Sweden). The absorbance was documented via a UV–Vis spectrophotometer (Model SP-752, China). For ¹³C NMR analysis, 50 mg of LRWP-Ap was dissolved in deuteroxide and analyzed by a Bruker Avance 600 MHz spectrometer (Bruker Corporation, MA, US). The homogeneity and molecular weight of LRWP-Ap were determined via HPGPC on a Shimadzu LC-10AT HPLC system with a TSK-Gel G3000PWXL column (TOSOH, Tokyo, Japan) and an RID-10 A detector.

2.2. Laboratory animals

C57BL/6 J male mice (20 ± 2 g) were sourced from Changchun Yis Laboratory Animal Technology LLC, animal license No. SYXK (JI) 2019-0015. All the mice were kept in controlled environmental conditions (22–24 °C, 60–65% relative humidity, and a 12/12-h dark/light cycle) in a well-lit room, where they had unrestricted access to nourishment and water. All procedures related to animals were approved by the Ethics Committee of Jilin University (Ethics No. 2022401).

2.3. Experimental design for LRWP-Ap treatment

The subjects used in the experiment were allotted at random into 5 groups (n = 6): Control, Model (DSS), and LRWP-Ap + DSS at 10, 25, and 50 mg/kg. An additional group in which LRWP-Ap (50 mg/kg) was administered alone were also evaluated during the experimental process to assess liver and kidney toxicity under the designed experimental conditions.

During the modeling period (days 1–7), the mice in the model group and the LRWP-Ap (10, 25, and 50 mg/kg) + DSS groups were provided with unrestricted access to sterile drinking water containing 3% DSS (MP Biomedicals, CA, USA) to induce colitis, while those in the control group were given purged aqua instead. The LRWP-Ap + DSS treatment groups were gavaged daily with 10, 25, and 50 mg/kg of LRWP-Ap solution, whereas the control and model groups received an equal volume of saline for 7 consecutive days. On the 8th day, all the mice granted unrestricted access to pure water. Daily observations were made noting the subjects' body weight, fecal consistency, and a kit (Solarbio, Beijing, China) was employed for the detection of occult blood in the feces to monitor the disease activity index (DAI) of each experimental animal(Cho et al., 2011). On day 9, the serum separated from orbital blood was collected, left to clot at 4 °C and centrifuged. The mice were euthanized, followed by the excision of the colon for length determination. The proximal colonic segments and its contents, in addition to other tissues, were gathered and preserved at −80 °C until subsequent analysis.

2.4. Cytokine analysis

The supernatant from the proximal colonic segment homogenates and serum were prepared as described above and assessed via the Luminex Bio-Plex system (Milliplex Analyst, version 5.1) via Luminex multicytokine microarray assay and were further determined via an ELISA kit (Elabscience, Hubei, China).

2.5. Western blotting

Total protein was isolated from proximal colonic segments using RIPA lysis buffer (Beyotime, Shanghai, China) with protease (Beyotime, Shanghai, China) and phosphatase inhibitors (Beyotime, Shanghai, China), and quantified with a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were loaded onto a 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, MA, USA). The membrane was blocked with a Rapid Closure Solution (GeneFist, Shanghai, China) for 30 min, then incubated overnight at 4°Cwith primary antibodies against ZO-1, Occludin, Claudin-1, and β-actin (Proteintech, Hubei, China). After washing, the membranes were incubated with secondary sheep anti-rabbit IgG-HRP antibodies (Proteintech, Hubei, China) at room temperature for 2 h.

2.6. H&E staining

Distal colonic segments, liver, and kidney tissues were preserved in 4% paraformaldehyde for over 24 h, embedded in paraffin, and sectioned at 4 μm. These sections were stained with hematoxylin and eosin (Servicebio, Hubei, China) and observed under a light microscope. Liver and kidney damage, colonic inflammation, and pathological damage were assessed. Histological scoring was based on outlined procedures(Y. Peng et al., 2019).

2.7. Immunohistochemical (IHC) analysis

Colon sections were de-waxed and restored with a citric acid-based antigen retrieval reagent (Servicebio, Hubei, China). After rinsing with PBS, they were blocked with 3% hydrogen peroxide (ANNJET, Shandong, China) and incubated with 3% BSA (Servicebio, Hubei, China) for 30 min at room temperature. Primary antibodies against IL17A (1:500) and FoxP3 (1:100) (Servicebio, Hubei, China) were applied, and the sections were incubated overnight at 4 °C. Following PBS washing, HRP-labeled secondary antibodies (Servicebio, Hubei, China) were added for a 50-min incubation at room temperature. The slides were stained with diaminobenzidine (DAB) and counterstained with hematoxylin to visualize nuclei, then blocked and imaged under a light microscope.

2.8. Immunofluorescence

The colon paraffin sections were treated as described above (method 2.7). Then, the sections were incubated overnight at 4 °C, utilizing primary antibodies (Servicebio, Hubei, China) against iNOS (1:5000) and Arg-1(1:8000). After washing, HRP-labeled secondary antibodies (Servicebio, Hubei, China) were co-cultured with samples at room temperature for 50 min. DAPI was employed for nuclear staining.

2.9. 16 S rRNA assessment

Intestinal genomic DNA was isolated according to standard protocols (TIANGEN, Beijing, China), and the genomic DNA served as the template for PCR amplification targeting the V3-V4 hypervariable segment of the bacterial 16 S rRNA gene. A compact DNA library was generated for paired-end sequencing utilizing the Illumina NovaSeq platform. Initial sequence data were processed and purified using Trimmomatic v0.33. The primer sequences were thendetected and excised with Cutadapt version 1.9.1. Denoised reads were obtained via QIIME2 2.2020 software. With QIIME2 2020.6 software, chimeric reads were removed via the merging method, succeeded by species identification and abundance assessment. Biodiversity assessment, significant species enrichment assessment and correlation analysis were performed using BMKCloud (www.biocloud.net) with the aim of discerning the variations in the gut microbiota composition across distinct sample groups.

2.10. Short chain fatty acid (SCFA) analysis

Add an appropriate amount of sample to a 1.5 mL centrifuge tube, then mix with 500 μL of water and 100 mg of glass beads. Homogenize for 1 min and centrifuge at 12,000 rpm for 10 min at 4 °C. Collect 200 μL of the supernatant, add 100 μL of 15% phosphoric acid, 20 μL of a 375 μg/mL internal standard (4-methylvaleric acid) solution, and 280 μg/mL of ether. Homogenize for 1 min, centrifuge at 12,000 rpm for 10 min at 4 °C, and collect the supernatant for quantifying SCFA concentration in feces by gas chromatography.

For standards, mix acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in water to create a 100 mg/mL stock solution, then dilute with water to prepare a series of working standards for the six acids.

2.11. Statistical analysis

One-way ANOVA was employed to compare differences between groups. The statistical computations were conducted using GraphPad Prism version 10.0.2, with P < 0.05 and P < 0.01 as indicative of significant statistical differences. All the data are represent the means ± standard deviations, and all the experiments were conducted at least three times.

3. Results

3.1. Preparation and characterization of LRWP-Ap

The yiled of LRWP-Ap is 0.37% relative to air-dried L. ruthenicum berry, and its total carbohydrate, uronic acid and protein contents were respectively 97.32%, 77.58%, and 1.09%. As shown in Fig. 1C, LRWP-Ap is a homogeneous polysaccharide with an approximate mean molecular mass of 4.34 kDa and a polydispersity index (PDI) of 1.69 (Fig. 1C). Monosaccharide composition analysis revealed that LRWP-Ap was mainly consists of galacturonic acid, arabinose and galactose, accounting for molar percentages of 75.81%, 15.41%, and 8.78%, respectively. 13C-NMR spectrum (Fig. 2, Table 1) suggested that LRWP-Ap is mainly composed of α-(1 → 5)-L-Araf, β-(1 → 3)-D-Galp, β-(1 → 6)-D-Galp, β-(1 → 3,6)-D-Galp and α-(1 → 4)-D-GalpA residues. This indicates that LRWP-Ap is a pectic polysaccharide in which galactose and arabinose might be composed of arabinogalactan (AG), which are related to their homogalacturonic acid (HG) domains in noncovalent form or as side branches of HG.

Fig. 1.

Elution profiles of L. ruthenicum Murr. polysaccharides. (A) Elution profile of LRWP on a DEAE-Cellulose column; (B) Elution profile of LRWP-Ap on a DEAE-Sepharose Fast Flow column; (C) Elution profile of LRWP-Ap on a TSK-Gel G3000PW_XL column.

Fig. 2.

¹³C NMR spectrum of LRWP-Ap.

Table 1.

¹³C-NMR chemical shifts of LRWP-Ap.

Fraction	Glycosidic linkage	Chemical shift (δ, ppm)
		C-1	C-2	C-3	C-4	C-5	C-6/COOH
LRWP-Ap	→5)-α-L-Araf-(1→	111.67	84.52	79.11	86.46	68.91
	α-L-Araf-(1→	109.93	83.57	78.84	86.75	63.21
	→3)-β-D-Galp-(1→	106.33	72.42	82.71	71.42	77.54	63.47
	→6)-β-D-Galp-(1→	105.55	73.09	75.43	72.52	76.70	71.57
	→3,6)-β-D-Galp-(1→	105.09	72.35	83.27	70.66	77.34	71.97
	→4)-α-D-GalpA-(1→	100.48	68.82	70.78	79.82	71.47	177.03

3.2. LRWP-Ap alleviates DSS-induced colitis in murine models

In order to explore whether LRWP-Ap could attenuate DSS-induced colitis, body weight changes, colon length, DAI score, and histological damage in different group of mice were explored. Findings indicated a significant decrease in body weight, significant shortening of the colon, and a sustained increase in the DAI score in the DSS group comparing to those in the control group (Fig. 3B–E). By contrast, low-dose LRWP-Ap (10 mg/kg) only slightly alleviated these symptoms comparing to those in the DSS group, and LRWP-Ap at higher dosages (25 and 50 mg/kg) significantly mitigated the body weight reduction and colonic contraction and suppressed the increase in the DAI score caused by DSS.

Fig. 3.

Effects of LRWP-Ap on body weight, DAI score, and organ parameters in mice. (A) Experimental design diagram: both the model group and the LRWP-Ap + DSS treated groups received drinking water containing 3% DSS, while the LRWP-Ap + DSS treated groups were also given the LRWP-Ap solution (10, 25 and 50 mg/kg) by daily gavage for 7 days. (B) Percent change in the body weight of the mice from day 1 to day 8. (C) Changes in the DAI scores of the mice from day 1 to day 8. (D) Images of the mouse colons. (E) Length of the mouse colons. (F) Mouse spleen images. (G) Mouse spleen index; spleen index = spleen weight (mg)/mouse body weight (g). Serum levels of ALT (H) in the mice and AST (I) in the mice. (J) Images of HE-stained mouse livers and kidneys. L–A: LRWP-Ap. Data are expressed as the mean ± standard deviation (n = 6). ∗∗P < 0.01 vs. the control group. #P < 0.05 and ##P < 0.01 vs. the model group.

The primary site affected by ulcerative colitis is the colorectal region; however, it may be exacerbated by various of extracolonic conditions, including those of the spleen(Zhao et al., 2023). In the DSS group, the spleen notably increased in size, and the splenic index was greater than that in the control group (Fig. 3F and G). However, LRWP-Ap administration decreased the size of the spleen and the splenic index, which alleviated these adverse changes.

In addition, liver lesions are frequently observed as systemic indicators of ulcerative colitis outside the gastrointestinal tract(Albillos et al., 2020). The levels of ALT and AST were slightly increased in the DSS group compared with those in the control group. Manwhile, the ALT and AST levels decreased upon LRWP-Ap intervention, but there was no significant difference between the LRWP-Ap group and the control group (Fig. 3H and I). Moreover, H&E staining of the liver and kidney revealed no significant lesions in any of the groups (Fig. 3J), indicating that LRWP-Ap has no significant toxicity in the testing range.

Consequently, the effects of LRWP-Ap on histopathological changes within the colon of mice suffering from colitis were investigated via H&E staining. The results indicated that the administration of DSS led to significant disorganization of the colonic mucosal structure, as evidenced by a massive decrease in the quantity of goblet cells within the mucosal layer and the disappearance of crypts, along with inflammatory cell infiltration from the mucosa to the muscularis propria (Fig. 4A). However, there was a notable enhancement in the structure of the colonic mucosa, accompanied by an increase in the quantity of goblet cells and the absence of significant inflammatory cell infiltration, and a notable decrease in histologic scores was observed in the LRWP-Ap (50 mg/kg) treatment group (Fig. 4B).

Fig. 4.

Effects of LRWP-Ap on pathological changes and TJ protein expression and on the balance of Arg-1/iNOS in mouse colon tissues. (A&B) H&E staining of mouse colon tissue. H&E staining (A) and colonic tissue damage score (B). (C–F) Western blot analysis of TJ protein and β-actin protein expression in colon tissues. Grayscale intensity statistics for Claudin 1 (D), Occludin (E), and ZO-1 (F) protein expression. (G–I) Immunofluorescence images of Arg-1, iNOS, and DAPI in paraffin-embedded colon tissue sections from each group. Relative fluorescence intensity of Arg-1 (H) and iNOS (I). L–A: LRWP-Ap. The data are expressed as the means ± standard deviations (n = 3, randomly selected from 6). ∗P < 0.05 and ∗∗P < 0.01 vs. the control group. #P < 0.05 and ##P < 0.01 vs. the model group.

3.3. LRWP-Ap enhances intestinal barrier function in mice suffering from colitis

The intestinal barrier is essential for maintaining intestinal health. To explore the impact of LRWP-Ap on the intestinal barrier in IBD mice, the expression of intestinal TJ proteins in mice was analyzed via Western blotting. The findings indicated a notable reduction in the level of ZO-1, Occludin and Claudin 1 in DSS-administration mice compared with that in control mice (Fig. 4C–F). However, LRWP-Ap treatment enhanced the ZO-1, Occludin, and Claudin 1 protein level in the mouse colon.

Immunofluorescence analysis of mouse colon tissues revealed that LRWP-Ap alleviated the increase in average iNOS fluorescence intensity induced by DSS treatment, whereas the Arg-1 fluorescence intensity was greater in LRWP-Ap-administration mice than in DSS-administration mice (Fig. 4G–I). These findings suggested that LRWP-Ap could increase the Arg-1/iNOS balance and thus attenuate iNOS-induced intestinal damage.

3.4. LRWP-Ap regulates intestinal immune homeostasis

The balance of Treg/Th17 within the intestinal lining is crucial for maintaining the homeostasis the intestinal microecosystem. To investigate the effect of LRWP-Ap on Treg/Th17 equilibrium, we determined the average optical density of FoxP3 and IL-17 A in mouse colon tissues via immunohistochemistry. The findings suggested that compared with the control group, the DSS group presented a significantly greater average optical density of IL-17 A and a lower average optical density of FoxP3 (Fig. 5A–C).

Fig. 5.

Effects of LRWP-Ap on FoxP3 and IL-17 A levels in mouse colon tissues. Immunohistochemical staining of FoxP3 and IL-17 A in colon tissues from each group. The average optical density values of FoxP3 (B) and IL-17 A (C) are shown. L-A represents LRWP-Ap. The data are expressed as the means ± standard deviations (n = 3, randomly selected from 6). ∗P < 0.05 vs. the control group.

Specifically, the factors that can partially influence IBD severity and the effects on the release of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) in colon tissues and serum were evaluated via Luminex assays and further verified via ELISA. DSS promoted the release of the proinflammatory cytokines IL-1β, TNF-α and IL-6 and inhibited the production of the anti-inflammatory cytokine IL-10 (Fig. 6A–P). In contrast, the DSS-induced upregulation of inflammatory cytokines and downregulation of the anti-inflammatory cytokine IL-10 were alleviated by LRWP-Ap.

Fig. 6.

Effects of LRWP-Ap on the levels of inflammatory factors in the serum and colonic tissues of colitis model mice, as determined by the Luminex assay and ELISAs, respectively. Multiplex assays (Luminex) for IL-1β, IL-6, TNF-α, and IL-10 in mouse serum (A–D) and in mouse colon tissues (E–H) (n = 3, randomly selected from 6). ELISAs for cytokines in the serum (I–L) and in mouse colon tissues (M–P) (n = 6). L–A: LRWP-Ap. The data are expressed as the means ± standard deviations. ∗P < 0.05 and ∗∗P < 0.01 vs. the control group. #P < 0.05 and ##P < 0.01 vs. the model group.

3.5. LRWP-Ap modulates the gut microbiota composition and SCFA levels

An imbalanced gut microbiota is a typical characteristic feature of IBD. To investigate the effect of LRWP-Ap on the intestinal microbiota of IBD mice, 16 S rRNA sequencing was employed to analyze the gut microbiota of the mice. Subsequently, a β-Diversity assessment was performed via the unweighted UniFrac algorithm to perform principal coordinate analysis (PCoA). The findings showed the distinct clustering and separation of the control and model groups, indicating that the intestinal bacterial communities in these two groups had different compositions (Fig. 7A). However, following LRWP-Ap administration, the gut microbiota's structure increasingly converged with that observed in the control group.

Fig. 7.

Effects of LRWP-Ap on the diversity of the intestinal flora in mice. (A) PCoA of the intestinal flora of the mice in each group. (B) Intestinal flora abundance at the phylum level in different samples. (C) Intestinal flora abundance at the genus level among different samples. (D) LEfSe analysis of the characteristic flora of the control, DSS and LRWP-Ap groups. C: control, M: model, L–A: LRWP-Ap (n = 5, randomly selected from 6).

We subsequently evaluated the potential compositional differences in the gut microbiota between the LRWP-Ap-treated and DSS groups. The results indicated that the mouse gut microbiota community changed significantly at the phylum level, with the total abundance of Firmicutes and Bacteroidota accounting for over 80% of the total community (Fig. 7B). Compared with the control group, the model group presented a notable decrease in the abundance of Firmicutes and a notable rise in the abundance of Bacteroidota and Proteobacteria. The reduction in the abundance of Firmicutes and the rise in the abundance of Bacteroidota, while decreasing the abundance of Proteobacteria, was reversed by treatment with LRWP-Ap. The taxonomic communities were similar between the LRWP-Ap-treated group and the control group. We also compared the community composition of the LRWP-Ap-treated and DSS groups at the genus level (Fig. 7C). The DSS group showed a significantly reduced abundance of Lactobacillus compared to the control group. In contrast, the LRWP-Ap administration failed to increase the abundance of Lactobacillus but markedly elevated the abundances of Dubosiella, Allobaculum, and Turicibacter.

The intestinal microbiota of the mice was analyzed via linear discriminant analysis effect size (LEfSe) to pinpoint distinct microbial components that differed among all the groups, and the findings indicated that the dominant phyla within the DSS group, compared to those in the control group, were Bacteroidota and Proteobacteria; the dominant taxa in the DSS group were Bacteroidales, Enterobacterales, as well as Burkholderiales (Fig. 7D). The dominant phylum in the LRWP-Ap treatment group was Firmicutes, the predominant phylum in the LRWP-Ap treatment group, compared to that in the DSS group, was Danitomycetes, with Erysipelotrichales and Clostridia-UCG-014 emerging as the dominant taxa.

The concentrations of seven SCFAs, including fatty acids, acetic acid, propanoic acid, butanoic acid, isobutyric acid, isovaleric acid, valeric acid, and pentanoic acid, within the samples of feces were evaluated via targeted metabolomics. DSS treatment significantly reduced the levels of all SCFAs except propanoic acid and isovaleric acid in the intestine (Fig. 8A). In contrast, although LRWP-Ap did not increase the levels of isobutyric or isovaleric acids, the total SCFA content, especially propanoic acid and butanoic acid, in the LRWP-Ap-treated group was significantly greater than that in the DSS group. The combined analysis of the gut microbiota and SCFA content suggested that DSS disrupted the gut microbial structure, reducing the abundance of bacteria that produce butyrate, including Erysipelotrichaceae, Allobaculum, and Turicibacter (Fig. 8B). However, LRWP-Ap effectively enhanced the abundance of bacteria that produce propionic acid and butyric acid, such as Akkermansia and Faecalibaculum, which are depleted in mice suffering from IBD (Fig. 8C).

Fig. 8.

Effects of LRWP-Ap on the structure and metabolites of the mouse intestinal flora. (A) Heatmap of differentially abundant metabolite clustering among different samples. (B) Correlation plot of the gut flora and metabolites between the control and DSS groups. (C) Correlation plot of the intestinal flora with metabolites between the DSS and LRWP-Ap groups. L–A: LRWP-Ap (n = 5, randomly selected from 6).

4. Discussion

The prevalence of IBD is on the rise, posing a significant public health concern worldwide. The exact etiology and pathogenesis of IBD remain unclear, and there is currently no cure. Existing treatments are limited in efficacy and often have significant adverse effects, prompting the exploration of natural substances that are safer and more efficient for IBD treatment. Compared with traditional treatments, polysaccharides have attracted attention because of their effectiveness in alleviating colitis and greater safety profile. This study explored whether the pectic polysaccharide fraction of LRWP-Ap from L. ruthenicum can reduce intestinal inflammation of mice with IBD, providing an important basis for the development of new IBD bioregulators.

Our results showed that LRWP-Ap may increase DSS-induced decreases in TJ protein levels. The intestinal epithelial barrier is crucial for maintaining gut homeostasis(Turner, 2009). Intestinal epithelial cells, the mucin layer, and tight junction proteins help maintain this barrier(Capaldo et al., 2017). Additionally, our results revealed that LRWP-Ap can shift the DSS-induced Arg-1/iNOS imbalance. As two important indicators of metabolism, Arg-1 affects repair during the maintenance and recovery of damaged intestinal mucosal(X. Xu et al., 2023), LRWP-Ap increased the DSS-induced reduction in Arg-1; iNOS, which leads to excessive nitric oxide production and tissue damage, can influence intestinal mucosal injury, is typically low in normal colonic tissue, and increases in response to exposure to pathogens or inflammation but is decreased by LRWP-Ap. The results described above suggest the potential of LRWP-Ap to modulate intestinal inflammation by upregulating TJ protein expression and regulating arginine metabolism.

The Treg/Th17 balance in IBD tends to favor Th17 cells and triggers excessive inflammatory responses in the gut(S. Luo et al., 2019; Wen et al., 2021; W. Zhang et al., 2019). Th17 cells are major players in autoimmune diseases, such as IBD, and can produce proinflammatory cytokines (IL-17 A)(Chen et al., 2022; Jiang et al., 2023), whereas Tregs can release anti-inflammatory cytokines (IL-10) to suppress a broad spectrum of immune responses and inflammation(Furusawa et al., 2013; Smith et al., 2013). In this study, LRWP-Ap treatment ameliorated the DSS-induced Treg/Th17 imbalance. Additionally, abnormal production of inflammatory cytokines poses a significant risk factor for IBD development and significantly affects colonic and systemic inflammation. LRWP-Ap significantly reduced DSS-induced increases in the concentrations of inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Anti-TNF-α antibodies have been clinically applied for IBD therapy(Harbord et al., 2017), and the efficacy of LRWP-Ap in reversing DSS-induced TNF-α upregulation highlights its potential as a therapeutic agent against IBD. Furthermore, the level of IL-10, which ameliorates colonic damage in UC patients, remained elevated in DSS-treated mice given high doses of LRWP-Ap, altogether suggesting that LRWP-Ap can alleviate colitis by maintaining immune homeostasis through immunomodulation.

Research has indicated that the gut microbiota dysbiosis and microbial barrier disruption are important triggers of IBD. The major gut microbiota components, Firmicutes and Bacteroidetes, are depleted in individuals with colitis, leading to reduced biodiversity and ecological imbalance(Arumugam et al., 2011; Richard and Sokol, 2019). In DSS-induced colitis, Proteobacteria significantly increase in abundance. LRWP-Ap supplementation counteracted the alterations in the changes in the abundance of these three major phyla, restoring the Firmicutes/Bacteroidetes ratio disrupted by DSS, which is a crucial indicator of the maintenance of normal gut homeostasis. At the genus level, LRWP-Ap also enriched beneficial bacteria such as Allobaculum, Dubosiella, and Turicibacter, which play crucial roles in improving metabolism and reducing inflammation, thus alleviating colitis(X. Liu et al., 2022; Mo et al., 2022; Wan et al., 2021; Y. Zhang et al., 2021).

Regulating gut microbiota metabolite levels is another key approach to treat colitis. Polysaccharides, as nondigestible carbohydrates, are not absorbed within the gut but undergo fermentation by specific anaerobic bacteria within the gut to produce SCFAs such as acetic acid, propionic acid, and butyric acid(X. Xu et al., 2023). Different gut bacteria tend to generate different SCFAs. Firmicutes are major butyric acid producers, whereas Bacteroidetes primarily produce acetic acid and propionic acid. Our results revealed that DSS significantly reduced butyric acid levels and increased propionic acid levels in the gut, likely due to DSS-induced disruption of the intestinal microbiota, decreasing Firmicutes abundance and increasing Bacteroidetes abundance. LRWP-Ap effectively increased SCFA levels within mice treated with DSS, possibly mediated by the restoration of the Firmicutes/Bacteroidetes proportion and enrichment of bacteria that produce SCFA, including Allobaculum, Akkermansia, and Faecalibaculum. SCFAs lower the pH of the intestine, inhibit harmful bacterial growth and enhance the colonization of beneficial bacteria, forming a positive feedback loop that supports gut health and maintains microbiota stability and diversity(Kałużna-Czaplińska et al., 2017).

Notably, gut dysbiosis, loss of beneficial bacteria, enrichment of pathogens, and reduced biodiversity affect the Treg/Th17 balance in IBD, favoring Th17 cells and triggering excessive gut inflammation. Beneficial bacteria and their microbial metabolites, including SCFAs can induce Treg differentiation, enhance anti-inflammatory cytokine secretion, suppress intestinal inflammation, and improve mucosal immune responses in IBD(Furusawa et al., 2013; Y. J. Liu et al., 2020). Additionally, SCFAs, as major energy sources for intestinal epithelial cells, promote intestinal barrier function and contribute to the modulating metabolism and inflammation(Dong et al., 2019; Wang et al., 2019). Therefore, the regulation of intestinal inflammation by LRWP-Ap is a multiple-function mode of action. On the one hand, LRWP-Ap is capable of modulating the Treg/Th17 balance by elevating the levels of beneficial intestinal bacteria and SCFAs, including butyric acid and acetic acid, supporting function of the barrier in the intestines and regulating the levels of inflammatory factors to maintain intestinal homeostasis. On the other hand, some immune cells (such as Tregs or Th17 cells) in the intestine express polysaccharide receptors, and polysaccharides can directly act on these related receptors, such as TLR-like receptors, to regulate the inflammatory response(Yang et al., 2022).

The functionality of polysaccharides is influenced by their molecular weight, monosaccharide units, the nature of glycosidic bond, degree of branching, and three-dimensional spatial structure. Our structural examination revealed that LRWP-Ap is a pectic polysaccharide with an estimated average molecular weight of 4.34 kDa. Some pectin structures have been found to have anti-inflammatory effects(Dongmei Wu et al., 2021). This anti-inflammatory effect of LRWP-Ap could be ascribed to the effects of structural components such degree of methyl-esterification (DM), arabinogalactan (AG) and homogalacturonic acid (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II)(Beukema et al., 2020; Sabater et al., 2019). Moreover, consistent with other studies showing that pectin abundantly present in the β1-6 and β1-3 galactoside bonds has a probiotic effect(M. Y. Zhang and Cai, 2023), LRWP-Ap also has a probiotic effect. In addition, pectin with a lower molecular weight is more easily utilized by the gut microbiota(Mao et al., 2019). However, other studies have shown that the arabinose side chain in pectin promotes the binding of pectin to galactoglucan lectin-3, which induces the release of proinflammatory factors, including TNF-α, to achieve anticancer effects(D. Wu et al., 2020). The differences in the bidirectional regulation of anti-inflammatory/proinflammatory activities might be attributed to the specific characteristics of certain pectic polysaccharides, which may also originate from differences in activity due to differences in the source of pectin, molecular weight, bond composition, and spatial structure(Niu et al., 2023). Further studies are currently exploring this research topic in depth.

5. Conclusion

The pectic polysaccharide LRWP-Ap from the fruit of L. ruthenicum exhibited good activity against DSS-induced colitis in mice, and the relevant mechanism involved multiple activities related to inflammation, intestinal barrier protection, immunomodulation, the gut microbiota and metabolism. These findings suggest that LRWP-Ap is a promising functional and nutritional food for alleviating intestinal inflammation and protecting intestinal barrier function.

CRediT authorship contribution statement

Dai Dong: Investigation, Writing – original draft. Hailiang Wang: Supervision, Methodology. Hongtao Bi: isolation and characterization. Yu Li: Software, Data curation. Tingting Gao: Data curation. Jingyue Feng: Software, Data curation. Guoqiang Li: isolation and characterization. Shiqi Guo: Data curation. Hongyan Yuan: Data curation. Weihua Ni: Conceptualization, Writing – review & editing.

Funding

The Department of Science and Technology of Jilin Province (Grant No. 20240101247JC), the Innovation Platform Program of Qinghai Province (Grant No. 2021-ZJ-T02).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Dr. Yeonhwa Park

Data availability

Data will be made available on request.