Use of clic5 gene in preparation of drugs for preventing and / or treating intestinal inflammation

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a CLIC5 gene in preparation of a drug for preventing and / or treating intestinal inflammation. The application provides application of the CLIC5 gene in preparation of a drug for preventing and / or treating intestinal inflammation, wherein a nucleotide sequence of the CLIC5 gene is shown as SEQ ID NO:1. The application finds that intestinal epithelial cell-specific knockout of the CLIC5 can reduce a serum endotoxin level, improve intestinal epithelial barrier function, increase a content of butyric acid, reduce a serum inflammatory factor level, and also reduce a gene expression level of an inflammatory factor in an ileum and a colon. The application first discloses that intestinal epithelial cell-specific knockout of the CLIC5 can effectively prevent or treat intestinal inflammation and reduce a body inflammatory factor level, thereby providing a new target and method for treating human intestinal inflammation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the CLIC5 gene in the preparation of drugs for the prevention and / or treatment of intestinal inflammation. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic disease that causes inflammation of the intestines, seriously threatening people's health and placing a significant burden and cost on the healthcare system. The pathogenesis of intestinal inflammation has a genetic predisposition, and is influenced by a complex interplay of factors, including Western diets, smoking, infections, antibiotic use, and gut microbiota dysbiosis and immune system dysfunction. For example, in IBD, the intestinal barrier function is disrupted, leading to microbial translocation, excessive activation of the mucosal immune system, and the release of pro-inflammatory factors. The harmful effects of intestinal inflammation are far-reaching. Locally, it manifests as abdominal pain, diarrhea, and a significantly increased risk of colorectal cancer; systemically, it causes malnutrition, osteoporosis, fatigue, and significantly increases the risk of thrombosis, liver and gallbladder diseases, and joint disorders. Furthermore, intestinal inflammation is strongly associated with a variety of diseases, including autoimmune diseases, metabolic diseases such as obesity and fatty liver, mental illnesses, and neurological disorders.

[0003] Currently, despite advancements in the treatment of intestinal inflammation using biologics, small molecule drugs, and combination therapies, most treatments remain ineffective in controlling intestinal inflammation, especially in patients with chronic or refractory cases. Furthermore, intestinal inflammation involves multi-pathway interactions, and existing small molecule drugs lack sufficient targeting for its treatment.

[0004] Intracellular chloride channels (CLIC) are present in the cytoplasm, cell membrane, and various organelle membranes, playing an important role in the development of cardiovascular diseases, cancer, and neurodegenerative diseases. The inventors previously discovered that overexpression of CLIC5 in skeletal muscle can accelerate the repair process after muscle injury, combat obesity and metabolic syndrome induced by a high-fat diet, and effectively alleviate hepatic fat deposition. However, there are currently no reports of CLIC5 being associated with intestinal inflammation. Summary of the Invention

[0005] To address the lack of drug targets in existing technologies capable of preventing and / or treating intestinal diseases, this invention provides the application of the CLIC5 gene in the preparation of drugs for the prevention and / or treatment of intestinal inflammation, specifically including the following technical solutions:

[0006] The CLIC5 gene is used in the preparation of drugs for the prevention and / or treatment of intestinal inflammation, wherein the nucleotide sequence of the CLIC5 gene is shown in SEQ ID NO:1.

[0007] Preferably, the drug is used to specifically knock out or inhibit the expression of the CLIC5 gene in intestinal epithelial cells.

[0008] The present invention also provides the use of reagents for inhibiting or silencing CLIC5 gene expression in the preparation of medicaments for the prevention and / or treatment of intestinal inflammation, wherein the nucleotide sequence of the CLIC5 gene is shown in SEQ ID NO:1.

[0009] Preferably, the prevention and / or treatment of intestinal inflammation includes one or more of the following: reducing serum endotoxin levels, reducing serum pro-inflammatory factor levels, reducing inflammatory factor levels in the ileum, reducing inflammatory factor levels in the colon, improving intestinal epithelial barrier function, and increasing butyrate content in colonic contents.

[0010] The present invention also provides a medicament for the prevention and / or treatment of intestinal inflammation, comprising a reagent and excipient for inhibiting or silencing CLIC5 gene expression, wherein the nucleotide sequence of the CLIC5 gene is shown in SEQ ID NO:1.

[0011] Preferably, the excipients include pharmaceutically acceptable excipients.

[0012] Preferably, in the application or drug described above, the intestinal inflammation includes I) and / or II):

[0013] 1) Intestinal inflammation caused by a high-fat diet;

[0014] II) Colitis.

[0015] Preferably, the colitis includes colitis caused by sodium dextran sulfate.

[0016] The present invention also provides a biological material for inhibiting or silencing CLIC5 gene expression, wherein the nucleotide sequence of CLIC5 gene is shown in SEQ ID NO:1.

[0017] Preferably, the biological material comprises sgRNA that knocks out the CLIC5 gene, as shown in SEQ ID NO:2 and / or SEQ ID NO:3.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention belongs to the field of biomedical technology, specifically relating to the application of the CLIC5 gene in the preparation of drugs for the prevention and / or treatment of intestinal inflammation. This invention provides the application of the CLIC5 gene in the preparation of drugs for the prevention and / or treatment of intestinal inflammation, the nucleotide sequence of which is shown in SEQ ID NO:1. Through a series of experiments, this invention discovered that in mice with intestinal epithelial cell-specific CLIC5 knockout as experimental animals, under normal dietary conditions, intestinal epithelial cell-specific CLIC5 knockout can reduce serum endotoxin levels, improve intestinal epithelial barrier function, and increase butyrate production. In experimental mice fed a high-fat diet, intestinal epithelial cell-specific CLIC5 knockout can reduce serum inflammatory factor levels and decrease the gene expression levels of inflammatory factors in the ileum and colon. In a mouse colitis model constructed with dextran sulfate sodium, intestinal epithelial cell-specific CLIC5 knockout can reduce serum inflammatory factor levels. This invention reveals for the first time that intestinal epithelial cell-specific CLIC5 knockout can effectively prevent or treat intestinal inflammation and reduce the level of inflammatory factors in the body, providing a new target and method for the treatment of human intestinal inflammation. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 The results of agarose gel electrophoresis of mice in different groups in Example 1;

[0022] Figure 2 This example illustrates the effect of CLIC5 knockout in intestinal epithelial cells on the expression level of colonic tight junction protein under normal dietary conditions in Example 2. WT NCD represents wild-type mice fed a normal diet, while IKO NCD represents CLIC5 knockout mice in intestinal epithelial cells fed a normal diet. Detailed Implementation

[0023] This invention provides the use of the CLIC5 gene in the preparation of medicaments for the prevention and / or treatment of intestinal inflammation, wherein the nucleotide sequence of the CLIC5 gene is shown in SEQ ID NO:1.

[0024] SEQ ID NO: 1: 5'-atgacggactcagcgacaactaatggggacgacagggaccccgagatcgagctct-3'.

[0025] In one embodiment, the drug is used to specifically knock out or inhibit the expression of the CLIC5 gene in intestinal epithelial cells. After specific knockout or inhibition of the CLIC5 gene expression in intestinal epithelial cells, intestinal inflammation can be effectively prevented or treated, and the level of inflammatory factors in the body can be reduced. In one embodiment, the intestinal inflammation includes I) and / or II): 1) intestinal inflammation caused by a high-fat diet; II) colitis. In one embodiment, the colitis includes colitis caused by sodium dextran sulfate.

[0026] This invention also provides the application of reagents for inhibiting or silencing CLIC5 gene expression in the preparation of drugs for preventing and / or treating intestinal inflammation, wherein the nucleotide sequence of the CLIC5 gene is shown in SEQ ID NO:1. As one embodiment, the prevention and / or treatment of intestinal inflammation includes one or more of the following: reducing serum endotoxin levels, reducing serum pro-inflammatory factor levels, reducing inflammatory factor levels in the ileum, reducing inflammatory factor levels in the colon, improving intestinal epithelial barrier function, and increasing butyrate content in colonic contents. As one embodiment, the reagent for inhibiting or silencing CLIC5 gene expression can be a CLIC5 gene knockout reagent. As one embodiment, the CLIC5 gene knockout reagent can prevent intestinal inflammation in mice. As one embodiment, when the drug is applied to mice, intestinal epithelial cell-specific knockout of the CLIC5 gene can reduce serum endotoxin levels, improve intestinal epithelial barrier function, and increase butyrate content in colonic contents under the premise of feeding a normal diet. As one embodiment, when the drug is applied to a mouse colitis model constructed with dextran sulfate sodium, intestinal epithelial cell-specific knockout of the CLIC5 gene can reduce the level of inflammatory factors in mouse serum. In one embodiment, the intestinal inflammation includes I) and / or II): 1) intestinal inflammation caused by a high-fat diet; II) colitis. In one embodiment, the colitis includes colitis caused by sodium dextran sulfate.

[0027] This invention also provides a medicament for the prevention and / or treatment of intestinal inflammation, comprising a reagent and excipients for inhibiting or silencing CLIC5 gene expression, the nucleotide sequence of which is shown in SEQ ID NO:1. As one embodiment, the medicament comprises a biological product. As one embodiment, the biological product comprises a drug prepared from biological materials such as microorganisms, cells, and various animal and human tissues and fluids obtained through biotechnology such as genetic engineering, cell engineering, protein engineering, and fermentation engineering, for the prevention and / or treatment of intestinal inflammation. As one embodiment, the excipients comprise pharmaceutically acceptable excipients. As one embodiment, the excipients comprise any one or more of excipients, solvents, coating agents, sustained-release agents, powder matrices, and injectable fusible media. As one embodiment, the dosage form of the medicament comprises tablets, injections, capsules, ointments, creams, granules, suppositories, or aerosols. As one embodiment, the intestinal inflammation comprises I) and / or II): 1) intestinal inflammation caused by a high-fat diet; II) colitis. As one embodiment, the colitis comprises colitis caused by sodium dextran sulfate.

[0028] The present invention also provides a biological material for inhibiting or silencing CLIC5 gene expression, wherein the nucleotide sequence of the CLIC5 gene is shown in SEQ ID NO:1. As one embodiment, the biological material comprises an sgRNA that knocks out the CLIC5 gene, wherein the sgRNA is shown in SEQ ID NO:2 and / or SEQ ID NO:3.

[0029] SEQ ID NO: 2: 5'-CTTAGTATAGTAACAGCGGTTGG-3';

[0030] SEQ ID NO: 3: 5'-GTAGATGTTACGACGAGGTAAGG-3'.

[0031] This invention does not specifically limit the method for knocking out the CLIC5 gene; any gene knockout method well-known to those skilled in the art can be used, such as using the Cre / LoxP recombinase system to knock out the CLIC5 gene. Preferably, the CLIC5 gene was constructed by Cyagen (Guangzhou) Biotechnology Co., Ltd. loxp / loxp The transgenic mouse was constructed using a method consistent with the authorized patent ZL202311139530.X. The preferred source of the Villin1-Cre transgenic mouse in this invention is Jackson Laboratory (catalog number 021504). In the embodiments of this invention, Villin1-Cre transgenic mice and CLIC5 are preferably used. loxp / loxp Transgenic mice were mated to obtain CLIC5 intestinal epithelial cell-specific knockout mice.

[0032] To further illustrate the present invention, the application of the CLIC5 gene provided by the present invention in the preparation of drugs for the prevention and / or treatment of intestinal inflammation is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1: Preparation of CLIC5 intestinal epithelial cell-specific knockout mice

[0034] This embodiment refers to the method in patent ZL202311139530.X to construct CLIC5. loxp / loxp Transgenic mice. Specifically, they were constructed by Cyagen (Guangzhou) Biotechnology Co., Ltd., and the construction process is as follows:

[0035] The CLIC5 gene is located on mouse chromosome 17 (accession number: NCBI Reference Sequence: NM_172621; Ensembl: ENSMUSG00000023959). The CLIC5 gene has six exons, with exon 2 selected as the conditional knockout region. Two sgRNAs were designed based on introns 1 and 2. The sgRNA sequences are as follows:

[0036] sgRNA-1:5'-CTTAGTATAGTAACAGCGGTTGG-3' (SEQ ID NO: 2);

[0037] sgRNA-2:5'-GTAGATGTTACGACGAGGTAAGG-3' (SEQ ID NO: 3);

[0038] Villin1-Cre transgenic mice (i.e., Vil1-cre transgenic mice) and CLIC5 loxp / loxp Transgenic mice were mated, and the offspring were genotyped using the mouse tail genome. The primer sequences are as follows:

[0039] loxp-F:5'-CGGAGTCATCCTTTAGTGCCTTAC-3' (SEQ ID NO:4);

[0040] loxp-R:5'-CTGCTCGGCTTCCTTGTCTAATA-3' (SEQ ID NO:5);

[0041] Cre-F:5'-CCATAGGAAGCCAGTTTCCCTTC-3' (SEQ ID NO:6);

[0042] Cre-R:5'-TTCCAGGTATGCTCAGAAAACGC-3' (SEQ ID NO:7);

[0043] After obtaining the transgenic mice, a 0.5 cm long tail tip was cut off to obtain the tail. The tail was added to a lysis buffer containing proteinase K (DirectPCR Lysis Reagent, Viagen, Cat#102-T) and lysed at 55°C for 5-6 hours to obtain a tail lysis buffer containing the genome. PCR detection was performed using the tail lysis buffer.

[0044] Amplification was performed using PCR. The PCR amplification system (10 μL) consisted of: 5 μL 2×PCR reagent; 0.25 μL upstream primer; 0.25 μL downstream primer; 4.1 μL ddH2O; and 0.4 μL rat tail lysis buffer. The PCR reaction conditions were: 95℃ for 5 min; (95℃ for 30 s, 60℃ for 30 s, 72℃ for 45 s), 35 cycles; 72℃ for 5 min, then held at 4℃.

[0045] The PCR products were detected by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown. According to Figure 1It can be seen that numbers 1, 2, 5, 8, 11, 12, 13, 15, 17, 20, and 23 are intestinal epithelial cell-specific knockout CLIC5 mice (IKO), with the genotype Villin1-Cre:CLIC5. loxp / loxp 3, 4, 6, 7, 9, 10, 14, 18, 19, 21, and 22 are wild-type mice (WT) with the CLIC5 genotype. loxp / loxp .

[0046] Example 2: Effects of CLIC5 knockout on intestinal epithelial cells under normal dietary conditions on intestinal inflammation.

[0047] This embodiment uses 8-week-old CLIC5 mice (hereinafter referred to as IKO mice) and wild-type mice (hereinafter referred to as WT mice) that were fed a normal diet, prepared in Example 1. In this embodiment, IKO mice and WT mice were fed a normal diet according to conventional feeding methods, with free access to food. The normal diet is the feed commonly used to feed C57BL / 6 mice and was purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0048] The experimental methods are as follows: IKO and WT mice were fasted for 12 hours before slaughter. Blood was collected from IKO and WT mice by enucleation. The blood was centrifuged at 4500×g for 15 min, and the supernatant was aliquoted and stored at -80℃. Serum pro-inflammatory factors, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), and interleukin-1β (IL-1β), were detected, as well as serum endotoxin LPS. The kits used were purchased from Nanjing Jiancheng Biotechnology Institute (China), and the procedures were performed according to the kit instructions. The results are shown in Table 1.

[0049] Table 1. Effects of CLIC5 knockout on intestinal epithelial cells under normal dietary conditions on serum inflammatory factor and endotoxin levels (n=8)

[0050] project WT Group IKO group p-value Tumor necrosis factor α (TNF-α), pg / mL 58.93±4.86 56.26±4.38 0.311 Interferon-γ (IFN-γ), pg / mL 29.19±2.93 28.22±1.90 0.498 Interleukin-6 (IL-6), pg / mL 135.24±7.04 132.64±9.76 0.572 Interleukin-1β (IL1β), pg / mL 23.83±1.42 22.28±2.99 0.220 Lipopolysaccharide (LPS), EU / mL 0.32±0.04 0.27±0.03 0.025

[0051] Note: n represents the number of samples.

[0052] As shown in Table 1, intestinal epithelial cell-specific knockout of CLIC5 had no effect on the levels of serum pro-inflammatory factors in mice, but significantly reduced the level of serum LPS (Table 1, P = 0.025). Serum LPS is one of the commonly used inflammatory markers in clinical practice, reflecting intestinal permeability.

[0053] Furthermore, after slaughtering IKO and WT mice, four representative samples were randomly selected from each treatment for analysis. Colonic tissue and contents were collected from the mice, flash-frozen in liquid nitrogen, and stored at -80°C for Western blot analysis or short-chain fatty acid analysis. The results are as follows: Figure 2As shown in Tables 2 and 3.

[0054] Table 2. Effects of CLIC5 knockout on colonic tight junction protein expression levels in intestinal epithelial cells under normal dietary conditions (n=4)

[0055] project WT Group IKO group p-value ZO-1 0.40±0.03 0.55±0.10 0.033 Occludin 0.70±0.14 1.14±0.33 0.053 Claudin-1 1.09±0.29 1.09±0.21 0.988

[0056] Note: n represents the number of samples.

[0057] Table 3. Effects of CLIC5 knockout on colonic short-chain fatty acid content under normal dietary conditions (μg / mg, n=8)

[0058] project WT Group IKO group p-value Acetic acid 4.06±0.30 4.05±0.83 0.992 propionic acid 0.88±0.14 0.87±0.14 0.883 butyric acid 0.70±0.22 1.12±0.38 0.039 Isobutyric acid 0.07±0.01 0.06±0.02 0.245 Valeric acid 0.06±0.02 0.06±0.01 0.755 Isovalerate 0.06±0.02 0.04±0.03 0.249

[0059] Note: n represents the number of samples.

[0060] Depend on Figure 2 As shown in Tables 2-3, the Western blot test or short-chain fatty acid analysis was consistent with Table 1. Intestinal epithelial cell-specific knockout of CLIC5 significantly increased the protein expression level of ZO-1 in mouse colon tissue (Table 2, P = 0.033), and the protein expression level of Occludin showed an increasing trend (Table 2, P = 0.053), indicating a significant improvement in intestinal epithelial barrier function. Short-chain fatty acids can promote tight junction protein expression, improve intestinal barrier integrity, and inhibit intestinal inflammatory responses. Further analysis in this example revealed that intestinal epithelial cell knockout of CLIC5 significantly increased the butyrate content in colonic contents (Table 3, P = 0.039).

[0061] In summary, under normal dietary conditions, knocking out CLIC5 in intestinal epithelial cells improves intestinal epithelial barrier function and reduces intestinal inflammation.

[0062] Example 3: The alleviating effect of CLIC5 knockout on intestinal epithelial cells under high-fat diet on intestinal inflammation.

[0063] Eight-week-old CLIC5 mice (hereinafter referred to as IKO mice) and wild-type mice (hereinafter referred to as WT mice), which were fed a normal diet and prepared in Example 1, were used in the experiment. Eight mice were in each group. The eight-week-old IKO and WT mice were fed a high-fat diet (60% fat calories, purchased from Beijing Huafukang Biotechnology Co., Ltd., product number H10060) according to standard feeding methods. The mice had free access to food, and distilled water was provided as their daily drinking water. The feeding period lasted 12 weeks. After the experiment, the mice were fasted for 12 hours. Following the method described in Example 2, they were anesthetized and euthanized, and serum samples were collected for the detection of serum pro-inflammatory factors TNF-α, IFN-γ, and IL-6.

[0064] Ileal and colonic tissues were collected, and the mRNA expression levels of TNF-α, IL6, IL1β, and IL10 were detected using qRT-PCR. Primer sequences are shown in Table 4. The qRT-PCR amplification system (10 μL) consisted of: 2×TaqPCR mastermix: 5 μL; forward primer: 0.3 μL; reverse primer: 0.3 μL; cDNA: 4 μL; ddH2O: 0.4 μL. The qRT-PCR reaction conditions were: 95℃ for 5 min, 95℃ for 15 s, annealing at (as shown in Table 4) for 15 s, 72℃ for 15 s, 2–4: 35 cycles, held at 4℃.

[0065] Table 4 Primer sequences for qRT-PCR (5'-3')

[0066]

[0067] Table 5. Effects of CLIC5 knockout on serum inflammatory cytokine levels in intestinal epithelial cells under high-fat diet conditions (n=8)

[0068] project WT Group IKO group p-value Tumor necrosis factor α (TNF-α), pg / mL 49.86±4.27 38.97±2.71 <0.001 Interferon-γ (IFN-γ), pg / mL 37.25±2.07 30.70±3.36 <0.001 Interleukin-6 (IL-6), pg / mL 149.12±3.89 133.03±17.84 0.037

[0069] Note: n represents the number of samples.

[0070] As shown in Table 5, under a high-fat diet, compared with wild-type mice, knockout of CLIC5 in intestinal epithelial cells significantly reduced the levels of serum TNF-α (P<0.001), IFN-γ (P<0.001), and IL6 (P=0.037).

[0071] Table 6. Effects of CLIC5 knockout on intestinal inflammatory gene expression levels in intestinal epithelial cells under high-fat diet conditions (n=8)

[0072]

[0073] Note: n represents the number of samples.

[0074] As shown in Table 6, qRT-PCR results indicated that knocking out CLIC5 in intestinal epithelial cells significantly reduced the mRNA expression level of IL6 in the ileum and colon of mice (P = 0.001 and 0.007, respectively).

[0075] Example 4: The alleviating effect of CLIC5 knockout in intestinal epithelial cells on the inflammatory response in a colitis model.

[0076] Eight-week-old IKO and WT mice, prepared as described in Example 1, were used in each group (n=8 per group). The mice's drinking water was replaced with a prepared 2.5% sodium dextran sulfate (DSS) solution, and they were fed the standard diet as described in Example 2, with free access to food. A colitis model was established. The sterile water supplemented with DSS was changed every two days for eight days. On day 9, blood was collected from the mice using enucleation, and serum was collected for the detection of serum pro-inflammatory factors TNF-α, IFN-γ, IL6, and IL1β. The results are shown in Table 7.

[0077] Table 7. Effects of CLIC5 knockout in intestinal epithelial cells on serum inflammatory factor levels in a colitis model (n=8)

[0078] project WT Group IKO group p-value Tumor necrosis factor α (TNF-α), pg / mL 71.49±7.84 49.33±5.09 <0.001 Interferon-γ (IFN-γ), pg / mL 40.74±3.30 28.67±3.48 <0.001 Interleukin-6 (IL-6), pg / mL 154.40±10.30 126.73±8.84 <0.001 Interleukin-1β (IL1β), pg / mL 31.78±2.68 24.33±2.47 <0.001

[0079] Note: n represents the number of samples.

[0080] As shown in Table 7, knocking out CLIC5 in intestinal epithelial cells significantly reduced the level of serum pro-inflammatory factors in a mouse colitis model (P<0.001).

[0081] In conclusion, intestinal epithelial cell-specific knockout of CLIC5 can enhance intestinal epithelial barrier function, reduce intestinal inflammation, and improve the overall inflammatory response of the body. CLIC5 has potential target roles in the prevention or treatment of intestinal inflammation.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of an agent for inhibiting or silencing CLIC5 gene expression in the preparation of a medicament for the prevention and / or treatment of intestinal inflammation, wherein the nucleotide sequence of the CLIC5 gene is shown in SEQ ID NO:1; The reagent used to inhibit or silence CLIC5 gene expression is sgRNA, and the sgRNA is SEQ ID NO:2 and SEQ ID NO:3; The intestinal inflammation mentioned is colitis.

2. The application as described in claim 1, characterized in that, The colitis mentioned is colitis caused by sodium dextran sulfate.

3. The application as described in claim 1, characterized in that, The prevention and / or treatment of intestinal inflammation includes one or more of the following: reducing serum endotoxin levels, reducing serum pro-inflammatory factor levels, reducing inflammatory factor levels in the ileum, reducing inflammatory factor levels in the colon, improving intestinal epithelial barrier function, and increasing butyrate content in colonic contents.

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