Application of indole-3-acetaldehyde and its salt in products for repairing intestinal damage in animals

Abstract

This invention discloses the application of indole-3-acetaldehyde and its salts in products for repairing intestinal damage in animals, relating to the technical field of repairing intestinal damage in animals, thereby improving intestinal damage induced by vomitoxin. Animal experiments have demonstrated that treatment with products containing indole-3-acetaldehyde significantly reduces the degree of vomitoxin-induced intestinal damage, significantly increases the activity of intestinal stem cells, improves intestinal villus height and permeability, promotes intestinal stem cell proliferation and differentiation, and enhances the proliferation level of intestinal epithelial cells. This invention provides new ideas for the research and development of products for repairing vomitoxin-induced intestinal damage and contributes to the development of new nutritional regulation methods for treating vomitoxin-induced intestinal damage.

Description

Technical Field

[0001] This invention relates to the technical field of repairing intestinal damage in animals, and more particularly to the application of indole-3-acetaldehyde and its salt in products for repairing intestinal damage in animals. Background Technology

[0002] Deoxynivalenol (DON) is a mycotoxin synthesized by Fusarium wilt bacteria. It is a common contaminant in grains and feed in my country, widely found in wheat, barley, corn, and other grains, seriously endangering food and feed safety and human and animal health. After oral ingestion, DON enters the intestines, which are the primary organ affected by it. Within the intestines, DON toxicity leads to a series of adverse consequences, including damage to intestinal stem cells, which play a crucial role in the proliferation and differentiation of intestinal epithelial cells. Repairing DON-induced intestinal stem cell damage is an effective way to reduce the harm caused by DON. Current strategies for controlling DON mainly include using antifungal agents and biodegradable enzymes at the raw material level, and blocking or reducing the damage to the body through nutritional strategies at the end. It is evident that while existing technologies reduce DON intake or nutritional supplementation to decrease its impact on animals, they cannot effectively repair DON-induced intestinal stem cell damage, and current technologies have not yet investigated how to effectively repair DON-induced intestinal stem cell damage.

[0003] In response to the problem of the inability to effectively repair intestinal damage in animals caused by vomitoxin, there is an urgent need to discover a safe and effective ingredient that can be used in products that repair vomitoxin-induced intestinal damage. Summary of the Invention

[0004] The purpose of this invention is to provide an application of indole-3-acetaldehyde and its salt in products for repairing intestinal damage in animals, thereby improving intestinal damage in animals.

[0005] To achieve the above objectives, the present invention provides the application of indole-3-acetaldehyde and its salt in products for repairing intestinal damage in animals.

[0006] Compared with existing technologies, this invention discovers a novel application of indole-3-acetaldehyde and its salts, which can be used in products that repair vomitoxin-induced intestinal damage. Animal experiments have demonstrated that treatment with products containing indole-3-acetaldehyde and its salts significantly reduces the degree of vomitoxin-induced intestinal damage, significantly increases intestinal stem cell activity, improves intestinal villus height and permeability, promotes intestinal stem cell proliferation and differentiation, and enhances intestinal epithelial cell proliferation. This invention provides new insights for the research and development of products that repair vomitoxin-induced intestinal damage and contributes to the development of new nutritional regulation methods for treating vomitoxin-induced intestinal damage.

[0007] Furthermore, it also includes the use of the combination of indole-3-acetaldehyde and its salt with taurine in products for repairing intestinal damage in animals.

[0008] In this invention, the synergistic effect of indole-3-acetaldehyde or a pharmaceutically acceptable salt of indole-3-acetaldehyde with taurine bile acid in the product is more beneficial for repairing intestinal damage caused by vomiting toxins.

[0009] Furthermore, based on the product weight, the effective amount of indole-3-acetaldehyde and its salt added to the product is 60-120 mg / kg, used to repair intestinal damage in animals.

[0010] Furthermore, the effective dose of the indole-3-acetaldehyde and its salt in animals is 15-30 mg / kg, based on the weight of the animals being raised.

[0011] Furthermore, the mass ratio of indole-3-acetaldehyde and its salt to taurine cholic acid is 1:0.5-1.

[0012] Furthermore, the repair of animal intestinal damage includes at least one of increasing the height of intestinal villi, reducing intestinal permeability, increasing the activity of intestinal stem cells, promoting the proliferation and differentiation of intestinal stem cells, and increasing the proliferation level of intestinal epithelial cells.

[0013] Furthermore, the reduction in intestinal permeability includes reducing the animal's serum D-lactic acid levels.

[0014] Furthermore, the enhancement of intestinal stem cell activity includes increasing the Lgr5 expression level and the number of intestinal stem cells in animal jejunal stem cells.

[0015] Furthermore, the promotion of intestinal stem cell proliferation and differentiation includes at least one of the following:

[0016] Increase the expression level of the MUC2 gene and the number of villi goblet cells in animal jejunal goblet cells;

[0017] Increase the expression level of LYZ, a marker protein of Paneth cells in the jejunal crypts of animals, and increase the number of Paneth cells;

[0018] Increase the expression level of the ChgA gene in animal intestinal endocrine cells.

[0019] Furthermore, the improvement in intestinal epithelial cell proliferation includes increasing the Ki67 expression level and the number of intestinal proliferating cells in animal jejunal epithelial proliferating cells.

[0020] Furthermore, the product uses indole-3-acetaldehyde and its salt as additives, or a combination of indole-3-acetaldehyde and its salt with taurine cholic acid as additives, to prepare a pharmaceutical preparation, animal health product, or functional feed for repairing intestinal damage in animals.

[0021] Compared with existing technologies, indole-3-acetaldehyde and its salts, or a combination of indole-3-acetaldehyde and its salts with taurine cholic acid, can be used to formulate pharmaceutical preparations, animal health products, or functional feeds with specific effects, facilitating the transportation, storage, and use of the products. Attached Figure Description

[0022] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1(A) is an HE staining image of mouse jejunal tissue in Example 1; Figure 1(B) is a statistical graph of villus height and crypt depth of mice in Example 1.

[0024] Figure 2 This is a statistical chart showing the serum D-lactic acid levels of mice in the control group, the vomitoxin group, and the combined indole-3-acetaldehyde and vomitoxin group in Example 1.

[0025] Figure 3(A) is an immunostaining image of Lgr5, a marker of mouse jejunal stem cells, in Example 1. Figure 3(B) is the ratio of the number of intestinal stem cells to the number of intestinal crypts in mice in Example 1.

[0026] Figure 4(A) is an immunostaining image of Ki67, a marker of proliferating cells in the mouse jejunum, in Example 1. Figure 4(B) is the ratio of the number of proliferating epithelial cells in the mouse jejunum to the number of crypts in Example 1.

[0027] Figure 5(A) is a picture of mouse jejunum after AB / PAS staining in Example 1; Figure 5(B) is the ratio of the number of goblet cells to the number of villi in mouse jejunum in Example 1.

[0028] Figure 6(A) is an immunostaining image of LYZ markers in the mouse jejunum Panthen cells in Example 1; Figure 6(B) is the ratio of the number of Panthen cells in the mouse jejunal crypts to the number of villi in Example 1.

[0029] Figure 7 This is a statistical graph showing the relative mRNA expression levels of the jejunal goblet cell marker gene MUC2, the Paneth cell marker gene LYZ, and the enteroendocrine cell marker gene ChgA in mice in Example 1.

[0030] Figure 8(A) is an HE staining image of mouse jejunal tissue in Example 2; Figure 8(B) is a statistical graph of villus height and crypt depth of mice in Example 2.

[0031] Figure 9(A) is a statistical graph of the relative mRNA expression level of the Lgr5 gene in mice in Example 2; Figure 9(B) is an immunostaining graph of Lgr5, a marker of mouse jejunal stem cells, in Example 2.

[0032] Figure 10(A) is an immunostaining image of Ki67, a marker of proliferating cells in the mouse jejunum, in Example 2; Figure 10(B) is the ratio of the number of proliferating epithelial cells in the mouse jejunum to the number of intestinal crypts in Example 2.

[0033] Figure 11(A) is an immunostaining image of LYZ markers in the mouse jejunum Panthen cells in Example 2, and Figure 11(B) is the ratio of the number of Panthen cells in the mouse jejunum crypts to the number of crypts in Example 2.

[0034] Figure 12 This is a microscope image of the organoid in Example 3.

[0035] Figure 13 This is a statistical chart of the average number of buds produced by organoids in Example 3.

[0036] Figure 14 This is a statistical graph of the organoid surface area in Example 3.

[0037] Figure 15 This is a statistical graph showing the relative mRNA expression level of the Lgr5 gene in Example 3.

[0038] Figure 16 This is an immunostaining image of LYZ, a marker for Paneth cells in intestinal organoids, from Example 3.

[0039] Figure 17 This is a statistical graph showing the relative mRNA expression level of the Lgr5 gene in MODE-K cells.

[0040] Figure 18 These are fluorescence micrographs of stained MODE-K cells.

[0041] Figure 19(A) is an HE staining image of mouse jejunal tissue in Example 5; Figure 19(B) is a statistical graph of villus height and crypt depth of mice in Example 5.

[0042] Figure 20 This is a statistical graph showing the relative mRNA expression levels of the Lgr5 gene in mice in Example 5.

[0043] Figure 21 This is an immunostaining image of Ki67, a marker of proliferating cells in mice, from Example 5.

[0044] Figure 22 This is a statistical graph showing the ratio of the number of proliferating epithelial cells in the mouse jejunum to the number of intestinal crypts in Example 5.

[0045] Figure 23 This is a statistical graph showing the relative mRNA expression levels of the jejunal goblet cell marker gene MUC2 and the Paneth cell marker gene LYZ in mice in Example 5. Detailed Implementation

[0046] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] This invention provides the application of indole-3-acetaldehyde and its salt in products for repairing intestinal damage in animals.

[0048] This invention discovers a novel application of indole-3-acetaldehyde and its salts, which can be used in products for repairing vomitoxin-induced intestinal damage. More specifically, this invention relates to the use of indole-3-acetaldehyde or its pharmaceutically acceptable salts in products for repairing vomitoxin-induced intestinal damage in animals.

[0049] The inventors discovered that when intestinal epithelial cells absorb large amounts of vomitoxin, it triggers ribosome damage, significantly inhibiting cell proliferation and differentiation, reducing the synthesis of intestinal tight junction proteins, disrupting the intestinal barrier, and increasing intestinal permeability. This further leads to infections caused by large amounts of vomitoxin and foreign bacteria and viruses, damaging the animal's body. Current research has investigated the effects of indole derivatives on improving animal health, intestinal stem cells, and growth performance under stable conditions; however, it has not addressed whether indole derivatives repair vomitoxin-induced infection damage to the animal matrix, such as vomitoxin-induced intestinal damage and intestinal stem cell damage, or how they repair such damage.

[0050] This invention demonstrates through animal experiments that treatment with products containing indole-3-acetaldehyde and its salts can significantly reduce the degree of vomitoxin-induced intestinal damage, significantly increase the activity of intestinal stem cells, improve intestinal villus height and permeability, promote intestinal stem cell proliferation and differentiation, and enhance the proliferation level of intestinal epithelial cells. This invention provides new insights for the research and development of products for repairing vomitoxin-induced intestinal damage and contributes to the development of new nutritional regulation methods for treating vomitoxin-induced intestinal damage.

[0051] Furthermore, the present invention also includes the use of the combination of indole-3-acetaldehyde and its salt with taurine in products for repairing intestinal damage in animals. In the present invention, the taurine is selected from at least one of taurine-β-cholic acid, taurine-α-cholic acid, and taurine-ω-cholic acid, preferably taurine-β-cholic acid.

[0052] In this invention, the synergistic effect of indole-3-acetaldehyde or a pharmaceutically acceptable salt of indole-3-acetaldehyde with taurine bile acid in the product is more beneficial for repairing intestinal damage caused by vomiting toxins.

[0053] In some embodiments, the effective amount of indole-3-acetaldehyde and its salt in the product, based on the product weight, is 60-120 mg / kg, for repairing intestinal damage in animals. For example, the effective amount of indole-3-acetaldehyde and its salt in the product, based on the product weight, is 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg, or 120 mg / kg.

[0054] In some embodiments, the effective dose of indole-3-acetaldehyde and its salt in animals is 15-30 mg / kg based on the weight of the raised animals. For example, the effective dose of indole-3-acetaldehyde and its salt in animals is 15 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg, 22 mg / kg, 24 mg / kg, 26 mg / kg, 28 mg / kg, or 30 mg / kg based on the weight of the raised animals.

[0055] In this invention, when the above-mentioned product contains the specific effective amount of indole-3-acetaldehyde, or when the amount of indole-3-acetaldehyde meets the above-mentioned scheme based on animal weight, it is more beneficial to repair vomitoxin-induced intestinal damage in animals. Preferably, based on the weight of the raised animals, the effective dose of indole-3-acetaldehyde and its salt in animals is 18-23 mg / kg.

[0056] In some embodiments, the mass ratio of indole-3-acetaldehyde and its salt to taurine cholic acid is 1:0.5-1.

[0057] In this invention, when the mass ratio of indole-3-acetaldehyde and its salt to taurine cholic acid in the product meets the above-mentioned range, it is more beneficial for repairing vomitoxin-induced intestinal damage in animals. For example, the mass ratio of indole-3-acetaldehyde and its salt to taurine cholic acid is 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, or 1:1. Preferably, the mass ratio of indole-3-acetaldehyde and its salt to taurine cholic acid is 1:0.8-1.

[0058] In some embodiments, repairing intestinal damage in animals includes at least one of increasing intestinal villus height, reducing intestinal permeability, increasing intestinal stem cell activity, promoting intestinal stem cell proliferation and differentiation, and increasing the proliferation level of intestinal epithelial cells.

[0059] Furthermore, reducing intestinal permeability includes lowering serum D-lactate levels in animals. In this invention, serum D-lactate levels reflect the permeability of an animal's intestines. Low serum D-lactate levels indicate low intestinal permeability and a relatively intact intestinal barrier. When animals ingest excessive amounts of vomitoxin due to feed contamination, intestinal permeability increases, leading to a significant rise in serum D-lactate levels. Feeding animals products containing indole-3-acetaldehyde and its salts significantly reduces serum D-lactate levels, indicating that treatment with indole-3-acetaldehyde and its salts reduces intestinal permeability and restores a more intact intestinal barrier.

[0060] Furthermore, enhancing intestinal stem cell activity includes increasing the expression level of Lgr5 in animal jejunal stem cells and the number of intestinal stem cells. In this invention, the expression level of the Lgr5 marker protein and the number of jejunal stem cells in animals induced by vomitoxin were significantly reduced, while indole-3-acetaldehyde and its salt could increase the expression level of Lgr5 and the number of stem cells in animals induced by vomitoxin. This indicates that feeding animals with products containing indole-3-acetaldehyde and its salt can effectively repair vomitoxin-induced intestinal damage and enhance the activity of animal intestinal stem cells.

[0061] Furthermore, promoting the proliferation and differentiation of intestinal stem cells includes at least one of the following:

[0062] Increase the expression level of the MUC2 gene and the number of villi goblet cells in animal jejunal goblet cells;

[0063] Increase the expression level of LYZ, a marker protein of Paneth cells in the jejunal crypts of animals, and increase the number of Paneth cells;

[0064] Increase the expression level of the ChgA gene in animal intestinal endocrine cells.

[0065] In this invention, intestinal stem cells can differentiate into different types of intestinal epithelial cells, such as goblet cells, Paneth cells, and enteroendocrine cells. When animals ingest large amounts of vomitoxin, it leads to a decrease in the number of vomitoxin goblet cells and the expression level of the MUC2 gene, a decrease in the number of Paneth cells and the expression level of the LYZ gene, or a decrease in the expression level of the ChgA gene. However, by feeding products containing indole-3-acetaldehyde and its salts, it is possible to increase the number of vomitoxin goblet cells and the expression level of the MUC2 gene; increase the number of Paneth cells and the expression level of the LYZ gene; and increase the expression level of the ChgA gene, thereby repairing vomitoxin-induced intestinal damage in animals and promoting the proliferation of intestinal stem cells.

[0066] Furthermore, enhancing intestinal epithelial cell proliferation includes increasing the Ki67 expression level and the number of proliferating intestinal epithelial cells in animals. In this invention, the expression level of the Ki67 marker protein and the number of proliferating cells in the jejunal epithelial cells of animals induced by vomitoxin were significantly reduced, while indole-3-acetaldehyde and its salt could increase the Ki67 expression level and the number of proliferating cells after vomitoxin induction, indicating that indole-3-acetaldehyde and its salt can effectively repair vomitoxin-induced intestinal damage in animals, enhance intestinal stem cell activity, and increase the proliferation level of intestinal epithelial cells.

[0067] In some embodiments, the product uses indole-3-acetaldehyde and its salts as additives, or a combination of indole-3-acetaldehyde and its salts with taurine as an additive, to formulate a pharmaceutical preparation, animal health product, or functional feed for repairing intestinal damage in animals. In this invention, when indole-3-acetaldehyde and its salts are used as additives, or when a combination of indole-3-acetaldehyde and its salts with taurine is used as an additive, it can be formulated into a pharmaceutical preparation, animal health product, or functional feed with specific effects, facilitating the transportation, storage, and use of the product.

[0068] In the following examples and comparative examples, vomitoxin was purchased from Sigma-Aldrich, and indole-3-acetaldehyde was purchased from Jizhi Biochemical. Other raw materials, unless otherwise specified, are commercially available.

[0069] Example 1: Effect of indole-3-acetaldehyde on vomitoxin-induced intestinal stem cell damage in mice

[0070] 1.1 Feeding and Management of Experimental Animals

[0071] Eighteen 6-8 week old C57BL / 6J mice (weighing 17-18g) were randomly divided into three treatment groups: a control group, a vomitoxin group, and a combined indole-3-acetaldehyde and vomitoxin group, with six mice in each group. All mice in the three treatment groups were housed at a temperature of 22-25℃ and humidity of 40-50%, with free access to food and water. The control group mice were administered phosphate-buffered saline (PBS) by gavage daily; the vomitoxin group mice were administered 2 mg / kg vomitoxin (400 μg / mL) by gavage daily; and the combined indole-3-acetaldehyde and vomitoxin group mice were administered 20 mg / kg indole-3-acetaldehyde (4 μg / μL) and 2 mg / kg vomitoxin by gavage daily. The experiment lasted 14 days, and all mice were sacrificed on the morning of day 14.

[0072] 1.2 Analysis of jejunal morphology

[0073] After euthanizing mice, jejunal tissue was collected and fixed in 4 wt% paraformaldehyde for 30 minutes, then embedded in paraffin and sectioned for HE staining. The stained sections were photographed under a microscope, and villus height and crypt depth were measured using ImageJ software.

[0074] 1.3 Intestinal permeability measurement

[0075] Blood was collected after the mice were euthanized, and serum was obtained by centrifugation at 3000 rpm for 15 minutes. The D-lactic acid level in the serum was measured using an ELISA kit to reflect the intestinal permeability of the mice.

[0076] 1.4 Assay of intestinal stem cell activity

[0077] Mouse jejunal paraffin blocks were sectioned, and the protein expression level of the stem cell marker protein Lgr5 was detected using immunofluorescence and Lgr5 antibody (Origene).

[0078] 1.5 Measurement of intestinal epithelial proliferation level

[0079] Mouse jejunal paraffin blocks were sectioned, and the protein expression level of the proliferating cell marker protein Ki67 was detected using immunofluorescence and Ki67 antibody (Proteintech).

[0080] 1.6 Determination of intestinal stem cell differentiation level

[0081] Mouse jejunal paraffin sections were sectioned, and the number of goblet cells was detected using AB / PAS staining. The protein expression level of the Paneth cell marker protein LYZ was detected using immunostaining and LYZ antibody (Abcam). The expression levels of the goblet cell marker gene MUC2, the Paneth cell marker gene LYZ, and the enteroendocrine cell marker gene ChgA were detected using quantitative real-time PCR. The primer sequences for each gene are shown in Table 1.

[0082] Table 1

[0083]

[0084] 1.7 Statistical Methods

[0085] One-way ANOVA was used in GraphPad Prism 8.0.1 software to compare differences between different treatment groups. Tukey's method was used for multiple comparisons. Different lowercase letters in the superscript of the same row indicate significant differences (P < 0.05).

[0086] 1.8 Test Results

[0087] The results of mouse jejunal morphology analysis are shown in Figures 1(A) and 1(B). Figure 1(A) is an HE staining image of mouse jejunal tissue, showing that the villus height in the indole-3-acetaldehyde combined with vomitoxin group was higher than that in the vomitoxin group. Figure 1(B) is a statistical graph of villus height and crypt depth in mice; the villus heights in the control group, vomitoxin group, and indole-3-acetaldehyde combined with vomitoxin group were 257 μm, 162 μm, and 206 μm, respectively, and the crypt depths were 93 μm, 87 μm, and 92 μm, respectively. There was no significant difference in crypt depth among the three treatment groups, but the villus height in the vomitoxin group was significantly lower than that in the control group and the indole-3-acetaldehyde combined with vomitoxin group, indicating that vomitoxin can induce jejunal injury in mice. The villus height in the indole-3-acetaldehyde combined with vomitoxin group was significantly higher than that in the vomitoxin group, indicating that indole-3-acetaldehyde can improve vomitoxin-induced jejunal injury in mice.

[0088] Intestinal permeability test results as follows Figure 2 As shown. Figure 2This is a statistical graph showing the serum D-lactate levels in mice from three treatment groups. The serum D-lactate levels in the control group, the vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group were 0.78 μmol / mL, 1.56 μmol / mL, and 1.01 μmol / mL, respectively. The serum D-lactate level in the vomitoxin group was higher than that in the control group, indicating that the intestinal barrier of the mice in the vomitoxin group was damaged under the influence of vomitoxin, leading to increased intestinal permeability and elevated serum D-lactate levels. The serum D-lactate level in the indole-3-acetaldehyde combined with vomitoxin group was significantly lower than that in the vomitoxin group, indicating that indole-3-acetaldehyde can repair vomitoxin-induced intestinal damage in mice, improve intestinal permeability, and enhance the intestinal barrier.

[0089] The results of the intestinal stem cell activity test are shown in Figures 3(A) and 3(B). Figure 3(A) is an immunostaining diagram of the intestinal stem cell marker Lgr5. It can be seen that the number of positive cells (black arrows) in the vomitoxin group was less than that in the indole-3-acetaldehyde and vomitoxin combined group, and the expression level of the jejunal stem cell marker protein Lgr5 was decreased in the vomitoxin group. Figure 3(B) shows the ratio of the number of intestinal stem cells to the number of intestinal crypts in the three treatment groups. The ratios of the number of intestinal stem cells to the number of intestinal crypts in the control group, the vomitoxin group, and the indole-3-acetaldehyde and vomitoxin combined group were 2.53, 0.96, and 1.87, respectively.

[0090] The expression level of the jejunal stem cell marker protein Lgr5 and the number of stem cells in the vomitoxin group were significantly lower than those in the control group, indicating that vomitoxin reduced the activity of jejunal stem cells in mice. In contrast, the expression level of Lgr5 and the number of stem cells in the indole-3-acetaldehyde combined with vomitoxin group were significantly higher than those in the vomitoxin group, indicating that indole-3-acetaldehyde can improve the problem of reduced intestinal stem cell activity caused by vomitoxin.

[0091] The results of the intestinal epithelial proliferation level assay are shown in Figures 4(A) and (B). Figure 4(A) is an immunostaining image of the proliferation marker Ki67. It can be seen that the number of epithelial proliferating cells (Ki67-positive cells) in the control group and the indole-3-acetaldehyde combined with vomitoxin group is greater than that in the vomitoxin group. Figure 4(B) is the ratio of the number of proliferating cells to the number of crypts in the mouse jejunal epithelium. The ratios of the number of proliferating cells to the number of intestinal crypts in the control group, the vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group are 38.66, 18.55, and 30.24, respectively. Vomitoxin can reduce the expression level of the proliferating cell marker protein Ki67 and the number of proliferating cells in the mouse jejunal epithelium, while indole-3-acetaldehyde can increase the expression level of Ki67 and the number of proliferating cells induced by vomitoxin, indicating that indole-3-acetaldehyde can improve the problem of reduced intestinal epithelial proliferation caused by vomitoxin.

[0092] Intestinal stem cells can differentiate into different types of intestinal epithelial cells, such as goblet cells, Paneth cells, and enteroendocrine cells. The results of intestinal stem cell differentiation level testing are shown in Figures 5(A), 5(B), 6(A), and 6(B). Figure 7 As shown in Figure 5(A), the mouse jejunum was stained with AB / PAS. It can be seen that the number of goblet cells in the control group and the combined indole-3-acetaldehyde and vomitoxin group was greater than that in the vomitoxin group. Figure 5(B) shows the ratio of goblet cells to villi in the mouse jejunum. The ratios for the control group, the vomitoxin group, and the combined indole-3-acetaldehyde and vomitoxin group were 17.68, 10.69, and 15.69, respectively. Vomitoxin significantly reduced the number of goblet cells in the mouse jejunum, while indole-3-acetaldehyde increased the number of goblet cells, indicating that indole-3-acetaldehyde can alleviate the effect of vomitoxin on intestinal stem cell differentiation.

[0093] Figure 6(A) shows the immunostaining pattern of the Panthen cell marker LYZ. It can be seen that the number of Panthen cells in the crypts of the control group and the indole-3-acetaldehyde combined with vomitoxin group was greater than that of the vomitoxin group. Figure 6(B) shows the ratio of the number of Panthen cells to the number of villi in the jejunal crypts of mice. The ratios for the control group, the vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group were 4.53, 2.06, and 3.47, respectively. Vomitoxin significantly reduced the expression level of the Panthen cell marker protein LYZ and the number of Panthen cells in the jejunal crypts of mice, while indole-3-acetaldehyde significantly increased the expression level of the Panthen cell marker protein LYZ and the number of Panthen cells induced by vomitoxin in mice. This indicates that indole-3-acetaldehyde can alleviate the effect of vomitoxin on intestinal stem cell differentiation.

[0094] Figure 7 This is a statistical graph showing the relative mRNA expression levels of the jejunal goblet cell marker gene MUC2, the Paneth cell marker gene LYZ, and the enteroendocrine cell marker gene ChgA in mice from three treatment groups. The relative expression levels of the MUC2 gene in the control group, the vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group were 1.01, 0.67, and 1.05, respectively; the relative expression levels of the LYZ gene were 1.05, 0.41, and 0.75, respectively; and the relative expression levels of the ChgA gene were 1.02, 0.49, and 0.88, respectively. Vomitoxin significantly reduced the expression levels of the jejunal goblet cell marker genes MUC2, Paneth cell marker gene LYZ, and enteroendocrine cell marker gene ChgA in mice, while indole-3-acetaldehyde increased the expression levels of MUC2, LYZ, and ChgA genes after vomitoxin induction, indicating that indole-3-acetaldehyde can alleviate the effect of vomitoxin on intestinal stem cell differentiation.

[0095] The results of Example 1 show that indole-3-acetaldehyde can effectively alleviate vomitoxin-induced intestinal damage in mice, increase the height of intestinal villi, reduce intestinal permeability, enhance the activity of intestinal stem cells, promote the proliferation and differentiation of intestinal stem cells, and increase the proliferation level of intestinal epithelial cells.

[0096] Example 2: Comparison of the effects of indole-3-acetaldehyde and indole-3-aldehyde in alleviating vomitoxin-induced intestinal stem cell damage

[0097] 2.1 Feeding and Management of Experimental Animals

[0098] Thirty-two 6-8 week old C57BL / 6J mice (weighing 17-18g) were randomly divided into four treatment groups: control group, vomitoxin group, indole-3-aldehyde and vomitoxin combination group, and indole-3-acetaldehyde and vomitoxin combination group, with eight mice in each group. All mice in the four treatment groups were housed at a temperature of 22-25℃ and humidity of 40-50%, with free access to food and water. The control group mice were administered PBS by gavage daily; the vomitoxin group mice were administered 2 mg / kg vomitoxin (400 μg / mL) by gavage daily; the indole-3-aldehyde and vomitoxin combination group mice were administered 20 mg / kg indole-3-aldehyde (4 μg / μL, Jizhi Biochemical) and 2 mg / kg vomitoxin by gavage daily; and the indole-3-acetaldehyde and vomitoxin combination group mice were administered 20 mg / kg indole-3-acetaldehyde (4 μg / μL) and 2 mg / kg vomitoxin by gavage daily. The formal experiment lasted for 7 days, and all mice were sacrificed on the morning of the 7th day.

[0099] 2.2 Analysis of jejunal morphology

[0100] The test was performed according to the method in 1.2 of Example 1.

[0101] 2.3 Assay of intestinal stem cell activity

[0102] The expression level of Lgr5, a marker gene for mouse jejunal stem cells, was detected using quantitative real-time PCR. Primer sequences for Lgr5 and β-actin are shown in Table 2. Mouse jejunal paraffin sections were prepared, and the protein expression level of the stem cell marker protein Lgr5 was detected using immunofluorescence and an Lgr5 antibody (Origene).

[0103] Table 2

[0104]

[0105] 2.4 Measurement of intestinal epithelial proliferation level

[0106] The test was conducted according to the method described in 1.5 of Example 1.

[0107] 2.5 Determination of intestinal stem cell differentiation level

[0108] Mouse jejunal paraffin blocks were sectioned, and the protein expression level of Paneth cell marker protein LYZ was detected using immunostaining techniques and LYZ antibody (Abcam).

[0109] 2.6 Statistical Methods

[0110] One-way ANOVA was used in GraphPad Prism 8.0.1 software to compare differences between different treatment groups. Tukey's method was used for multiple comparisons. Different lowercase letters in the superscript of the same row indicate significant differences (P < 0.05).

[0111] 2.7 Test Results

[0112] The morphological analysis results of the jejunum are shown in Figures 8(A) and 8(B). Figure 8(A) is an HE staining image of mouse jejunal tissue, showing that the villus height of mice in the indole-3-acetaldehyde and vomitoxin combination group was higher than that in the indole-3-aldehyde and vomitoxin combination group. Figure 8(B) is a statistical graph of villus height and crypt depth of mice in the four treatment groups. The villus height of mice in the control group, vomitoxin group, indole-3-aldehyde and vomitoxin combination group, and indole-3-acetaldehyde and vomitoxin combination group were 261 μm, 140 μm, 154 μm, and 200 μm, respectively, and the crypt depths were 101 μm, 85 μm, 92 μm, and 96 μm, respectively. There was no significant difference in crypt depth among the four treatment groups. There was no significant difference in villus height among the vomitoxin group and the indole-3-aldehyde combined with vomitoxin group. However, the villus height of mice in the indole-3-acetaldehyde combined with vomitoxin group was significantly higher than that in the indole-3-aldehyde combined with vomitoxin group. This indicates that indole-3-aldehyde did not improve jejunal injury in mice induced by vomitoxin, while indole-3-acetaldehyde could improve jejunal injury in mice.

[0113] The results of the intestinal stem cell activity test are shown in Figures 9(A) and 9(B). Figure 9(A) is a statistical graph of the relative mRNA expression level of the Lgr5 gene. The relative expression levels of the Lgr5 gene in the control group, the vomitoxin group, the indole-3-aldehyde and vomitoxin combination group, and the indole-3-acetaldehyde and vomitoxin combination group were 1.00, 0.45, 0.55, and 0.87, respectively. Figure 9(B) is an immunostaining graph of the stem cell marker Lgr5. It can be seen that the number of positive cells (black arrows) in the indole-3-acetaldehyde and vomitoxin combination group was higher than that in the vomitoxin group and the indole-3-aldehyde and vomitoxin combination group. This indicates that indole-3-aldehyde has no significant effect on the expression levels of the Lgr5 gene and protein, a marker of mouse jejunal stem cells, in mice induced by vomitoxin; however, indole-3-acetaldehyde can increase the expression levels of both the Lgr5 gene and protein. Obviously, indole-3-acetaldehyde can improve the problem of reduced intestinal stem cell activity caused by vomitoxin, while indole-3-aldehyde cannot solve this problem.

[0114] The results of intestinal epithelial proliferation are shown in Figures 10(A) and 10(B). Figure 10(A) is an immunostaining image of the proliferation marker Ki67. It can be seen that the number of epithelial proliferating cells (Ki67-positive cells) in the indole-3-acetaldehyde and vomitoxin combined group was higher than that in the vomitoxin group and the indole-3-acetaldehyde and vomitoxin combined group. Figure 10(B) shows the ratio of the number of proliferating cells in the jejunal epithelium to the number of intestinal crypts in mice. The ratios of the number of proliferating cells in the jejunal epithelium to the number of intestinal crypts in the control group, the vomitoxin group, the indole-3-acetaldehyde and vomitoxin combined group, and the indole-3-acetaldehyde and vomitoxin combined group were 34.66, 12.55, 13.24, and 28.55, respectively. In mice induced by vomitoxin, indole-3-aldehyde had no significant effect on the expression level of Ki67, a marker protein of proliferating cells in the jejunal epithelium, or on the number of proliferating cells. Indole-3-acetaldehyde, however, increased the expression level of Ki67 and the number of proliferating cells. This indicates that indole-3-acetaldehyde can improve the problem of reduced intestinal epithelial proliferation caused by vomitoxin, while indole-3-aldehyde cannot solve this problem.

[0115] The results of intestinal stem cell differentiation levels are shown in Figures 11(A) and 11(B). Figure 11(A) is an immunostaining image of the Panthenocyte marker LYZ, showing that the number of Panthenocytes in the indole-3-acetaldehyde combined with vomitoxin group was higher than that in the vomitoxin group and the indole-3-acetaldehyde combined with vomitoxin group. Figure 11(B) shows the ratio of the number of Panthenocytes in the jejunal crypts to the number of crypts in mice. The ratios for the control group, the vomitoxin group, the indole-3-acetaldehyde combined with vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group were 4.60, 1.95, 2.30, and 3.35, respectively. Clearly, indole-3-acetaldehyde had no significant effect on the number of Panthenocytes in the jejunal crypts of mice induced by vomitoxin; however, indole-3-acetaldehyde increased the number of Panthenocytes in the crypts. This indicates that indole-3-acetaldehyde can improve the problem of reduced intestinal stem cell differentiation levels caused by vomitoxin, while indole-3-acetaldehyde cannot solve this problem.

[0116] The above experimental results indicate that, under the influence of vomitoxin, indole-3-aldehyde has no effect on alleviating damage to intestinal stem cells, while indole-3-acetaldehyde can effectively alleviate damage to intestinal stem cells, increase the height of intestinal villi, enhance the activity of intestinal stem cells, promote the proliferation and differentiation of intestinal stem cells, and increase the proliferation level of intestinal epithelial cells.

[0117] Example 3: Effect of indole-3-acetaldehyde on damage to intestinal organoid stem cells in mice challenged with vomitoxin

[0118] 3.1 Isolation of mouse small intestinal crypts and organoid culture

[0119] A 4-6 week old C57BL / 6J mouse was selected. After sacrifice, approximately 10 cm of small intestine tissue was longitudinally dissected and the contents were washed with pre-chilled Duchenne phosphate-buffered saline (DPBS). After removing the mesentery, the intestinal tissue was cut into 5×5 mm pieces and washed 10 times with pre-chilled DPBS. The intestinal tissue pieces were then resuspended in Gentle Cell Dissociation Reagent (STEMCELL Technologies) and incubated at room temperature for 25 minutes. Subsequently, the tissue pieces were washed with pre-chilled DPBS containing 0.1 wt% bovine serum albumin, and the supernatant was filtered through a 70-micron filter. The crypts were collected by centrifugation at 290×g for 5 minutes and finally resuspended in Corning gel and seeded into 24-well cell culture plates. After incubating the cell culture plates at 37°C for 10 minutes, 500 μL of IntestiCult containing 1% penicillin-streptomycin was added. TM The organoids were cultured in STEMCELL Technologies organoid growth medium, which was changed every 3-4 days.

[0120] 3.2 Treatment for Intestinal Organoid Experiments

[0121] After passage, organoids were divided into three groups: a control group, a vomitoxin group, and a combined indole-3-acetaldehyde and vomitoxin group, with four wells in each group. The control group received PBS; the vomitoxin group was treated with vomitoxin (Sigma-Aldrich) at a concentration of 250 ng / mL; and the combined indole-3-acetaldehyde and vomitoxin group was treated with both vomitoxin and indole-3-acetaldehyde (Jizhi Biochemical), with vomitoxin at a concentration of 250 ng / mL and indole-3-acetaldehyde at a concentration of 100 μM. The treatment time for all three groups was 48 hours.

[0122] 3.3 Growth status of intestinal organoids

[0123] After processing, the organoids were photographed using an upright microscope to count the number of buds, and then the surface area of ​​the organoids was counted using ImageJ.

[0124] 3.4 Assay of intestinal stem cell activity

[0125] Organoids were collected, RNA was extracted, and cDNA was reverse transcribed. The expression level of the intestinal stem cell marker gene Lgr5 was detected using quantitative real-time PCR. The primer sequences for Lgr5 and β-actin were the same as in Example 2, as detailed in Table 2.

[0126] 3.5 Determination of intestinal stem cell differentiation level

[0127] Intestinal organoids were collected, fixed in 4% paraformaldehyde for 30 minutes, and then embedded in paraffin. The paraffin blocks of organoids were sectioned, and the protein expression level of the Paneth cell marker protein LYZ was detected using immunofluorescence and the LYZ antibody (Abcam).

[0128] 3.6 Statistical Methods

[0129] One-way ANOVA was used in GraphPad Prism 8.0.1 software to compare differences between different treatment groups. Tukey's method was used for multiple comparisons. Different lowercase letters in the superscript of the same row indicate significant differences (P < 0.05).

[0130] 3.7 Test Results

[0131] Results of intestinal organoid growth status as follows Figure 12-13 As shown. Figure 12 These are microscopic images of organoids. It can be seen that the number of flower-like organoids in the indole-3-acetaldehyde and vomitoxin combined group is greater than that in the vomitoxin group, and the organoids are also larger. Figure 13 The chart shows the average number of organoid buds. The average number of organoid buds in the control group, the vomitoxin group, and the combined indole-3-acetaldehyde and vomitoxin group were 4.17, 2.36, and 3.48, respectively. Figure 14 This is a statistical chart of organoid surface area. The organoid surface areas of the control group, the vomitoxin group, and the combined indole-3-acetaldehyde and vomitoxin group were 3.90 × 10⁻⁶ and 3.90 × 10⁻⁶, respectively. 4 μm 2 1.87×10 4 μm 2 3.48×10 4 μm 2 Vomitoxin significantly reduced the number and surface area of ​​organoid buds, while the additional addition of indole-3-acetaldehyde significantly increased the number and surface area of ​​organoid buds.

[0132] Results of intestinal stem cell activity assay Figure 15 As shown, Figure 15 This is a statistical graph showing the relative mRNA expression levels of the Lgr5 gene. The relative expression levels of the Lgr5 gene in mice in the control group, the vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group were 1.02, 0.36, and 0.65, respectively. Clearly, the expression level of the stem cell marker gene Lgr5 was significantly reduced in the vomitoxin group, while the Lgr5 expression level in the indole-3-acetaldehyde combined with vomitoxin group was higher than that in the vomitoxin group. This indicates that vomitoxin can significantly reduce the expression level of the stem cell marker gene Lgr5, while the additional addition of indole-3-acetaldehyde can mitigate the effects of vomitoxin, significantly increase the expression level of organoid Lgr5, and thus enhance the activity of intestinal stem cells.

[0133] Results of intestinal stem cell differentiation level assay as follows Figure 16 As shown. Figure 16 The image shows an immunostaining pattern of LYZ, a Paneth cell marker in organoids. It can be seen that the protein expression level of LYZ in the vomitoxin group was lower than that in the indole-3-acetaldehyde combined with vomitoxin group. This indicates that vomitoxin challenge significantly reduced the expression of the Paneth cell marker LYZ, while indole-3-acetaldehyde treatment could mitigate the effects of vomitoxin, increasing LYZ protein expression and thus enhancing intestinal stem cell differentiation.

[0134] The results of Example 3 show that indole-3-acetaldehyde can alleviate vomitoxin-induced intestinal stem cell damage in mouse intestinal organoids, improve intestinal stem cell activity and stem cell differentiation level, thereby promoting the growth and development of intestinal organoids.

[0135] Example 4: Effect of indole-3-acetaldehyde on stem damage in MODE-K cells challenged with vomitoxin

[0136] 4.1 Culture of MODE-K cells, mouse small intestinal epithelial cells

[0137] MODE-K cells were purchased from Beina Biotechnology and cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a cell culture incubator with 5 vol% CO2 and 95 vol% relative humidity. After the cells adhered and grew to cover 80%–90% of the bottom surface, they were digested with 0.25 wt% trypsin. The cells were passaged 2–3 times and then seeded into 24-well cell plates. Experiments were performed when the cells covered 80%–90% of the bottom surface.

[0138] 4.2 MODE-K cell assay treatment

[0139] Cells were divided into three groups: a control group, a vomitoxin group, and a combined indole-3-acetaldehyde and vomitoxin group, with four wells in each group. The control group received PBS; the vomitoxin group was treated with vomitoxin (Sigma-Aldrich) at a concentration of 500 ng / mL; and the combined indole-3-acetaldehyde and vomitoxin group was treated with both vomitoxin and indole-3-acetaldehyde (Jizhi Biochemical), with vomitoxin at a concentration of 500 ng / mL and indole-3-acetaldehyde at a concentration of 100 μM. Samples were collected after 24 hours of co-treatment with all three groups.

[0140] 4.3 MODE-K Cell Stemness Detection

[0141] After treatment with indole-3-acetaldehyde and vomitoxin for 24 hours, RNA was extracted from three groups of cells and cDNA was reverse transcribed. The expression level of the stem cell marker gene Lgr5 was detected using real-time quantitative PCR. The primer sequences for Lgr5 and β-actin were the same as in Example 2, as detailed in Table 2.

[0142] 4.4 MODE-K cell proliferation detection

[0143] After treatment with indole-3-acetaldehyde and vomitoxin for 24 hours, the cell culture medium was discarded, and the cells were washed three times with pre-chilled phosphate-buffered saline (PBS). 500 μL of 4 wt% paraformaldehyde was added, and the cells were fixed at room temperature for 30 minutes. Cells were then treated with immunostaining permeabilization buffer (Beyotime) at room temperature for 15 minutes, followed by washing three times with pre-chilled PBS. Subsequently, the cells were incubated with Ki67 antibody (Proteintech) overnight at 4°C (12 h). The Ki67 antibody was removed, and the cells were washed three times with pre-chilled PBS. The cells were then incubated for 30 minutes with goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Alexa Fluor™ 488; Invitrogen) and DAPI (Invitrogen), followed by washing three times with pre-chilled PBS. The cells were then photographed under an inverted fluorescence microscope.

[0144] 4.5 Statistical Methods

[0145] One-way ANOVA was used in GraphPad Prism 8.0.1 software to compare differences between different treatment groups. Tukey's method was used for multiple comparisons. Different lowercase letters in the superscript of the same row indicate significant differences (P < 0.05).

[0146] 4.6 Test Results

[0147] MODE-K cell stemness test results are as follows Figure 17 As shown, Figure 17 This is a statistical graph showing the relative mRNA expression levels of the Lgr5 gene in MODE-K cells. The relative expression levels of the Lgr5 gene in the control group, the vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group were 1.07, 0.31, and 0.72, respectively. This indicates that vomitoxin significantly reduces the expression level of the stem cell marker gene Lgr5 in MODE-K cells, while indole-3-acetaldehyde treatment can reduce the effect of vomitoxin on MODE-K cells and significantly increase the expression level of Lgr5.

[0148] MODE-K cell proliferation detection results are as follows Figure 18 As shown, Figure 18These are fluorescence micrographs of MODE-K cells after Ki67 immunostaining. It can be seen that the expression level of the proliferation marker protein Ki67 in MODE-K cells of the vomitoxin group was lower than that of the indole-3-acetaldehyde combined with vomitoxin group. This indicates that vomitoxin can reduce the expression level of the proliferation marker protein Ki67 in MODE-K cells, while indole-3-acetaldehyde treatment can reduce the effect of vomitoxin on MODE-K cells and significantly increase the Ki67 expression level.

[0149] The experimental results in Example 4 show that indole-3-acetaldehyde can effectively repair the stemness loss of MODE-K cells induced by vomitoxin and promote cell proliferation.

[0150] Example 5: The synergistic effect of taurine-β-mouse cholic acid and indole-3-acetaldehyde in alleviating vomitoxin-induced intestinal stem cell damage.

[0151] 5.1 Feeding and Management of Experimental Animals

[0152] Thirty 5-6 week old Balb / c mice (weighing 15-16g) were randomly divided into five treatment groups: control group, vomitoxin group, indole-3-acetaldehyde and vomitoxin combination group, taurine-β-cholic acid and vomitoxin combination group, and taurine-β-cholic acid and indole-3-acetaldehyde and vomitoxin combination group, with 6 mice in each treatment group. Mice in the control group were administered PBS by gavage daily. Mice in the vomitoxin group were administered 2 mg / kg vomitoxin (400 μg / mL) (Sigma-Aldrich) by gavage daily. Mice in the indole-3-acetaldehyde and vomitoxin combination group were administered 20 mg / kg indole-3-acetaldehyde (4 μg / μL) and 2 mg / kg vomitoxin by gavage daily. Mice in the taurine-β-cholic acid and vomitoxin combination group were administered 20 mg / kg taurine-β-cholic acid (4 μg / μL, MedChemExpress) and 2 mg / kg vomitoxin by gavage daily. Mice in the taurine-β-cholic acid, indole-3-acetaldehyde, and vomitoxin combination group were administered 20 mg / kg taurine-β-cholic acid, 20 mg / kg indole-3-acetaldehyde, and 2 mg / kg vomitoxin by gavage daily. The formal experiment lasted 10 days. Mice were housed at a temperature maintained at 24-26℃ and humidity maintained at 50-60%; all mice had free access to food and water. All mice were euthanized on the morning of the 10th day of the formal experiment.

[0153] 5.2 Analysis of jejunal morphology

[0154] After slaughtering mice, jejunal tissue was collected and fixed overnight in 4% paraformaldehyde at 4°C. The tissue was then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The stained sections were photographed under a microscope, and villus height and crypt depth were measured using ImageJ software.

[0155] 5.3 Assay of intestinal stem cell activity

[0156] The expression level of the mouse jejunal stem cell marker gene Lgr5 was detected using real-time PCR. The primer sequences for Lgr5 and β-actin were the same as in Example 3, as detailed in Table 2.

[0157] 5.4 Measurement of intestinal epithelial proliferation level

[0158] Mouse jejunal paraffin blocks were sectioned, and the protein expression level of the proliferating cell marker protein Ki67 was detected using immunofluorescence and Ki67 antibody (Proteintech).

[0159] 5.5 Determination of intestinal stem cell differentiation level

[0160] The expression levels of goblet cell marker gene MUC2 and Paneth cell marker gene LYZ were detected using real-time PCR. The primer sequences for MUC2, LYZ and β-actin were the same as in Example 1, as detailed in Table 1.

[0161] 5.6 Statistical Methods

[0162] One-way ANOVA was used in GraphPad Prism 8.0.1 software to compare differences between different treatment groups. Tukey's method was used for multiple comparisons. Different lowercase letters in the superscript of the same row indicate significant differences (P < 0.05).

[0163] 5.7 Test Results

[0164] The morphological results of the mouse jejunum are shown in Figures 19(A) and 19(B). It can be seen that the villus height in the vomitoxin group was significantly lower than that in the control group, indicating that vomitoxin can induce jejunal damage in mice. The villus heights of the control group, vomitoxin group, indole-3-acetaldehyde combined with vomitoxin group, taurine-β-cholic acid combined with vomitoxin group, and taurine-β-cholic acid combined with indole-3-acetaldehyde combined with vomitoxin group were 275 μm, 150 μm, 195 μm, 175 μm, and 254 μm, respectively; the crypt depths were 99 μm, 91 μm, 95 μm, 94 μm, and 97 μm, respectively. Compared with the vomitoxin group, the taurine-β-mouse cholic acid combined with vomitoxin group showed no significant difference, while the villus height was significantly increased in the indole-3-acetaldehyde combined with vomitoxin group and the taurine-β-mouse cholic acid combined with indole-3-acetaldehyde combined with vomitoxin group. This indicates that taurine-β-mouse cholic acid did not improve jejunal injury in mice induced by vomitoxin, while indole-3-acetaldehyde and the combination of indole-3-acetaldehyde and taurine-β-mouse cholic acid both improved jejunal injury in mice. Among these, the combined treatment of indole-3-acetaldehyde and taurine-β-mouse cholic acid showed a better effect in improving jejunal injury.

[0165] Results of intestinal stem cell activity, such as Figure 20 As shown, the relative expression levels of the Lgr5 gene in mice in the control group, the vomitoxin group, the indole-3-acetaldehyde combined with vomitoxin group, the taurine-β-cholic acid combined with vomitoxin group, and the taurine-β-cholic acid combined with indole-3-acetaldehyde combined with vomitoxin group were 1.06, 0.41, 0.80, 0.62, and 0.99, respectively. Compared with the control group, vomitoxin significantly reduced the expression level of the Lgr5 gene, a marker of mouse jejunal stem cells. Compared with the vomitoxin group, both indole-3-acetaldehyde and taurine-β-cholic acid significantly increased the Lgr5 gene expression level, while the combined treatment of taurine-β-cholic acid and indole-3-acetaldehyde further increased the Lgr5 expression level. This indicates that the synergistic effect of indole-3-acetaldehyde and taurine-β-cholic acid is more beneficial in improving the problem of reduced intestinal stem cell activity caused by vomitoxin.

[0166] Results of intestinal epithelial proliferation level as follows Figure 21 and Figure 22 As shown. Figure 21 This is an immunostaining image of Ki67, a marker of proliferating cells. Figure 22 This is a statistical graph showing the ratio of proliferating cells in the jejunal epithelium to the number of intestinal crypts in mice. The ratios for the control group, vomitoxin group, indole-3-acetaldehyde combined with vomitoxin group, taurine-β-cholic acid combined with vomitoxin group, and taurine-β-cholic acid combined with indole-3-acetaldehyde combined with vomitoxin group were 24.67, 9.16, 16.22, 12.38, and 23.61, respectively. Compared with the control group, vomitoxin significantly reduced the number of proliferating cells in the jejunal epithelium. Compared with the vomitoxin group, taurine-β-cholic acid had no significant effect on the number of proliferating cells, while indole-3-acetaldehyde significantly increased the number of proliferating cells. Combined treatment with taurine-β-cholic acid and indole-3-acetaldehyde further increased the number of proliferating cells. This indicates that taurine-β-cholic acid did not improve intestinal epithelial proliferation in mice induced by vomitoxin; however, indole-3-acetaldehyde and the combination of indole-3-acetaldehyde and taurine-β-cholic acid could improve epithelial proliferation, thereby improving jejunal injury. Among these, the combined treatment with indole-3-acetaldehyde and taurine-β-cholic acid showed a more significant improvement.

[0167] Results of intestinal stem cell differentiation level as follows Figure 23As shown, the relative expression levels of MUC2 in the control group, vomitoxin group, indole-3-acetaldehyde combined with vomitoxin group, taurine-β-cholic acid combined with vomitoxin group, and taurine-β-cholic acid combined with indole-3-acetaldehyde combined with vomitoxin group were 1.12, 0.51, 0.72, 0.64, and 0.96, respectively; the relative expression levels of LYZ were 1.06, 0.33, 0.65, 0.60, and 0.93, respectively. Compared with the control group, vomitoxin significantly reduced the expression levels of the mouse jejunal goblet cell marker gene MUC2 and the Paneth cell marker gene LYZ. Compared with the vomitoxin group, both indole-3-acetaldehyde and taurine-β-cholic acid significantly increased the expression levels of MUC2 and LYZ genes, while the combined treatment of taurine-β-cholic acid and indole-3-acetaldehyde further increased the expression levels of MUC2 and LYZ. This indicates that the synergistic effect of indole-3-acetaldehyde and taurine-β-cholic acid is more beneficial in improving the problem of reduced intestinal stem cell differentiation caused by vomiting toxins.

[0168] The above experimental results show that, under the influence of vomitoxin, the combined treatment with taurine-β-mouse cholic acid and indole-3-acetaldehyde can greatly alleviate intestinal stem cell damage, promote stem cell proliferation and differentiation, and improve the intestinal barrier, and the effect is better than that of indole-3-acetaldehyde alone.

[0169] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of indole-3-acetaldehyde and its salt as active ingredients in the preparation of pharmaceutical formulations for repairing vomitoxin-induced intestinal damage in animals.

2. The application according to claim 1, characterized in that, It also includes the use of the combination of indole-3-acetaldehyde and its salt with taurine in pharmaceutical formulations for repairing intestinal damage in animals.

3. The application according to claim 1 or 2, characterized in that, Based on the weight of the pharmaceutical preparation, the effective amount of indole-3-acetaldehyde and its salt in the pharmaceutical preparation is 60-120 mg / kg, used for repairing intestinal damage in animals; and / or, The effective dose of the indole-3-acetaldehyde and its salt in animals is 15-30 mg / kg based on the weight of the animals being raised.

4. The application according to claim 2, characterized in that, The mass ratio of the indole-3-acetaldehyde and its salt to taurine cholic acid is 1:0.5-1.

5. The application according to claim 1 or 2, characterized in that, The repair of animal intestinal damage includes at least one of the following: increasing the height of intestinal villi, reducing intestinal permeability, increasing the activity of intestinal stem cells, promoting the proliferation and differentiation of intestinal stem cells, and increasing the proliferation level of intestinal epithelial cells.

6. The application according to claim 5, characterized in that, The reduction of intestinal permeability includes reducing the animal's serum D-lactic acid level.

7. The application according to claim 5, characterized in that, The enhancement of intestinal stem cell activity includes increasing the Lgr5 expression level and the number of intestinal stem cells in animal jejunal stem cells.

8. The application according to claim 5, characterized in that, The promotion of intestinal stem cell proliferation and differentiation includes at least one of the following: Increase the expression level of the MUC2 gene and the number of villi goblet cells in animal jejunal goblet cells; Increase the expression level of LYZ, a marker protein of Paneth cells in the jejunal crypts of animals, and increase the number of Paneth cells; Increase the expression level of the ChgA gene in animal intestinal endocrine cells.

9. The application according to claim 5, characterized in that, The improvement of intestinal epithelial cell proliferation includes increasing the Ki67 expression level and the number of intestinal proliferating cells in animal jejunal epithelial proliferating cells.

10. The application according to claim 1 or 2, characterized in that, The drug formulation uses a combination of indole-3-acetaldehyde and its salt with taurine as the active ingredient to prepare a drug formulation for repairing intestinal damage in animals.

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