Walnut Green Husk Polysaccharide Improve Gut Microbiota and Alleviate Intestinal Inflammation Caused by Immune Checkpoint Inhibitors

Abstract

In this study, the structure of Walnut green husk polysaccharides (WGHP) and their effects on immune checkpoint inhibitor induced colitis (ICIIC) and intestinal microbiota in mice were studied. The results showed that WGHP was composed of mannose (Man) 0.56%, rhamnose (Rha) 6.81%, galacturonic acid (GalA) 53.52%, glucose (Glc) 8.93%, galactose (Gal) 13.94%, arabinose (Ara) 15.88% and fucose (Fuc) 0.35%. The results of animal experiments showed that the intake of WGHP could not only effectively improve the phenotype of ICIIC in mice, but also significantly regulate the composition of intestinal flora and the content of short-chain fatty acids in mice, such as regulating the ratio of Firmicutes/Bacterotoides, Lachnospiraceae NK4A136 group, Lactobacillus, and increasing the content of butyric acid, acetic acid, and isobutyric acid to restore intestinal homeostasis. In addition, WGHP improves inflammation in mouse ICIIC by inhibiting the secretion of pro-inflammatory cytokines TNF-α and IL-1β, thereby activating the GPR43/PD-1/PD-L1 signaling pathway. Therefore, WGHP can be used as a functional polysaccharide for the prevention of ICIIC.

1. Introduction

Programmed cell death protein 1 (PD-1) inhibitors, a class of immune checkpoint inhibitors, have gained increasing prominence in cancer immunotherapy due to their ability to activate the host immune response. They are now widely used in clinical cancer treatment. A retrospective study on locally advanced head and neck squamous cell carcinoma found that the introduction of PD-1 inhibitor treatment can improve patients’ overall survival [1]. In order to further provide alternative therapies in clinical practice and reduce drug resistance, studies have found that the combination of theobromine with PD-1 can enhance the anti-tumor effect of low-dose apatinib combined with PD-1 inhibitors on hepatocellular carcinoma [2]. However, their clinical application is often associated with a range of adverse effects, particularly affecting the gastrointestinal system, with colitis being one of the most prevalent complications [3]. Epidemiological data indicate that approximately 25% of patients develop colitis within two months of initiating PD-1 inhibitor therapy, with diarrhea being the most common symptom, occasionally accompanied by mucus or blood in the stool. Furthermore, clinical studies have reported that 50% of patients receiving PD-1 inhibitors experience abdominal pain, fever, vomiting, and nausea. If left untreated, these symptoms can progress to severe, life-threatening complications [4].

The exact pathogenesis of ICIIC remains unclear, but emerging evidence suggests a strong association between regulatory T cells and the gut microbiome (GM). The GM is critically involved in the pathogenesis of colitis. Intestinal inflammation leads to metabolic disturbances in the GM, which in turn affects intestinal barrier integrity. The production of pro-inflammatory cytokines by immune cells, combined with an increase in pathogenic bacteria and the translocation of endotoxins, further aggravates the condition. A study on stool samples from 230 proctitis patients revealed a significant correlation between the microbial species present in their stools and the degree of disease improvement [5]. Short-chain fatty acids (SCFAs), which are primarily produced by Bacterotoides and Firmicutes [6], serve as important by-products of intestinal microbial metabolism. These compounds play crucial roles in regulating electrolyte balance, protecting the intestinal mucosal barrier, enhancing metabolism, and modulating intestinal immunity [6]. There is a strong link between SCFAs and the regulation of GM. Studies have shown that lactic acid bacteria can influence the intestinal microbiota, producing substantial amounts of SCFAs, which have been shown to effectively alleviate diarrhea symptoms in mice [7]. Although ICIIC shares many characteristics with inflammatory bowel disease, it is considered a distinct form of colitis due to its rapid onset and progression, potentially leading to severe complications, including intestinal perforation and death. Timely identification and management of ICIIC are essential to achieving the best possible clinical outcomes.

Walnut (Juglans regia), recognized as both a nutritious food and a traditional medicine, has been described in the Compendium of Materia Medica for its therapeutic properties, including clearing heat, detoxifying the body, moisturizing the intestines, and alleviating respiratory conditions such as asthma [8]. The walnut green husk, a by-product of walnut processing, constitutes a substantial portion of the plant. However, due to limited awareness, much of it is discarded as waste, leading to environmental pollution and resource loss. Thus, fully harnessing the medicinal components in walnut holds considerable economic and environmental value. Polysaccharides, a class of natural bioactive macromolecules, have garnered considerable attention due to their diverse biological activities, including anti-tumor and antiviral properties. WGHP, a polysaccharide extracted from walnut green husk, has demonstrated notable anti-inflammatory, anti-tumor, and immunomodulatory effects. Moreover, WGHP has been widely applied in the food industry [9]. Researchers illustrated that WGHP has the capacity to modulate the intestinal microbiota in murine models and mitigate liver damage induced by a high-fat diet. Subsequent research has demonstrated that WGHP provides significant protective benefits against liver inflammation and gluconeogenesis induced by ochratoxin A, as evidenced by experiments involving fecal microbiota transplantation [10]. The results indicate that WGHP reduces liver inflammation and gluconeogenesis through the modulation of the GM [11]. Despite these promising findings, research on WGHP remains limited, particularly regarding its potential in mitigating immune-related intestinal inflammation. To address this knowledge gap, we developed an ICIIC mouse model using PD-1 inhibitors to investigate the effects of WGHP on immune-related intestinal inflammation, with a specific focus on GM and SCFAs. Further research into WGHP’s impact on intestinal repair is essential for understanding its therapeutic potential.

2. Materials and Methods

2.1. Experimental Materials and Reagents

Walnut green husks were provided by the Heilongjiang Academy of Traditional Chinese Medicine. The following reagents were used: SCFAs (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China), enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-22 (IL-22) (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China), PD-1 inhibitors (MCE, Monmouth Junction, NJ, USA, HY-19991), corn oil (Biyuntian Biotechnology Co., Ltd., Shanghai, China), bicinchoninic acid (BCA) protein concentration assay kit (Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China), enhanced RIPA tissue lysate, anti-GAPDH antibody (BM3896), HRP-conjugated goat anti-rabbit IgG, anti-PD-L1/CD274 antibody, and anti-GPR43 antibody (Beijing Braitex Biotechnology Co., Ltd., Beijing, China), and protease phosphatase inhibitor cocktail (general-purpose, 50X) (Biyuntian Biotechnology Co., Ltd., Shanghai, China).

2.2. Preparation of WGHP

2.2.1. Extraction and Purification of WGHP

WGHP was extracted following the method described by previous investigators [12]. Briefly, dried walnut green husks were mixed with distilled water at a 1:2 ratio (w/v) and subjected to multiple rounds of decoction and concentration. Subsequently, four times the volume of 95% ethanol was added to the concentrated extract, followed by overnight incubation at 4 °C. The precipitate was collected via centrifugation at 4000 rpm for 15 min and subsequently washed with ether, acetone, and absolute ethanol in sequence. The resultant crude polysaccharides were then deproteinized and depigmented using papain, trichloroacetic acid (TCA), and a 5 mg/mL crude WGHP solution. The mixture was further purified using a D101 macroporous resin column and a DEAE Cellulose 52 ion exchange column, followed by dialysis using 3.5 kDa membranes to remove small molecules. The purified WGHP was subsequently freeze-dried for further use.

2.2.2. Chemical Characterization of WGHP

The WGHP content was quantified using the phenol-sulfuric acid method. A microplate reader (US) was utilized to measure absorbance at 490 nm, with glucose serving as the standard for generating a calibration curve. The WGHP content was determined by interpolating the absorbance values into the calibration curve equation, where the x-axis represented concentration and the y-axis represented absorbance. The TCA-papain method was used to deproteinize the total WGHP. The supernatant was taken to determine the protein content in the sugar solution, and the full wavelength of WGHP was measured using ultraviolet spectroscopy.

Fourier-transform infrared spectroscopy (FT-IR) was employed to identify the functional groups present in WGHP. Prior to analysis, 300 mg of KBr was oven-dried, and 1 mg of WGHP was mixed with 200 mg of dry KBr. The mixture was finely ground in an agate mortar and pressed into a pellet. FT-IR spectra were recorded within the wavenumber range of 400–4000 cm⁻¹ using an FT-IR spectrometer.

Using the characteristic that polysaccharides can be hydrolyzed into monosaccharides under the action of phenol and concentrated sulfuric acid, then quickly dehydrated to form aldose derivatives, and subsequently condensed with phenol to form an orange-yellow compound, we measured the total sugar content of WGHP.

The molecular weight of WGHP was determined using efficient gel chromatography detection. Standard solution: Prepare a 2% aqueous solution of Dextran T1, Dextran T4, Dextran T7, Dextran T10, Dextran T40, Dextran T70, and Dextran T100 at a concentration of 1 mg/mL, and detect them using the method described above.

Sample solution: Dissolve 3 mg of WGHP in 1 mL of distilled water to prepare a WGHP solution with a concentration of 3 mg/mL.

The monosaccharide composition of WGHP was analyzed using PMP-HPLC. The aqueous phase was filtered through a 0.45 μm microporous membrane before analysis using an Agilent Extend-C18 column (4.6 mm × 250 mm, 5 μm). Isocratic elution was performed over 60 min with a mobile phase consisting of 85% ammonium acetate (A) and 15% acetonitrile (B, v/v). The injection volume was 10 μL, the flow rate was maintained at 1 mL/min, and the detection wavelength was set at 250 nm. The column temperature was controlled at 30 °C.

2.3. Animal and Experimental Methods

Five-week-old male specific pathogen-free (SPF) grade BALB/c mice (19.2 ± 2.0 g) were obtained from the Second Affiliated Hospital of Harbin Medical University (Harbin, China). All animal experiments were conducted in accordance with the ethical guidelines and regulations of the People’s Republic of China and were approved by the Laboratory Animal Ethics Committee of Heilongjiang Academy of Traditional Chinese Medicine (license number 2015-64). The experiments were conducted at the Institute of Traditional Chinese Medicine, Heilongjiang Academy of Traditional Chinese Medicine, under controlled environmental conditions (temperature: 21 ± 2 °C; humidity: 50 ± 3%).

Following a one-week acclimation period, the mice were randomly assigned to five groups: normal control (NC, n = 20), model control (MC, n = 20), WGHP high-dose (QH, 400 mg/kg, n = 20), WGHP medium-dose (QM, 200 mg/kg, n = 20), and WGHP low-dose (QL, 100 mg/kg, n = 20). The experimental modeling and WGHP dosages were based on previous studies [13,14]. Except for the NC group, all mice received intraperitoneal injections of PD-1 inhibitors (5 mg/kg) every other day for a total of five injections. To prevent spontaneous recovery, PD-1 inhibitor administration continued every seven days during the treatment period. Starting on the tenth day after PD-1 inhibitor administration, the QL, QM, and QH groups received daily oral gavage of WGHP for 21 days, while the NC and MC groups received normal saline. During the treatment period, mouse behavior was observed, and body weight was recorded every three days (Figure 1). After 21 days of WGHP administration, the mice were fasted for 12 h and anesthetized with 30 mg/kg sodium pentobarbital. Blood samples were collected via the retro-orbital plexus into anticoagulant-free centrifuge tubes and centrifuged at 3000 rpm for 15 min at 4 °C to obtain serum, which was subsequently stored at −80 °C. Following euthanasia, colon length and body weight were measured. A 1 cm segment of the colon was fixed in 4% paraformaldehyde for histological analysis, while the remaining colon tissues and intestinal contents were stored in 2 mL cryovials at −80 °C for further examination.

2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of TNF-α, IL-1β, and IL-22 in serum samples were measured using commercially available ELISA kits, following the manufacturer’s instructions.

2.5. Histopathological Analysis

Colonic tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm-thick slices for histopathological analysis. Hematoxylin and eosin (H&E) staining was performed as follows: tissue sections were deparaffinized, rehydrated, and incubated with hematoxylin for 1–2 min, followed by eosin staining for 30 s. After rinsing under running water for 2 min, the sections were dehydrated with 95% ethanol (two changes), absolute ethanol (two changes), and xylene (three changes, 3 min each). The stained sections were mounted with a coverslip and examined under an upright light microscope (Nikon, Tokyo, Japan). Histopathological scoring of intestinal tissue damage was performed according to the criteria established in previous studies [15].

2.6. GM Analysis by High-Throughput Sequencing

Genomic DNA was extracted from fecal samples using a fecal DNA extraction kit (Tiangen Biotechnology Co., Ltd., Beijing, China) under sterile conditions. The V3-V4 region of the bacterial 16S ribosomal RNA (rRNA) gene was amplified using specific primers (341F: CCTAYGGGRBGCASCAG and 806R: GGACTACNNGGGTATCTAAT) in combination with FastPfu polymerase. The resulting amplicons were subjected to further analysis. Each polymerase chain reaction (PCR) mixture contained 10 ng of genomic DNA, 0.2 μM of each primer, and 15 μL of Phusion High-Fidelity PCR Master Mix. The PCR amplification protocol included an initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. The PCR products were purified using magnetic beads, pooled based on concentration, and analyzed. After pooling, target bands were recovered, and a sequencing library was constructed. The constructed library was quantified using Qubit and quantitative PCR (Q-PCR), and its quality was assessed. Sequencing was performed using the NovaSeq6000 (Inmena, Inamina, CA, USA) platform with PE250 machine measurement.

2.7. Bioinformatics Analysis

Sequences obtained from samples were filtered to remove barcode and primer sequences, and the raw sequencing data were processed using FLASH (Version 1.2.11), to generate raw tags [16]. Residual primer sequences were eliminated using Cutadapt software, and the filtered sequences were processed using fastp software (Version 0.23.1) to generate high-quality tags (Clean Tags) [17]. Chimera sequences were subsequently removed, and the final effective tags were identified by aligning them against the Silva database for 16S/18S rRNA sequences and the Unite database for ITS sequences to ensure the elimination of any chimeric sequences [18].

2.8. Determination of SCFAs

According to the method provided by previous researchers for measuring short-chain fatty acids, we prepared a standard mixed solution of short-chain fatty acids, adjusting the mixed solution to 100 mL with CH₃OH, and the internal standard to 50 mL with CH₃OH. Solutions except for the internal standard were diluted in multiples. 1 mL of each concentration solution was placed into a sample vial, and 50 μL of the internal standard was added. Cecal contents (0.5 g) were collected from ten mice per group. Each sample was homogenized with 2 mL of methanol and vortexed at 2500 rpm for 2 min. Following centrifugation at 5000 rpm for 10 min, 1 mL of the supernatant was collected, and 1 μL of sulfuric acid was added, followed by the introduction of 50 μL of an internal standard solution (2-ethylbutyric acid). The samples were further centrifuged at 13,000 rpm for 5 min at 4 °C, and the supernatants were carefully collected [19]. Samples were either stored on ice or frozen until the quantification of short-chain fatty acids (SCFAs) was performed using gas chromatography-mass spectrometry (GC-MS, Agilent 7890 A/5975 C). The analysis was conducted using an Elite-WAX capillary column (30 m × 0.25 mm × 0.25 μm) and an electron ionization (EI) detector.

2.9. Western Blot (WB)

Colonic tissues (50 mg) were weighed, homogenized, and lysed in 500 μL of enhanced radioimmunoprecipitation assay (RIPA) buffer (lysis buffer:protease inhibitor:phosphatase inhibitor = 50:1:1). The lysates were incubated on ice for 30 min, homogenized using a grinder, and centrifuged. The supernatant was collected, and protein concentrations were determined using a BCA assay. The extracted proteins were denatured and separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with 40 μg of protein loaded per lane [20]. Afterwards, a PVDF membrane was used to transfer the proteins. The 5% skim milk was used for 1.5 h for blocking, and then there were five 8 min washes with TBST. Overnight at 4 °C, the primary antibody was treated with the membrane. After 2 h of incubation with the secondary antibody in the dark, the signals were detected using enhanced chemiluminescence (ECL) reagent (GE Image Quant LAS 4000, Fairfield, CA, USA). Data quantification was carried out using ImageJ software (Version 1.41).

2.10. Data Analysis

Data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using SPSS 20, with Duncan’s multiple range test applied for comparisons. Graphs were generated using Origin 2019b and GraphPad Prism 9.5.1. A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Characterization of WGHP

Fourier transform infrared (FT-IR) spectroscopy revealed a prominent, broad absorption peak at 3362.14 cm⁻¹, indicative of characteristic carbohydrate groups and hydroxyl (-OH) stretching vibrations. The -OH group, known to act as a hydrogen donor, plays a crucial role in the antioxidant activity of walnut green peel polysaccharides. An additional peak at 2931.11 cm⁻¹ was associated with the stretching vibration of C-H bonds from the -CH₂ group present in the polysaccharide. A peak at 1742.55 cm⁻¹ reflected the stretching vibration of uronic acid, while a peak at 1618.08 cm⁻¹ suggested the presence of unmethylated carboxyl C=O groups. A peak at 1418.42 cm⁻¹ corresponded to C-H bending vibrations, and the peaks observed at 1151.04 cm⁻¹, 1077.98 cm⁻¹, and 1024.00 cm⁻¹ were attributed to C-O stretching vibrations. Additionally, the absorption peak at 762.83 cm⁻¹ was linked to the symmetrical stretching and contraction vibrations of the D-glucose pyranose ring (Figure 2A). We obtained WGHP with a total sugar content of 85.5%, a protein content of 3%, and a relative molecular mass of 3.89 × 10⁶ Da (Supplementary Documents S2 and S3). For specific data, see the Supplementary File.

The monosaccharide composition of WGHP was determined using high-performance liquid chromatography (HPLC), with retention times compared to those of monosaccharide standards (S1). WGHP is composed of Mannose (Man) 0.56%, Rhamnose (Rha) 6.81%, Galacturonic Acid (GalA) 53.52%, Glucose (Glc) 8.93%, Galactose (Gal) 13.94%, arabinose (Ara) 15.88% and Fucose (Fuc) 0.35% (Figure 2B).

3.2. Effect of WGHP on the Physiological Characteristics of Mice

Prior to modeling, all mice in the experimental groups exhibited normal physical conditions. Following model induction, all groups, except for the NC group, showed a reduction in body weight (BW) and exhibited signs of lethargy. However, after treatment with WGHP, both the general condition and BW of the mice significantly improved in comparison to the MC group. The changes in BW are illustrated in Figure 3A.

3.3. Anti-Inflammatory Effects of WGHP

As illustrated in Figure 3B–D, the concentrations of TNF-α and IL-1β in the α MC group were higher than those observed in the high, medium, and low WGHP treatment groups. In contrast, IL-22 concentrations were lower in the MC group. These results confirm the successful establishment of the enteritis model. In the WGHP-treated groups (QH, QM, QL), the levels of TNF-α and IL-1β were lower (p < 0.05), while IL-22 concentrations were elevated (p < 0.05). The 200 mg/kg WGHP dosage demonstrated the most substantial improvement.

3.4. Effect of WGHP on Intestinal Tissue Structure

Analysis of colon length indicated that the MC group exhibited a significantly reduced colon length (p < 0.05), while the colon length in the WGHP-treated groups was similar to that of the NC group (Figure 4A,B). To elucidate the protective effects of WGHP on the intestinal barrier, a histopathological analysis of colon tissue was performed. As shown in Figure 4C, colon tissue from the NC group exhibited clear folds, intact intestinal epithelium, and a single layer of columnar cells. The epithelial cells were well-organized, tightly arranged, and devoid of shedding. The crypts were intact and neatly aligned, while the lamina propria was rich in goblet cells, with no evidence of lymphocyte infiltration. In contrast, the MC group displayed epithelial erosion in the mucosal layer (green arrows), inflammatory cell infiltration, nuclear condensation, and local lymphocytic infiltration in the lamina propria (red arrows). Necrosis of epithelial and intestinal gland cells, characterized by nuclear fragmentation and hyperchromatic staining, was also observed (black arrows) (Figure 4D). The QL group showed limited epithelial cell necrosis, with some nuclear condensation and mild connective tissue hyperplasia in the lamina propria (gray arrows), as well as focal lymphocyte infiltration (red arrows) (Figure 4E). The QM group exhibited a structurally normal colon with no significant inflammatory changes (blue arrows) (Figure 4F). In the QH group, isolated necrotic epithelial cells and cell detachment were observed (black arrows), along with nuclear condensation and connective tissue hyperplasia (gray arrows). The muscle layer appeared normal, with uniformly stained and well-aligned muscle fibers (Figure 4G). As the MC group received no treatment, it exhibited the most severe inflammation, resulting in the highest histological score. Following WGHP administration, intestinal inflammation showed improvement, and the histological score approached that of the NC group (Figure 4H).

3.5. Effect of WGHP on Microbial Composition

3.5.1. α Diversity Analysis

In alpha diversity analysis, Shannon, Simpson, Chao1, and Ace are commonly used. These indices can calculate microbial richness through different algorithms, so in this experiment, we used Shannon, Simpson, Chao1, and Ace to perform an in-depth analysis of microbial richness. In the MC group, the observed-species, Chao1, and Shannon indices were all lower than those in the NC group, indicating that there were differences in community species within the MC group. The observed-species, Chao1, and Shannon indices in each WGHP group approached those of the NC group, suggesting that WGHP can effectively regulate the gut microbiota. There was no difference in the Simpson index, indicating that the community diversity was similar across groups under this algorithm. The lowest value of Good’s coverage index was 0.999, indicating that the sequencing results are reliable (

Table 1. Alpha diversity index of intestinal microbiota in five groups of mice.

Group	Diversity Index
	observed_species	shannon	simpson	chao1	ACE	PD_whole_tree	Good’s Coverage
NC	390.86 ± 8.91	5.60 ± 0.38	0.97 ± 0.01	401.23 ± 9.72	402.16 ± 2.48	27.67 ± 3.33	0.999
MC	329.67 ± 6.21	4.13 ± 0.95	0.96 ± 0.04	347.86 ± 2.28	345.39 ± 7.54	19.62 ± 1.36	0.999
QL	340.66 ± 8.15	5.34 ± 0.86	0.95 ± 0.06	375.77 ± 2.88	375.54 ± 5.14	21.78 ± 2.36	1.000
QM	387.89 ± 8.87	5.58 ± 0.32	0.93 ± 0.08	395.86 ± 3.18	401.42 ± 6.31	26.83 ± 4.13	0.999
QH	379.98 ± 9.65	5.47 ± 0.54	0.95 ± 0.03	385.77 ± 4.88	395.51 ± 1.64	25.06 ± 5.25	1.000
p-value	0.023167658	0.012318247	0.013752217	0.033571218	0.005806078	0.018293395

).

3.5.2. Changes in Microbial Community Abundance

Venn diagram analysis was employed to illustrate the shared and unique OTUs across multiple samples. The Venn diagram shows that treatment with WGHP increases the number of operational taxonomic units (OTUs) compared to the MC group, which favors the NC group (Figure 5A).

3.5.3. β Diversity Analysis

The β-diversity index calculates the distances between samples by utilizing the evolutionary relationships and abundance information of sequences in each sample, reflecting whether there are significant differences in microbial communities among samples. In this experiment, PCoA (Principal Coordinates Analysis) based on Bray Curtis distances showed clear separation among the NC group, MC group, and WGHP treatment group, indicating that the WGHP treatment group and the MC group had a significant effect on the gut microbiota of mice (Figure 5B). In addition, Linear Discriminant Analysis Effect Size (LEfSe) was used to identify biomarkers in the microbiota of different treatment groups. Compared with the MC group, the WGHP treatment group and the NC group had a higher proportion of characteristic bacterial species, with Lactobacillales, Lactobacillaceae, Lactobacillus johnsonii, and Limosilactobacillus serving as the main bacteria. In the MC group, Clostridia, Lachnospiraceae_NK4A136_group, and Lachnospiraceae had higher LDA scores than the other groups (Figure 5C,D).

3.5.4. Species Annotation and Assessment

Based on the top 10 abundant species in each sample at different taxonomic levels (phylum, genus), draw a histogram of relative abundance in Perl using the SVG function.

At the phylum level (Figure 5E, the average abundance of Bacteroides was higher in the NC group and WGHP treatment groups. The relative abundances of Firmicutes and Bacterotoides were the highest among all flora in the NC, MC, QL, QM, QH groups, with significant differences in the relative abundances of Firmicutes (Figure 5F, Bacterotoides (Figure 5G, and Firmicutes/Bacterotoides ratio components (Figure 5H).

At the genus level, the differences between the groups showed significant differences between the NC group and the MC group, including Lachnospiraceae NK4A136 group, Lactobacillus, Limosilactobacillus, Candidatus Saccharimonas, Enterorhabdus, GCA-900066575, Roseburia, and Colidextribacter. Lachnospiraceae NK4A136 group, Enterorhabdus, Candidatus saccharimonas, and Anaerotruncus showed significant differences between MC and WGHP groups. Importantly, WGHP treatment resulted in decreased levels of Eubacterium xylanophilum group, Lachnospiraceae NK4A136, Clostridia and Lachnospirales (Figure 6). These results indicate that WGHP exerts a protective effect on the intestinal microbiota.

3.5.5. Effect of WGHP on SCFAs Concentrations

We plotted linear equations for short-chain fatty acids to calculate the short-chain fatty acids in each group of mice. By examining the peak areas of six short-chain fatty acids (acetic acid, propionic acid, isobutyric acid, butyric acid, etc.) and performing RSD calculations, the results are shown in

Table 2. Short-chain fatty acid precision.

Standard Product	Average Peak Area	RSD
Acetic acid	15,319,391.1	0.91%
Propionic acid	5,101,232.2	0.93%
Isobutyric acid	1,398,921.3	0.99%
Butyric acid	14,194,962.5	0.98%
Isovaleric acid	1,803,557.3	1.13%
Valeric acid	1,636,488.2	1.18%
2-Ethylbutyric acid	624,120.3	0.90%

, with RSD values ranging from 0.90% to 1.18%, indicating good precision. Using GC-MS, samples and standards were sequentially analyzed for data acquisition, as shown in Figure 7A–F. All six acids were well separated from diethyl butyrate, and the sample retention times were similar to those of the standards, confirming the correctness of the analytical method. The content of short-chain fatty acids in the model group was significantly lower than that in the blank group (p < 0.05). Compared to the model group, the WGHP group showed a notable recovery, with the medium-dose WGHP group exhibiting more pronounced recovery, suggesting that a dose of 200 mg/kg WGHP best modulates the short-chain fatty acids in mice with immune colitis induced by PD-1 inhibitors.

3.5.6. Effect of WGHP on Related Proteins in Intestinal Tissue

WB analysis was employed to assess the expression levels of GPR43, PD-1, and PD-L1 proteins in the colon. The results revealed that the expression of GPR43, PD-1, and PD-L1 in the traditional Chinese medicine group was significantly higher (p < 0.05). These findings suggest that WGHP may activate the GPR43/PD-1/PD-L1 pathway and alleviate enteritis (Figure 7G–J).

4. Discussion

Chinese medicinal polysaccharides have functions such as promoting the growth of immune organs and immune cells, activating immune cells, and facilitating the release of immune factors. However, the immune-regulating characteristics of each medicinal polysaccharide are different. Researchers have found that Polysaccharides of Lycium chinense can activate dendritic cells and re-polarize macrophages, exhibiting enhanced immune regulatory effects. Interestingly, galacturonic acid in the total polysaccharides of goji accounts for 76.18% of the acidic polysaccharides [21]. The results of Wen et al.’s study indicate that Polygonatum polysaccharides can enhance the proliferation and phagocytic activity of macrophages, through HPGPC, HPLC, and other analyses, it was detected that Polygonatum polysaccharides have a molecular weight of 3.2 kDa, with glucose (55.4%) and galactose (44.6%) being the main monosaccharide components [22]. In this study, we found that WGHP are acidic heteropolysaccharides composed of mannose, rhamnose, galacturonic acid, glucose, galactose, arabinose, and fucose, with a GalA content of 53.52%. Based on this, we have reason to believe that GalA in WGHP is closely associated with immune cells in the process of regulating mouse immunity. Interestingly, in this study, the regulatory effect was best at the medium dose of WGHP. We speculate that this is because the medium dose of WGHP happens to be the optimal dose for regulating ICIIC, while increasing the dose triggers a strong response from immune cells and organs, causing slight over-adjustment in the body. Therefore, the effect of a high dose of WGHP on regulating ICIIC is less favorable than that of the medium dose.

PD-1 is predominantly expressed in activated immune cells and serves as an autostable immune checkpoint. PD-1 inhibitors disrupt this immune regulation, leading to overactivation of immune cells and triggering intestinal inflammation. Clinical studies have shown that patients develop gastrointestinal inflammation within six months of discontinuing PD-1 inhibitors. Follow-up assessments revealed not only watery diarrhea but also involvement of the colon and ileum, with some patients presenting lymphoplasmacytic infiltration and basal plasmacytosis, with colonic inflammation being the most affected [23]. Our study found that inflammation was correlated with an increase in colon length, and the colon length in the WGHP treatment groups showed a notable improvement.

Maintaining a balanced intestinal flora is essential for overall animal homeostasis, with dysbiosis often manifesting as diarrhea, which may lead to colonic inflammation. Relevant investigators analyzed biochemical and pathological that WGHP significantly alleviated inflammation and dysfunction associated with glucose metabolism, lipid metabolism, and oxidative stress, while reversing disturbances in the intestinal flora and reducing systemic inflammation [24]. Based on this, we hypothesize that WGHP modulates inflammation through its impact on the intestinal microbiota. Our study found that the composition of the microbiota in the MC group was significantly changed compared to the NC group. The microbiota of enteritis animals is mainly composed of Firmicutes and Bacterotoides, among which an increase in the proportion of Firmicutes usually leads to the progression of enteritis, and an increase in the ratio of Firmicutes/Bacterotoides affects the composition of intestinal microbiota and the inflammatory response plays an important role in enteritis [3]. Here we also found that the ratio of Firmicutes/Bacterotoides in ICIIC mice increased, and the whole intestinal homeostasis was disturbed. Short-chain fatty acids are primary indirect nutrients produced by the intestinal flora and have a strong physiological regulatory effect. Clinical studies have reported reduced microbial diversity in patients with ICIIC, especially in families such as Marinifilaceae, Desulfovibrionaceae, Lactobacillaceae, and Streptococcaceae, compared to healthy individuals who exhibit greater diversity, notably in Peptostreptococcaceae [25]. We observed that WGHP regulates Lachnospiraceae NK4A136 group, Lactobacillus, Limosilactobacillus, Candidatus Saccharimonas, Enterorhabdus, Roseburia, Colidextribacter and other abundance values alleviate intestinal inflammatory responses. It is worth noting that there was no significant difference in the effect of regulating microbiota between the QM group and the QH group, suggesting that a good modulation effect had been achieved at the dose of QM. Indigestible polysaccharides are fermented by intestinal microbiota into SCFAs, which are essential for maintaining colonic integrity and metabolism [26]. Our results suggest that WGHP enhances SCFAs concentrations, particularly acetic acid (AA), butyric acid (BA), and valeric acid, indicating that WGHP may promote SCFAs production. In this experiment, the reason why the model group produced more Lachnospiraceae NK4A136 than the other groups is that the intestinal microbiota homeostasis in the model group was disrupted. This disruption is reversible. Previous researchers have found that gastrointestinal reactions can be alleviated after discontinuing PD-1 inhibitors for a certain period. This suggests that during the period when the model mice are intermittently exposed to PD-1 inhibitors, their gut microbiota actively works to restore homeostasis. Therefore, this phenomenon is normal. Additionally, Lachnospiraceae NK4A136 is considered one of the butyrate producers. To maintain intestinal homeostasis, the gut actively responds by producing Lachnospiraceae NK4A136. However, other butyrate-producing bacteria in the gut are relatively few, and overall, this is insufficient to significantly increase the butyrate content. As a result, in the subsequent measurement of short-chain fatty acids, the butyrate content in the model group was lower than in the other groups.

The anti-inflammatory properties of SCFAs are likely mediated via the metabolite-sensing G protein-coupled receptor, free fatty acid receptor 2 (GPR43), which is expressed in intestinal epithelial and immune cells, effectively regulating intestinal inflammation [27]. Increased expression of GPR43 in the intestine has been shown to mitigate colonic inflammation [27]. The PD-1/PD-L1 axis is a complex signaling pathway that plays an important role in immune system cells. Clinical researchers have explored the effects of PD-1/PD-L1 on the gut microbiota of mice through fecal microbiota transplantation and found that transplantation of fecal matter from healthy individuals can modulate the PD-1/PD-L1 signaling pathway and the gut microbiota of mice, reducing inflammation in the body [28].

Researcher measured soluble PD-1 and PD-L1 levels in the peripheral blood of colitis patients, finding a correlation between the expression of PD-1/PD-L1 and the severity of colitis [29]. Furthermore, the onset and progression of ICIIC is closely related to inflammatory factors in the body. Therefore, we observed that WGHP can, on one hand, mediate short-chain fatty acid regulation of intestinal immune homeostasis and gut microbiota by increasing the amount of GPR43 receptors, and on the other hand, increase the expression of downstream proteins PD-1 and PD-L1, promoting key pathways that improve immunity, reducing the levels of pro-inflammatory factors, forming a bidirectional regulation, and effectively alleviating ICIIC symptoms. In this experiment, based on various evidence such as the expression levels of key proteins in different groups of mice and in vivo inflammatory factors, we found that a medium dose of WGHP had the optimal regulatory effect, while a high dose of WGHP led to over-regulation. Observations of indicators showed that the treatment group exhibited mild inflammation, unstable gut microbiota, and deviation in the intensity of microbial regulation. Since a healthy gut consistently maintains microbial and immune homeostasis, an appropriate dose of WGHP can help the entire gut maintain better stability, whereas under high-dose WGHP, the body makes slight counter-regulatory adjustments to maintain the gut microbiota and immune system in a relatively stable state, aiming to correct deviations from homeostasis. Therefore, we speculate that this phenomenon is likely caused by over-regulation induced by high doses of WGHP in mice. In conclusion, WGHP may reduce proctitis induced by PD-1 inhibitors by modulating the intestinal microbiota, enhancing SCFAs production, and activating the GPR43/PD-1/PD-L1 pathway.

5. Conclusions

This study demonstrates that Walnut green husk polysaccharides significantly alleviates colitis induced by PD-1 inhibitors. The underlying mechanism is likely related to the digestion of Walnut green husk polysaccharides in the colon, leading to the production of short chain fatty acids, which activate the GPR43/PD-1/PD-L1 pathway, thereby reducing the levels of pro-inflammatory factors. These results indicate that Walnut green husk polysaccharides can effectively mitigate gastrointestinal reactions caused by immune checkpoint inhibitors, making it a promising therapeutic approach for immune checkpoint inhibitor-induced colitis. While this study offers strong support for the potential of Walnut green husk polysaccharides in colitis management, several research areas remain unexplored. Future investigations should assess the long-term therapeutic efficacy and safety of Walnut green husk polysaccharides, as well as its role in modulating complex signaling pathways and proteins. These directions will be essential for developing more effective treatments for immune checkpoint inhibitor induced colitis.