Application of indole-3-acetaldehyde and salt thereof in product for repairing animal intestinal injury

Abstract

The invention discloses application of indole-3-acetaldehyde and salt thereof in a product for repairing animal intestinal injury, relates to the technical field of animal intestinal injury repairing, and aims to improve animal intestinal injury induced by vomitoxin. Animal experiments prove that after being treated by a product containing indole-3-acetaldehyde, the vomitoxin-induced intestinal injury degree can be remarkably reduced, the activity of intestinal stem cells can be remarkably improved, the villus height and permeability in intestines can be improved, proliferation and differentiation of the intestinal stem cells can be promoted, and the proliferation level of intestinal epithelial cells can be improved. A new thought can be provided for research and development of products for repairing the vomitoxin-induced intestinal injury, and a new nutrition regulation and control means can be developed for treating the vomitoxin-induced intestinal injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of repairing animal intestinal injury, and particularly relates to application of indole-3-acetaldehyde and salts thereof in a product for repairing animal intestinal injury. BACKGROUND

[0002] Deoxynivalenol (DON) is a mycotoxin synthesized by Fusarium, is one of the common grain and feed pollutants in China, and widely exists in wheat, barley, corn and other grains, seriously endangering food and feed safety and human and animal health. After oral intake of deoxynivalenol, it enters the intestinal tract, and the intestinal tract is the primary organ of deoxynivalenol damage. In the intestinal tract, deoxynivalenol toxicity can cause a series of adverse consequences such as intestinal stem cell damage, and the intestinal stem cell plays a crucial role in intestinal epithelial cell proliferation and differentiation. Repairing deoxynivalenol-induced intestinal stem cell damage is an effective method to reduce the harm of deoxynivalenol. The current prevention and control strategies for deoxynivalenol mainly include the use of mold inhibitors and biodegradation enzyme preparations at the raw material end, and the use of nutritional strategies to block or reduce the damage of toxins to the body at the terminal. It can be seen that the prior art reduces the intake of deoxynivalenol or reduces the damage of deoxynivalenol to animals through nutritional supplementation, but cannot effectively repair the deoxynivalenol-induced intestinal stem cell damage, and the prior art also does not study how to effectively repair the deoxynivalenol-induced intestinal stem cell damage.

[0003] In view of the problem that the animal intestinal injury caused by deoxynivalenol cannot be effectively repaired, it is urgent to find a safe and effective ingredient which can be applied to a product for repairing deoxynivalenol-induced intestinal injury. SUMMARY

[0004] The present application aims to provide application of indole-3-acetaldehyde and salts thereof in a product for repairing animal intestinal injury, so as to improve animal intestinal injury.

[0005] In order to achieve the above-mentioned purpose, the present application provides application of indole-3-acetaldehyde and salts thereof in a product for repairing animal intestinal injury.

[0006] Compared with the prior art, the application finds a new application of indole-3-acetaldehyde and its salt, and the indole-3-acetaldehyde and its salt can be applied to products for repairing vomitoxin-induced intestinal injury. The application proves through animal tests that, after being treated by the product containing the indole-3-acetaldehyde and its salt, the degree of vomitoxin-induced intestinal injury can be significantly reduced, the intestinal stem cell activity can be significantly increased, the intestinal villus height and permeability can be improved, the intestinal stem cell proliferation and differentiation can be promoted, and the intestinal epithelial cell proliferation level can be improved. The application can provide a new idea for the research and development of products for repairing vomitoxin-induced intestinal injury, and is helpful to develop a new nutritional regulation means for treating vomitoxin-induced intestinal injury.

[0007] Further, the application also includes the application of the combination of the indole-3-acetaldehyde and its salt and taurocholic acid in products for repairing animal intestinal injury.

[0008] In the application, the indole-3-acetaldehyde or the pharmaceutically acceptable salt of the indole-3-acetaldehyde in the product is synergistic with the taurocholic acid, and is more conducive to repairing the intestinal injury caused by vomitoxin.

[0009] Further, the effective amount of the indole-3-acetaldehyde and its salt added in the product for repairing animal intestinal injury is 60-120 mg / kg, based on the mass of the product.

[0010] Further, the effective dose of the indole-3-acetaldehyde and its salt in the animal is 15-30 mg / kg, based on the mass of the animal.

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

[0012] Further, the repairing of the animal intestinal injury includes at least one of the following: improving the intestinal villus height of the animal, reducing the intestinal permeability of the animal, improving the intestinal stem cell activity of the animal, promoting the intestinal stem cell proliferation and differentiation of the animal, and improving the intestinal epithelial cell proliferation level of the animal.

[0013] Further, the reducing of the intestinal permeability of the animal includes reducing the serum D-lactic acid level of the animal.

[0014] Further, the improving of the intestinal stem cell activity of the animal includes improving the Lgr5 expression level of the jejunal stem cell of the animal and the number of intestinal stem cells of the animal.

[0015] Further, the promoting of the intestinal stem cell proliferation and differentiation of the animal at least includes any one of the following: improving the expression level of the MUC2 gene of the jejunal goblet cell of the animal and the number of villus goblet cells of the animal; improving the expression level of the LYZ protein of the jejunal crypt Paneth cell marker of the animal and the number of Paneth cells of the animal; The expression level of ChgA gene in the intestinal endocrine cells of the animal is improved.

[0016] Further, the improved intestinal epithelial cell proliferation level comprises improving the Ki67 expression level of the jejunum epithelial proliferative cells and the number of intestinal proliferative cells of the animal.

[0017] Further, the product is prepared into a pharmaceutical preparation, an animal health product or a functional feed for repairing the intestinal injury of the animal by using indole-3-acetaldehyde and its salt as an additive or by using a combination of indole-3-acetaldehyde and its salt and taurocholic acid as an additive.

[0018] Compared with the prior art, when indole-3-acetaldehyde and its salt is used as an additive or when a combination of indole-3-acetaldehyde and its salt and taurocholic acid is used as an additive, the pharmaceutical preparation, the animal health product or the functional feed with specific effects can be prepared, and the transportation, storage and use of the product are facilitated. BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings: FIG. 1(A) is a HE staining diagram of the jejunum tissue of the mice in Example 1; and FIG. 1(B) is a statistical diagram of the villus height and crypt depth of the mice in Example 1.

[0020] Figure 2 FIG. 2 is a statistical diagram of the serum D-lactic acid level of the mice in Example 1.

[0021] FIG. 3(A) is an Lgr5 immunostaining diagram of the jejunum stem cell marker of the mice in Example 1; and FIG. 3(B) is a ratio of the number of intestinal stem cells to the number of intestinal crypts of the mice in Example 1.

[0022] FIG. 4(A) is a Ki67 immunostaining diagram of the proliferative cell marker of the jejunum of the mice in Example 1; and FIG. 4(B) is a ratio of the number of jejunum epithelial proliferative cells to the number of crypts of the mice in Example 1.

[0023] FIG. 5(A) is a picture of the jejunum of the mice after AB / PAS staining in Example 1; and FIG. 5(B) is a ratio of the number of villus goblet cells to the number of villi of the jejunum of the mice in Example 1.

[0024] FIG. 6(A) is an LYZ immunostaining diagram of the Paneth cell marker of the jejunum of the mice in Example 1; and FIG. 6(B) is a ratio of the number of crypt Paneth cells to the number of villi of the jejunum of the mice in Example 1.

[0025] Figure 7Figure 7 is a statistical graph of the relative mRNA expression levels of the small intestine goblet cell marker gene MUC2, the Paneth cell marker gene LYZ, and the intestinal endocrine cell marker gene ChgA gene of the mice in Example 1.

[0026] Figure 8 (A) is a HE staining graph of the small intestine tissue of the mice in Example 2; Figure 8 (B) is a statistical graph of the villus height and crypt depth of the mice in Example 2.

[0027] Figure 9 (A) is a statistical graph of the relative mRNA expression levels of the Lgr5 gene of the mice in Example 2; Figure 9 (B) is an immunostaining graph of the small intestine stem cell marker Lgr5 of the mice in Example 2.

[0028] Figure 10 (A) is an immunostaining graph of the small intestine proliferative cell marker Ki67 of the mice in Example 2; Figure 10 (B) is the ratio of the small intestine epithelial proliferative cell number to the intestinal crypt number of the mice in Example 2.

[0029] Figure 11 (A) is an immunostaining graph of the small intestine Paneth cell marker LYZ of the mice in Example 2; Figure 11 (B) is the ratio of the small intestine crypt Paneth cell number to the crypt number of the mice in Example 2.

[0030] Figure 12 Figure 12 is a microscope graph of the organoids in Example 3.

[0031] Figure 13 Figure 13 is a statistical graph of the average number of outgrowth of the organoids in Example 3.

[0032] Figure 14 Figure 14 is a statistical graph of the surface area of the organoids in Example 3.

[0033] Figure 15 Figure 15 is a statistical graph of the relative mRNA expression levels of the Lgr5 gene in Example 3.

[0034] Figure 16 Figure 16 is an immunostaining graph of the intestinal organoid Paneth cell marker LYZ in Example 3.

[0035] Figure 17 Figure 17 is a statistical graph of the relative mRNA expression levels of the Lgr5 gene in the MODE-K cells.

[0036] Figure 18 Figure 18 is a fluorescence microscope picture of the stained MODE-K cells.

[0037] Figure 19 (A) is a HE staining graph of the small intestine tissue of the mice in Example 5; Figure 19 (B) is a statistical graph of the villus height and crypt depth of the mice in Example 5.

[0038] Figure 20is a statistical graph of the relative mRNA expression level of Lgr5 gene of the mice in Example 5.

[0039] Figure 21 is a graph of the immunostaining of the proliferation cell marker Ki67 of the mice in Example 5.

[0040] Figure 22 is a statistical graph of the ratio of the number of epithelial proliferative cells to the number of intestinal crypts in the jejunum of the mice in Example 5.

[0041] Figure 23 is a statistical graph of the relative mRNA expression level of the jejunum goblet cell marker gene MUC2 and the Paneth cell marker gene LYZ of the mice in Example 5. DETAILED DESCRIPTION

[0042] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clearly understood, the present application will be further described in detail below in conjunction with the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0043] The present application provides an application of indole-3-acetaldehyde and its salt in a product for repairing intestinal injury of animals.

[0044] The present application finds a new application of indole-3-acetaldehyde and its salt, and indole-3-acetaldehyde or its pharmaceutically acceptable salt can be applied in a product for repairing emetic toxin-induced intestinal injury. Further, the present application specifically relates to an application of indole-3-acetaldehyde or its pharmaceutically acceptable salt in a product for repairing emetic toxin-induced intestinal injury of animals.

[0045] The inventors have found that when the intestinal epithelial cells absorb a large amount of emetic toxin, ribosome damage is triggered, cell proliferation and differentiation are significantly inhibited, tight junction protein synthesis in the intestinal tract is reduced, the intestinal barrier is damaged, the intestinal permeability is increased, and further a large amount of emetic toxin and foreign bacteria and viruses cause infection and damage to the animal body. The prior art has studied the influence of indole derivatives on improving the health status of animals, animal intestinal stem cells and animal growth performance under stable state conditions; however, whether the indole derivatives can repair the infection and damage to the animal body induced by emetic toxin, such as intestinal injury and intestinal stem cell injury induced by emetic toxin, has not been involved.

[0046] The animal experiment proves that the degree of intestinal injury induced by vomitoxin can be significantly reduced, the activity of intestinal stem cells can be significantly increased, the height and permeability of intestinal villi can be improved, the proliferation and differentiation of intestinal stem cells can be promoted, and the proliferation level of intestinal epithelial cells can be improved after the product containing indole-3-acetaldehyde and salts thereof is treated. The present application can provide a new idea for the research and development of products for repairing vomitoxin-induced intestinal injury, and help to develop new nutritional regulation means for treating vomitoxin-induced intestinal injury.

[0047] Further, the present application also includes the use of the combination of indole-3-acetaldehyde and salts thereof and taurocholic acid in the product for repairing animal intestinal injury. In the present application, the taurocholic acid is selected from at least one of tauro-beta-muricholic acid, tauro-alpha-muricholic acid and tauro-omega-muricholic acid, and preferably tauro-beta-muricholic acid.

[0048] In the present application, indole-3-acetaldehyde or pharmaceutically acceptable salts of indole-3-acetaldehyde in the product is synergistic with taurocholic acid, which is more conducive to repairing intestinal injury caused by vomitoxin.

[0049] In some embodiments, the effective amount of indole-3-acetaldehyde and salts thereof added in the product is 60-120 mg / kg based on the mass of the product for repairing animal intestinal injury. For example, the effective amount of indole-3-acetaldehyde and salts thereof added in the product is 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg or 120 mg / kg based on the mass of the product.

[0050] In some embodiments, the effective dose of indole-3-acetaldehyde and salts thereof in animals is 15-30 mg / kg based on the mass of the animals fed. For example, the effective dose of indole-3-acetaldehyde and salts thereof 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 mass of the animals fed.

[0051] In the present application, when the product contains the above-mentioned specific effective amount of indole-3-acetaldehyde, or the amount of indole-3-acetaldehyde satisfies the above-mentioned scheme based on the mass of the animals, it is more conducive to repairing vomitoxin-induced intestinal injury of animals. Preferably, the effective dose of indole-3-acetaldehyde and salts thereof in animals is 18-23 mg / kg based on the mass of the animals fed.

[0052] In some embodiments, the mass ratio of indole-3-acetaldehyde and salts thereof to taurocholic acid is 1:0.5-1.

[0053] In the present application, when the mass ratio of indole-3-acetaldehyde and its salt to taurocholic acid in the product meets the above range, it is more conducive to repairing the intestinal damage of animals induced by vomitoxin. For example, the mass ratio of indole-3-acetaldehyde and its salt to taurocholic 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 taurocholic acid is 1:0.8-1.

[0054] In some embodiments, repairing the intestinal damage of animals comprises at least one of increasing the villus height in the intestine of animals, reducing the intestinal permeability, improving the intestinal stem cell activity, promoting the proliferation and differentiation of intestinal stem cells, and increasing the proliferation level of intestinal epithelial cells.

[0055] Further, reducing the intestinal permeability comprises reducing the serum D-lactic acid level of animals. In the present application, the serum D-lactic acid level can reflect the intestinal permeability of animals. When the D-lactic acid content in the serum of animals is low, it indicates that the intestinal permeability is low and the intestinal barrier is relatively complete. After animals ingest more vomitoxin due to feed pollution and other reasons, the intestinal permeability of animals will be increased, thereby significantly increasing the serum D-lactic acid level of animals. However, after feeding the product containing indole-3-acetaldehyde and its salt, the serum D-lactic acid level can be significantly reduced, indicating that the intestinal permeability of animals treated by indole-3-acetaldehyde and its salt is reduced and the intestinal barrier is relatively complete.

[0056] Further, improving the intestinal stem cell activity comprises increasing the Lgr5 expression level of jejunal stem cells of animals and the number of intestinal stem cells. In the present application, the Lgr5 expression level of jejunal stem cells of animals induced by vomitoxin and the number of stem cells are significantly reduced. However, indole-3-acetaldehyde and its salt can increase the Lgr5 expression level of animals induced by vomitoxin and the number of stem cells, indicating that after feeding the product containing indole-3-acetaldehyde and its salt, the intestinal damage of animals induced by vomitoxin can be effectively repaired and the intestinal stem cell activity of animals can be improved.

[0057] Further, promoting the proliferation and differentiation of intestinal stem cells at least comprises any one of the following: increasing the expression level of MUC2 gene of jejunal goblet cells of animals and the number of villus goblet cells; increasing the expression level of LYZ, a marker protein of jejunal crypt Paneth cells of animals, and the number of Paneth cells; increasing the expression level of ChgA gene of intestinal endocrine cells of animals.

[0058] In the present application, the intestinal stem cells can differentiate into different types of intestinal epithelial cells, such as goblet cells, Paneth cells and intestinal endocrine cells. After the animals ingest more vomitoxin, the number of villus goblet cells and the expression level of MUC2 gene decrease, the number of Paneth cells and the expression level of LYZ gene decrease, or the expression level of ChgA gene decreases. However, after feeding the product containing indole-3-acetaldehyde and its salt, the number of villus goblet cells and the expression level of MUC2 gene can be increased; the number of Paneth cells and the expression level of LYZ gene can be increased; and the expression level of ChgA gene can be increased, thereby repairing the intestinal damage of animals induced by vomitoxin and promoting the proliferation of intestinal stem cells.

[0059] Further, increasing the proliferation level of intestinal epithelial cells includes increasing the expression level of Ki67 of animal jejunum epithelial proliferative cells and the number of intestinal proliferative cells. In the present application, the expression level of Ki67 of animal jejunum epithelial proliferative cells and the number of proliferative cells after induction of vomitoxin are significantly reduced, while indole-3-acetaldehyde and its salt can increase the expression level of Ki67 and the number of proliferative cells after induction of vomitoxin, indicating that indole-3-acetaldehyde and its salt can effectively repair the intestinal damage of animals induced by vomitoxin, increase the activity of intestinal stem cells, and increase the proliferation level of intestinal epithelial cells.

[0060] In some embodiments, the product is prepared into a pharmaceutical preparation, an animal health product or a functional feed for repairing intestinal damage of animals, with indole-3-acetaldehyde and its salt as an additive, or with a combination of indole-3-acetaldehyde and its salt and taurocholic acid as an additive. In the present application, when indole-3-acetaldehyde and its salt are used as an additive, or when a combination of indole-3-acetaldehyde and its salt and taurocholic acid is used as an additive, a pharmaceutical preparation, an animal health product or a functional feed with specific effects can be prepared, facilitating the transportation, storage and use of the product.

[0061] In the following examples and comparative examples, vomitoxin is purchased from Sigma-Aldrich, and indole-3-acetaldehyde is purchased from Jituo Biochemical. Other raw materials not specifically described can be obtained from commercial channels.

[0062] Example 1: Effect of indole-3-acetaldehyde on intestinal stem cell damage induced by vomitoxin in mice 1.1 Feeding and management of test animals A total of 18 C57BL / 6J mice aged 6-8 weeks (mice weighing 17-18 g) were selected and randomly divided into three treatment groups: control group, emetic toxin group, and indole-3-acetaldehyde and emetic toxin combination group, with 6 mice in each treatment group. The temperature of all mice in the three treatment groups was maintained at 22-25°C, and the humidity was maintained at 40-50%, and they were all free to eat and drink water. The control group of mice was given phosphate buffer solution (PBS) by gavage every day, the emetic toxin group of mice was given 2 mg / kg emetic toxin (concentration of 400 μg / mL) by gavage every day, and the indole-3-acetaldehyde and emetic toxin combination group of mice was given 20 mg / kg indole-3-acetaldehyde (concentration of 4 μg / μL) and 2 mg / kg emetic toxin by gavage every day. All mice were sacrificed on the morning of the 14th day of the formal test.

[0063] 1.2 Jejunal morphological analysis After the mice were sacrificed, jejunal tissue was collected and fixed in 4wt% paraformaldehyde for 30 minutes, then paraffin-embedded and sectioned, and subjected to HE staining. The stained sections were placed under a microscope for photography, and the villus height and crypt depth were measured using ImageJ software.

[0064] 1.3 Intestinal permeability determination After the mice were sacrificed, blood was collected and centrifuged at 3000 rpm for 15 minutes to obtain serum, and the D-lactic acid level in the serum was determined using an ELISA kit (enzyme-linked) to reflect the intestinal permeability of the mice.

[0065] 1.4 Intestinal stem cell activity determination Jejunal wax blocks of mice were sectioned, and the protein expression level of the stem cell marker protein Lgr5 was detected using immunofluorescence technology and Lgr5 antibody (Origene).

[0066] 1.5 Intestinal epithelial proliferation level determination Jejunal wax blocks of mice were sectioned, and the protein expression level of the proliferation cell marker protein Ki67 was detected using immunofluorescence technology and Ki67 antibody (Proteintech).

[0067] 1.6 Intestinal stem cell differentiation level determination Jejunal wax blocks of mice were sectioned, and the number of goblet cells was detected using AB / PAS staining technology, and the protein expression level of the Paneth cell marker protein LYZ was detected using immunostaining technology and LYZ antibody (Abcam). The expression levels of the goblet cell marker gene MUC2, the Paneth cell marker gene LYZ, and the intestinal endocrine cell marker gene ChgA were detected using fluorescence quantitative PCR technology; the fluorescence quantitative PCR primer sequences of the genes are shown in Table 1.

[0068] Table 1 1.7 Statistical Methods 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).

[0069] 1.8 Test Results 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.

[0070] Intestinal permeability test results as follows Figure 2 As shown. Figure 2 This 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.

[0071] The results of the intestinal stem cell activity test are shown in FIG. 3(A) and FIG. 3(B). FIG. 3(A) is an immunostaining diagram of the intestinal stem cell marker Lgr5, and it can be seen that the number of stained positive cells (black arrows) in the emetic toxin group is less than that in the indole-3-acetaldehyde and emetic toxin combined group, and the expression level of the jejunal stem cell marker protein Lgr5 in the emetic toxin group mice is reduced. FIG. 3(B) is the ratio of the number of intestinal stem cells to the number of intestinal crypts in mice of the three treatment groups, and the ratio of the number of intestinal stem cells to the number of intestinal crypts in mice of the control group, the emetic toxin group, and the indole-3-acetaldehyde and emetic toxin combined group is 2.53, 0.96, and 1.87, respectively.

[0072] The expression level of the jejunal stem cell marker protein Lgr5 and the number of stem cells in the emetic toxin group were significantly lower than those in the control group, indicating that emetic toxin caused a decrease in the activity of jejunal stem cells in mice; and the expression level of Lgr5 and the number of stem cells in the indole-3-acetaldehyde and emetic toxin combined group were significantly higher than those in the emetic toxin group, indicating that indole-3-acetaldehyde could improve the problem of decreased intestinal stem cell activity caused by emetic toxin.

[0073] The results of the intestinal epithelial proliferation level test are shown in FIG. 4(A) and FIG. 4(B). FIG. 4(A) is an immunostaining diagram of the proliferation cell marker Ki67, and it can be seen that the number of epithelial proliferating cells (Ki67 corresponding stained positive cells) in the control group and the indole-3-acetaldehyde and emetic toxin combined group is more than that in the emetic toxin group. FIG. 4(B) is the ratio of the number of jejunal epithelial proliferating cells to the number of crypts in mice, and the ratio of the number of jejunal epithelial proliferating cells to the number of intestinal crypts in mice of the control group, the emetic toxin group, and the indole-3-acetaldehyde and emetic toxin combined group is 38.66, 18.55, and 30.24, respectively. Emetic toxin can reduce the expression level of the jejunal epithelial proliferating cell marker protein Ki67 and the number of proliferating cells, and indole-3-acetaldehyde can increase the expression level of Ki67 and the number of proliferating cells induced by emetic toxin, indicating that indole-3-acetaldehyde can improve the problem of reduced intestinal epithelial proliferation level caused by emetic toxin.

[0074] Intestinal stem cells can differentiate into different types of intestinal epithelial cells, such as goblet cells, Paneth cells, and intestinal endocrine cells. The results of the intestinal stem cell differentiation level test are shown in FIG. 5(A), FIG. 5(B), FIG. 6(A), FIG. 6(B), and Figure 7Figure 5(A) is a picture of mouse jejunum after AB / PAS staining treatment, it can be seen that the number of goblet cells in the control group and the indole-3-acetaldehyde combined with emetic toxin group is more than that in the emetic toxin group. Figure 5(B) is the ratio of the number of mouse jejunum villus goblet cells to the number of villi, the ratio of the number of mouse jejunum villus goblet cells to the number of villi in the control group, emetic toxin group, indole-3-acetaldehyde combined with emetic toxin group is 17.68, 10.69, 15.69 respectively. Emetic toxin significantly reduces the number of mouse jejunum villus goblet cells, while indole-3-acetaldehyde can increase the number of mouse jejunum villus goblet cells, which shows that indole-3-acetaldehyde can alleviate the influence of emetic toxin on intestinal stem cell differentiation.

[0075] Figure 6(A) is a picture of LYZ immunostaining of Paneth cell marker, it can be seen that the number of crypt Paneth cells in the control group and the indole-3-acetaldehyde combined with emetic toxin group is more than that in the emetic toxin group. Figure 6(B) is the ratio of the number of mouse jejunum crypt Paneth cells to the number of villi, the ratio of the number of mouse jejunum crypt Paneth cells to the number of villi in the control group, emetic toxin group, indole-3-acetaldehyde combined with emetic toxin group is 4.53, 2.06, 3.47 respectively. Emetic toxin significantly reduces the expression level of LYZ, a marker protein of mouse jejunum crypt Paneth cells, and the number of Paneth cells, while indole-3-acetaldehyde significantly increases the expression level of LYZ, a marker protein of mouse jejunum crypt Paneth cells, and the number of Paneth cells induced by emetic toxin, which shows that indole-3-acetaldehyde can alleviate the influence of emetic toxin on intestinal stem cell differentiation.

[0076] Figure 7 Figure 7 is a statistical chart of the relative mRNA expression levels of MUC2, LYZ and ChgA genes, which are goblet cell marker genes, Paneth cell marker genes and intestinal endocrine cell marker genes of jejunum of mice in three treatment groups. The relative expression amounts of MUC2 gene of mice in the control group, emetic toxin group and indole-3-acetaldehyde combined with emetic toxin group are 1.01, 0.67 and 1.05 respectively, the relative expression amounts of LYZ gene are 1.05, 0.41 and 0.75 respectively, and the relative expression amounts of ChgA gene are 1.02, 0.49 and 0.88 respectively. Emetic toxin significantly reduces the expression levels of MUC2, LYZ and ChgA genes, which are goblet cell marker genes, Paneth cell marker genes and intestinal endocrine cell marker genes of mouse jejunum, while indole-3-acetaldehyde can increase the expression levels of MUC2, LYZ and ChgA genes after emetic toxin induction, which shows that indole-3-acetaldehyde can alleviate the influence of emetic toxin on intestinal stem cell differentiation.

[0077] The test results of Example 1 show that indole-3-acetaldehyde can effectively alleviate the intestinal injury induced by emetic toxin in mice, increase the height of intestinal villi in animals, reduce the intestinal permeability, improve the activity of intestinal stem cells, promote the proliferation and differentiation of intestinal stem cells, and increase the proliferation level of intestinal epithelial cells.

[0078] Example 2: Comparison of the effects of indole-3-acetaldehyde and indole-3- aldehyde on alleviating the damage of intestinal stem cells induced by vomitoxin 2.1 Test animal feeding management A total of 32 C57BL / 6J mice aged 6-8 weeks (mouse weight: 17-18 g) were selected and randomly divided into four treatment groups: a control group, a vomitoxin group, an indole-3-aldehyde and vomitoxin combined group, and an indole-3-acetaldehyde and vomitoxin combined group, with 8 mice in each treatment group. The temperature of all mice in the four treatment groups was maintained at 22-25°C, and the humidity was maintained at 40-50%, and they were all free to eat and drink water; among them, the mice in the control group were given PBS by gavage every day, the mice in the vomitoxin group were given 2 mg / kg vomitoxin (concentration: 400 μg / mL) by gavage every day, the mice in the indole-3-aldehyde and vomitoxin combined group were given 20 mg / kg indole-3-aldehyde (concentration: 4 μg / μL, Jizhisheng Biochemical) and 2 mg / kg vomitoxin by gavage every day, and the mice in the indole-3-acetaldehyde and vomitoxin combined group were given 20 mg / kg indole-3-acetaldehyde (concentration: 4 μg / μL) and 2 mg / kg vomitoxin by gavage every day. All mice were sacrificed in the morning of the 7th day of the formal test.

[0079] 2.2 Jejunal morphological analysis The test was performed according to the method of 1.2 in Example 1.

[0080] 2.3 Intestinal stem cell activity determination The expression level of the mouse jejunal stem cell marker gene Lgr5 was detected by fluorescence quantitative PCR technology, and the Lgr5 and β-actin primer sequences are shown in Table 2. The mouse jejunal wax block was sectioned, and the protein expression level of the stem cell marker protein Lgr5 was detected by immunofluorescence technology and Lgr5 antibody (Origene).

[0081] Table 2 2.4 Intestinal epithelial proliferation level determination The test was performed according to the method of 1.5 in Example 1.

[0082] 2.5 Intestinal stem cell differentiation level determination The mouse jejunal wax block was sectioned, and the protein expression level of the Paneth cell marker protein LYZ was detected by immunostaining technology and LYZ antibody (Abcam).

[0083] 2.6 Statistical methods The differences between different treatment groups were compared by One-Way ANOVA in GraphPad Prism 8.0.1 software, and the multiple comparisons were performed by Tukey method, and the same row of numerical value with different lowercase letters indicated significant difference (P < 0.05).

[0084] 2.7 Test results The jejunum morphology analysis results are shown in FIG. 8(A) and FIG. 8(B). FIG. 8(A) is a HE staining diagram of mouse jejunum tissue, and it can be seen that the villus height of the mouse in the indole-3-acetaldehyde and emetic toxin combined group is higher than that in the indole-3-aldehyde and emetic toxin combined group. FIG. 8(B) is a statistical diagram of the villus height and crypt depth of mice in the four treatment groups. The villus height of the control group, emetic toxin group, indole-3-aldehyde and emetic toxin combined group, and indole-3-acetaldehyde and emetic toxin combined group mice is 261 μm, 140 μm, 154 μm and 200 μm, respectively, and the crypt depth is 101 μm, 85 μm, 92 μm and 96 μm, respectively. There is no significant difference in the crypt depth of the four treatment groups, and there is no significant difference in the villus height of the emetic toxin group and the indole-3-aldehyde and emetic toxin combined group, but the villus height of the mouse in the indole-3-acetaldehyde and emetic toxin combined group is significantly higher than that in the indole-3-aldehyde and emetic toxin combined group, which indicates that for mice induced by emetic toxin, indole-3-aldehyde does not improve the damage of mouse jejunum, while indole-3-acetaldehyde can improve the damage of mouse jejunum.

[0085] The intestinal stem cell activity test results are shown in FIG. 9(A) and FIG. 9(B). FIG. 9(A) is a statistical diagram of the relative mRNA expression level of Lgr5 gene, and the relative expression amount of Lgr5 gene of the control group, emetic toxin group, indole-3-aldehyde and emetic toxin combined group, and indole-3-acetaldehyde and emetic toxin combined group mice is 1.00, 0.45, 0.55 and 0.87, respectively. FIG. 9(B) is an immunostaining diagram of stem cell marker Lgr5, and it can be seen that the number of positive cells (black arrows) in the indole-3-acetaldehyde and emetic toxin combined group is higher than that in the emetic toxin group and the indole-3-aldehyde and emetic toxin combined group. It indicates that for mice induced by emetic toxin, indole-3-aldehyde has no significant effect on the expression level of mouse jejunum stem cell marker Lgr5 gene and protein; indole-3-acetaldehyde can improve the expression level of Lgr5 gene and protein. Obviously, indole-3-acetaldehyde can improve the problem of reduced intestinal stem cell activity caused by emetic toxin, while indole-3-aldehyde cannot solve the problem.

[0086] The intestinal epithelial proliferation level results are shown in FIG. 10(A) and FIG. 10(B). FIG. 10(A) is a Ki67 immunostaining diagram of proliferating cells, and it can be seen that the number of proliferating cells (staining positive cells corresponding to Ki67) in the indole-3-acetaldehyde and emetic toxin combination group is more than that in the emetic toxin group and the indole-3-aldehyde and emetic toxin combination group. FIG. 10(B) is the ratio of the number of proliferating cells in the jejunum epithelium of the mouse to the number of intestinal crypts. The ratio of the number of proliferating cells in the jejunum epithelium of the mouse to the number of intestinal crypts in the control group, the emetic toxin group, the indole-3-aldehyde and emetic toxin combination group, and the indole-3-acetaldehyde and emetic toxin combination group is 34.66, 12.55, 13.24, and 28.55, respectively. For the mice induced by emetic toxin, indole-3-aldehyde has no significant effect on the expression level of Ki67, the marker protein of the proliferating cells in the jejunum epithelium of the mouse, and the number of proliferating cells; indole-3-acetaldehyde can increase the expression level of Ki67 and the number of proliferating cells; and it indicates that indole-3-acetaldehyde can improve the problem of reduced intestinal epithelial proliferation level caused by emetic toxin, while indole-3-aldehyde cannot solve the problem.

[0087] The intestinal stem cell differentiation level results are shown in FIG. 11(A) and FIG. 11(B). FIG. 11(A) is a LYZ immunostaining diagram of Paneth cells, and it can be seen that the number of Paneth cells in the indole-3-acetaldehyde and emetic toxin combination group is more than that in the emetic toxin group and the indole-3-aldehyde and emetic toxin combination group. FIG. 11(B) is the ratio of the number of Paneth cells in the jejunum crypt of the mouse to the number of crypts. The ratio of the number of Paneth cells in the jejunum crypt of the mouse to the number of crypts in the control group, the emetic toxin group, the indole-3-aldehyde and emetic toxin combination group, and the indole-3-acetaldehyde and emetic toxin combination group is 4.60, 1.95, 2.30, and 3.35, respectively. Apparently, for the mice induced by emetic toxin, indole-3-aldehyde has no significant effect on the number of Paneth cells in the jejunum crypt of the mouse; indole-3-acetaldehyde can increase the number of Paneth cells. It indicates that indole-3-acetaldehyde can improve the problem of reduced intestinal stem cell differentiation level caused by emetic toxin, while indole-3-aldehyde cannot solve the problem.

[0088] The above test results show that, under the condition of emetic toxin attack, indole-3-aldehyde has no remission effect on intestinal stem cell damage, while indole-3-acetaldehyde can effectively alleviate intestinal stem cell damage, increase the height of the villi in the animal intestine, improve the activity of intestinal stem cells, promote the proliferation and differentiation of intestinal stem cells, and increase the proliferation level of intestinal epithelial cells.

[0089] Example 3: Effect of indole-3-acetaldehyde on intestinal organoid stem cell damage in emetic toxin-attacked mice 3.1 Isolation of mouse small intestinal crypts and culture of organoids One 4-6 week C57BL / 6J mouse was selected. After the mouse was sacrificed, about 10 centimeters of small intestine tissue was taken and cut longitudinally. The contents were rinsed with pre-cooled Dulbecco's Phosphate Buffered Saline (DPBS). After the mesentery was removed, the intestinal tissue was cut into small pieces of 5 x 5 mm and rinsed repeatedly with pre-cooled DPBS for 10 times. Then the intestinal tissue pieces were resuspended in enzyme dissociation reagent (Gentle Cell Dissociation Reagent; STEMCELL Technologies) and incubated at room temperature for 25 minutes. Then, the tissue pieces were rinsed with pre-cooled DPBS containing 0.1 wt% bovine serum albumin, and the supernatant was filtered through a 70 micron mesh. The crypts were collected by centrifugation at 290 x g for 5 minutes, and finally resuspended in Matrigel (Corning) and seeded in a 24-well cell culture plate. After the cell culture plate was placed in a 37°C cell culture incubator for 10 minutes, 500 μL of IntestiCult TM organoid culture medium (STEMCELL Technologies) was added, and the organoids were cultured, with the organoid culture medium being replaced every 3-4 days.

[0090] 3.2 Intestinal organoid experimental treatment After the organoids were passaged, they were divided into three groups: a control group, a vomitoxin group, and an indole-3-acetaldehyde and vomitoxin combination group, with 4 wells in each group. Among them, the control group was added with PBS; the vomitoxin group was treated with vomitoxin (Sigma-Aldrich) at a concentration of 250 ng / mL; and the indole-3-acetaldehyde and vomitoxin combination group was treated with vomitoxin and indole-3-acetaldehyde (Ji Zhisheng Biochemical) at a concentration of 250 ng / mL for vomitoxin and 100 μM for indole-3-acetaldehyde. The treatment time for the three treatment groups was 48 hours.

[0091] 3.3 Growth state of intestinal organoids After the treatment was completed, 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.

[0092] 3.4 Intestinal stem cell activity determination The organoids were collected, RNA was extracted, and cDNA was reverse transcribed, and the expression level of the intestinal stem cell marker gene Lgr5 was detected using fluorescent quantitative PCR technology. The Lgr5 and β-actin primer sequences were the same as in Example 2, and are shown in Table 2.

[0093] 3.5 Intestinal stem cell differentiation level determination Intestinal organoids were collected and fixed in 4% paraformaldehyde for 30 min, and then embedded with paraffin. The organoid wax blocks were sectioned, and the protein expression level of Paneth cell marker protein LYZ was detected by immunofluorescence technology and LYZ antibody (Abeam).

[0094] 3.6 Statistical methods The differences between different treatment groups were compared by One-Way ANOVA in GraphPad Prism 8.0.1 software, and the multiple comparisons were performed by Tukey method, and the same row of numerical shoulder marks different lowercase letters represented significant difference (P < 0.05).

[0095] 3.7 Test results The growth state results of intestinal organoids are shown in Figures 12-13 . Figure 12 is a microscope graph of organoids. It can be seen that the number of flower-like organoids in the indole-3-acetaldehyde combined with vomitoxin group is more than that in the vomitoxin group, and the volume of the organoids is also larger. Figure 13 is a statistical graph of the average number of organoids, and the average number of organoids of the control group, the vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group is 4.17, 2.36, and 3.48, respectively. Figure 14 is a statistical graph of the surface area of organoids, and the surface area of organoids of the control group, the vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group is 3.90 x 10 4 μm 2 , 1.87 x 10 4 μm 2 , and 3.48 x 10 4 μm 2 , respectively. Vomitoxin significantly reduces the number and surface area of organoids, and additional addition of indole-3-acetaldehyde can significantly improve the number and surface area of organoids.

[0096] The intestinal stem cell activity test results are shown in Figure 15 , Figure 15 is a statistical graph of the relative mRNA expression level of Lgr5 gene. The relative expression amount of Lgr5 gene of mice in the control group, the vomitoxin group, and the indole-3-acetaldehyde combined with vomitoxin group is 1.02, 0.36, and 0.65, respectively. Obviously, the expression level of stem cell marker gene Lgr5 in the vomitoxin group is significantly reduced, and the expression level of Lgr5 in the indole-3-acetaldehyde combined with vomitoxin group is higher than that in the vomitoxin group. It shows that vomitoxin can significantly reduce the expression level of stem cell marker gene Lgr5, and additional addition of indole-3-acetaldehyde can improve the influence caused by vomitoxin, significantly improve the expression level of Lgr5 of organoids, and thus improve the intestinal stem cell activity.

[0097] The results of the determination of the differentiation level of intestinal stem cells are shown in Table 2. Figure 16 Figure 16 is an immunostaining diagram of the organoid Paneth cell marker LYZ, and it can be seen that the protein expression level of the Paneth cell marker protein LYZ of the vomitoxin group is lower than that of the indole-3-acetaldehyde combined with vomitoxin group. It is indicated that after the attack of vomitoxin, the expression of Paneth cell marker protein LYZ is significantly reduced, and the treatment of indole-3-acetaldehyde can improve the influence caused by vomitoxin, thereby increasing the protein expression level of LYZ, so as to improve the differentiation level of intestinal stem cells.

[0098] The test results of Example 3 show that indole-3-acetaldehyde can alleviate the intestinal stem cell damage induced by vomitoxin in mouse intestinal organoids, improve the activity of intestinal stem cells and the differentiation level of stem cells, and further promote the growth and development of intestinal organoids.

[0099] Example 4: Effect of indole-3-acetaldehyde on the damage of MODE-K cell stemness by vomitoxin attack 4.1 Culture of mouse small intestinal epithelial cells MODE-K The MODE-K cells were purchased from Beina Biological Technology Co., Ltd. and were cultured in DMEM high-sugar medium containing 10% fetal bovine serum in a 5vol% CO2, 95vol% relative humidity 37°C cell incubator. After the cells adhered and grew to cover 80% to 90% of the bottom surface, they were digested with 0.25wt% trypsin, and the cells were subcultured for 2-3 times and then inoculated in a 24-well cell plate. When the cells grew to cover 80% to 90% of the bottom surface, the test was performed.

[0100] 4.2 Test treatment of MODE-K cells The inoculated cells were divided into three groups: a control group, a vomitoxin group, and an indole-3-acetaldehyde combined with vomitoxin group, with 4 wells in each group. Among them, the control group was added with PBS; the vomitoxin group was treated with vomitoxin (Sigma-Aldrich) at a concentration of 500 ng / mL; the indole-3-acetaldehyde combined with vomitoxin group was treated with vomitoxin and indole-3-acetaldehyde (Jizhisheng Biochemical Technology Co., Ltd.) at a concentration of 500 ng / mL for vomitoxin and 100 μM for indole-3-acetaldehyde. After 24 hours of treatment, the samples were collected.

[0101] 4.3 Detection of stemness of MODE-K cells After 24 hours of treatment of indole-3-acetaldehyde and vomitoxin, the RNA of the cells in the three groups was collected and reverse transcribed into cDNA. The expression level of the tight stem cell marker gene Lgr5 was detected by real-time fluorescent quantitative PCR technology. The primer sequences of Lgr5 and β-actin are the same as those in Example 2, and are shown in Table 2. ​

[0102] 4.4 MODE-K cell proliferation detection After 24 hours of indole-3-acetaldehyde and vomitoxin treatment, the cell culture medium was discarded, and the cells were washed with pre-cooled phosphate buffered saline (PBS) for 3 times, 500 μL of 4 wt% paraformaldehyde was added, and the cells were fixed at room temperature for 30 minutes. The cells were treated with immunostaining permeation solution (Bi Yun Tian) at room temperature for 15 minutes, and then washed with pre-cooled PBS for 3 times. Then, the cells were incubated with Ki67 antibody (Proteintech) at 4°C overnight (time is 12 h). The Ki67 antibody was removed, and the cells were washed with pre-cooled PBS for 3 times. Then, the cells were incubated with goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488; Invitrogen) and DAPI (Invitrogen) for 30 minutes, and the cells were washed with pre-cooled PBS for 3 times. The cells were placed under an inverted fluorescence microscope for photography.

[0103] 4.5 Statistical method The differences between different treatment groups were compared by One-Way ANOVA in GraphPad Prism 8.0.1 software, and the multiple comparisons were performed by Tukey method, and the same row of numerical shoulder marks different lowercase letters represented significant difference (P < 0.05).

[0104] 4.6 Test results The results of the stemness detection of MODE-K cells are shown in Figure 17 , and the results of the stemness detection of MODE-K cells are shown in Figure 17 is a statistical chart of the relative mRNA expression level of Lgr5 gene in MODE-K cells. The relative expression amount of Lgr5 gene of the control group, the vomitoxin group, the indole-3-acetaldehyde and vomitoxin combined group mice was 1.07, 0.31, 0.72 respectively. It is shown that vomitoxin significantly reduces the expression level of stem cell marker gene Lgr5 in MODE-K cells, and indole-3-acetaldehyde treatment can reduce the influence of vomitoxin on MODE-K cells, and can significantly improve the expression level of Lgr5.

[0105] The results of the proliferation detection of MODE-K cells are shown in Figure 18 , and the results of the proliferation detection of MODE-K cells are shown in Figure 18The fluorescence microscope pictures of MODE-K cells after Ki67 immunostaining. It can be seen that the expression level of proliferative cell marker protein Ki67 in the MODE-K cells of the vomitoxin group is lower than that of the indole-3-acetaldehyde combined with vomitoxin group, indicating that vomitoxin can reduce the expression level of proliferative cell marker protein Ki67 in the MODE-K cells, and indole-3-acetaldehyde treatment can reduce the impact of vomitoxin on the MODE-K cells, significantly increasing the expression level of Ki67.

[0106] The test results of Example 4 show that indole-3-acetaldehyde can effectively repair the loss of stemness of MODE-K cells induced by vomitoxin and promote cell proliferation.

[0107] Example 5: Effect of tauroursodeoxycholic acid and indole-3-acetaldehyde on synergistically alleviating intestinal stem cell damage induced by vomitoxin 5.1 Test animal feeding management In this test, 30 5-6 week old balb / c mice (mouse weight 15-16 g) were randomly divided into 5 treatment groups: control group, vomitoxin group, indole-3-acetaldehyde combined with vomitoxin group, tauroursodeoxycholic acid combined with vomitoxin group, and tauroursodeoxycholic acid combined with indole-3-acetaldehyde combined with vomitoxin group, 6 mice in each treatment group. The control group of mice was given PBS by gavage every day, the vomitoxin group of mice was given 2 mg / kg vomitoxin (concentration 400 μg / mL) (Sigma-Aldrich) by gavage every day, the indole-3-acetaldehyde combined with vomitoxin group of mice was given 20 mg / kg indole-3-acetaldehyde (concentration 4 μg / μL) and 2 mg / kg vomitoxin by gavage every day, the tauroursodeoxycholic acid combined with vomitoxin group of mice was given 20 mg / kg tauroursodeoxycholic acid (concentration 4 μg / μL, MedChemExpress) and 2 mg / kg vomitoxin by gavage every day, and the tauroursodeoxycholic acid combined with indole-3-acetaldehyde combined with vomitoxin group of mice was given 20 mg / kg tauroursodeoxycholic acid, 20 mg / kg indole-3-acetaldehyde and 2 mg / kg vomitoxin by gavage every day. The formal test lasted for 10 days. The temperature of the mice was maintained at 24-26℃, and the humidity was maintained at 50-60%; all mice were free to eat and drink water. All mice were sacrificed in the morning of the 10th day of the formal test.

[0108] 5.2 Jejunal morphology analysis After the mice were slaughtered, jejunal tissue was collected and fixed in 4% paraformaldehyde at 4℃ overnight, then paraffin-embedded and sectioned, and subjected to HE staining. The stained sections were placed under a microscope for photography, and the villus height and crypt depth were measured using ImageJ software.

[0109] 5.3 Intestinal stem cell activity determination The expression level of Lgr5, a marker gene of mouse jejunum stem cells, was detected by fluorescence quantitative PCR technology. The primer sequences of Lgr5 and β-actin were the same as those in Example 3, and details were shown in Table 2.

[0110] 5.4 Determination of intestinal epithelial proliferation level The mouse jejunum wax block was sectioned, and the protein expression level of Ki67, a marker protein of proliferating cells, was detected by immunofluorescence technology and Ki67 antibody (Proteintech).

[0111] 5.5 Determination of intestinal stem cell differentiation level The expression levels of MUC2, a marker gene of goblet cells, and LYZ, a marker gene of Paneth cells, were detected by fluorescence quantitative PCR technology. The primer sequences of MUC2, LYZ and β-actin were the same as those in Example 1, and details were shown in Table 1.

[0112] 5.6 Statistical method The differences between different treatment groups were compared by One-Way ANOVA in GraphPad Prism 8.0.1 software, and multiple comparisons were performed by Tukey method. The same row of numerical shoulder marks different lowercase letters represented significant difference (P < 0.05).

[0113] 5.7 Test results The mouse jejunum morphology results are shown in FIG. 19 (A) and FIG. 19 (B). It can be seen that the villus height of the emetic toxin group is significantly lower than that of the control group, indicating that emetic toxin can induce mouse jejunum injury. The villus height of the control group, the emetic toxin group, the indole-3-acetaldehyde and emetic toxin combined group, the taurine-β-muricholic acid and emetic toxin combined group, and the taurine-β-muricholic acid and indole-3-acetaldehyde and emetic toxin combined group is 275 μm, 150 μm, 195 μm, 175 μm, and 254 μm, respectively. The crypt depth is 99 μm, 91 μm, 95 μm, 94 μm, and 97 μm, respectively. Compared with the emetic toxin group, the taurine-β-muricholic acid and emetic toxin combined group has no significant difference, while the villus height of the indole-3-acetaldehyde and emetic toxin combined group and the taurine-β-muricholic acid and indole-3-acetaldehyde and emetic toxin combined group is significantly improved. It is indicated that for mice induced by emetic toxin, taurine-β-muricholic acid does not improve mouse jejunum injury, while indole-3-acetaldehyde and the combination of indole-3-acetaldehyde and taurine-β-muricholic acid can improve mouse jejunum injury. Among them, the combination of indole-3-acetaldehyde and taurine-β-muricholic acid has better improvement effect on jejunum injury.

[0114] The intestinal stem cell activity results are shown in FIG. 20 (A) and FIG. 20 (B). It can be seen that the expression level of Lgr5 in the emetic toxin group is significantly lower than that in the control group, indicating that emetic toxin can induce mouse jejunum injury. The expression level of Lgr5 in the control group, the emetic toxin group, the indole-3-acetaldehyde and emetic toxin combined group, the taurine-β-muricholic acid and emetic toxin combined group, and the taurine-β-muricholic acid and indole-3-acetaldehyde and emetic toxin combined group is 0.62, 0.31, 0.45, 0.36, and 0.54, respectively. Compared with the emetic toxin group, the taurine-β-muricholic acid and emetic toxin combined group has no significant difference, while the expression level of Lgr5 in the indole-3-acetaldehyde and emetic toxin combined group and the taurine-β-muricholic acid and indole-3-acetaldehyde and emetic toxin combined group is significantly improved. It is indicated that for mice induced by emetic toxin, taurine-β-muricholic acid does not improve mouse jejunum injury, while indole-3-acetaldehyde and the combination of indole-3-acetaldehyde and taurine-β-muricholic acid can improve mouse jejunum injury. Among them, the combination of indole-3-acetaldehyde and taurine-β-muricholic acid has better improvement effect on jejunum injury. Figure 20The relative expression of Lgr5 gene of mice in the control group, the vomitoxin group, the indole-3-acetaldehyde and vomitoxin combined group, the tauroursodeoxycholic acid and vomitoxin combined group, and the tauroursodeoxycholic acid and indole-3-acetaldehyde and vomitoxin combined group was 1.06, 0.41, 0.80, 0.62, and 0.99, respectively. Compared with the control group, the vomitoxin significantly reduced the expression level of the jejunum stem cell marker Lgr5 gene of the mice. Compared with the vomitoxin group, the indole-3-acetaldehyde and the tauroursodeoxycholic acid could significantly improve the expression level of the Lgr5 gene, and the tauroursodeoxycholic acid and the indole-3-acetaldehyde combined treatment could further improve the expression level of the Lgr5. It is illustrated that the indole-3-acetaldehyde and the tauroursodeoxycholic acid are more beneficial to improve the problem of the reduced intestinal stem cell activity caused by the vomitoxin in a synergistic manner.

[0115] The results of the intestinal epithelial proliferation level are shown in Figure 21 and Figure 22 . Figure 21 is a Ki67 immunostaining diagram of proliferative cells; Figure 22 is a statistical diagram of the ratio of the number of proliferative cells of the mouse jejunum epithelium to the number of intestinal crypts. The ratio of the number of proliferative cells of the jejunum epithelium to the number of intestinal crypts of the mice in the control group, the vomitoxin group, the indole-3-acetaldehyde and vomitoxin combined group, the tauroursodeoxycholic acid and vomitoxin combined group, and the tauroursodeoxycholic acid and indole-3-acetaldehyde and vomitoxin combined group was 24.67, 9.16, 16.22, 12.38, and 23.61, respectively. Compared with the control group, the vomitoxin significantly reduced the number of proliferative cells of the jejunum epithelium of the mice. Compared with the vomitoxin group, the tauroursodeoxycholic acid had no significant effect on the number of proliferative cells, the indole-3-acetaldehyde significantly increased the number of proliferative cells, and the tauroursodeoxycholic acid and the indole-3-acetaldehyde combined treatment could further increase the number of proliferative cells. It is illustrated that for the mice induced by the vomitoxin, the tauroursodeoxycholic acid cannot improve the intestinal epithelial proliferation level; and the indole-3-acetaldehyde, the combination of the indole-3-acetaldehyde and the tauroursodeoxycholic acid can increase the epithelial proliferation level, thereby improving the jejunum damage, and the improvement effect of the indole-3-acetaldehyde and the tauroursodeoxycholic acid combined treatment is more significant.

[0116] The results of the intestinal stem cell differentiation level are shown in Figure 23The relative expression amounts of MUC2 of the control group, the vomitoxin group, the indole-3-acetaldehyde combined with vomitoxin group, the tauroursodeoxycholic acid combined with vomitoxin group and the tauroursodeoxycholic acid combined with indole-3-acetaldehyde combined with vomitoxin group are 1.12, 0.51, 0.72, 0.64 and 0.96 respectively; the relative expression amounts of LYZ are 1.06, 0.33, 0.65, 0.60 and 0.93 respectively. Compared with the control group, the vomitoxin significantly reduces the expression levels of the mouse jejunum goblet cell marker gene MUC2 and the Paneth cell marker gene LYZ. Compared with the vomitoxin group, the indole-3-acetaldehyde and the tauroursodeoxycholic acid can significantly improve the expression levels of MUC2 and LYZ, and the tauroursodeoxycholic acid combined with the indole-3-acetaldehyde can further improve the expression levels of MUC2 and LYZ. It is illustrated that the indole-3-acetaldehyde and the tauroursodeoxycholic acid are more beneficial to improve the problem of reduced intestinal stem cell differentiation caused by vomitoxin in a synergistic manner.

[0117] The above test results show that under the condition of vomitoxin attack, the tauroursodeoxycholic acid combined with the indole-3-acetaldehyde can greatly alleviate the intestinal stem cell damage, promote the proliferation and differentiation of stem cells and improve the intestinal barrier, and the effect is better than that of using the indole-3-acetaldehyde alone.

[0118] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. The application of indole-3-acetaldehyde and its salt in products for repairing 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 salts with taurine in products for repairing intestinal damage in animals.

3. The application according to claim 1 or 2, characterized in that, Based on the product weight, the effective amount of indole-3-acetaldehyde and its salt in the product 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 product uses indole-3-acetaldehyde and its salts as additives, or a combination of indole-3-acetaldehyde and its salts with taurine cholic acid as additives, to prepare a pharmaceutical preparation, animal health product, or functional feed for repairing intestinal damage in animals.

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