Application of kynurenic acid in product for preventing and treating vomitoxin toxicity

Abstract

The invention discloses kynurenic acid with an effect of relieving vomitoxin toxicity, and belongs to the technical field of agricultural animals. The application proves that the kynurenic acid can effectively relieve the toxic effect induced by vomitoxin, and the kynurenic acid mainly has the effects of effectively promoting the weight gain of animals, improving the intestinal motility function, inhibiting the release of inflammatory factors, regulating intestinal flora and the like. The invention determines the effect of kynurenic acid in relieving vomitoxin toxicity, and the kynurenic acid has a wide application prospect in preparation of products for preventing and treating vomitoxin toxicity.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural animals, and particularly relates to application of kynurenic acid in preventing and treating the toxicity of vomitoxin. Background Art

[0002] Deoxynivalenol (DON), also known as deoxynivalenol, is a secondary metabolite produced by fungi such as Fusarium graminearum. It is widely found in grains, feed ingredients, and feed, and is currently one of the most seriously contaminated mycotoxins in the world. DON enters the human and animal body and causes symptoms of poisoning such as vomiting, diarrhea, weight loss, and decreased immune function. The intestine, as the primary organ for digestion and absorption in animals, is also the primary target organ for DON's toxic effects. DON disrupts the intestinal barrier function, leading to decreased growth performance in livestock and poultry, posing a serious threat to healthy livestock and poultry farming. However, there is currently a lack of effective agents to prevent and treat DON toxicity.

[0003] Kynurenic acid (KA), a tryptophan metabolite, is abundant in herbs such as mint, nettle, birch leaves, and horsetail, as well as in honey and broccoli. Pharmacological studies have shown that KA promotes intestinal barrier protein expression, fights inflammation, and promotes the growth of probiotics. Because the toxicological mechanisms of DON remain unclear, there are currently no reports on the role of kynurenic acid in mitigating DON toxicity and its application. Summary of the Invention

[0004] The present invention aims to provide a use of kynurenic acid in products for preventing and treating the toxicity of vomitoxin.

[0005] According to the above application provided by the present invention, the product uses kynurenic acid as an active ingredient and is prepared into a pharmaceutical preparation for preventing and treating the toxicity of vomitoxin.

[0006] According to the above application provided by the present invention, kynurenic acid is used in the preparation of animal health products, enteral nutrition agents, and feed additives for preventing and treating the toxicity of vomitoxin.

[0007] The present invention has the beneficial effects of discovering and demonstrating that kynurenic acid can effectively alleviate the toxic effects of vomitoxin, primarily by promoting animal weight gain, improving intestinal motility, inhibiting the release of inflammatory factors, and regulating intestinal flora. Kynurenic acid has broad application prospects in the preparation of products for the prevention and treatment of vomitoxin poisoning. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1This figure shows the effect of kynurenic acid on mitigating the effects of DON on mouse weight gain. The relative weight changes after challenge were calculated, with the starting weight of each group of mice before challenge (day 0) as the control (100%). Statistical differences in relative weight between the DON group and the control group are indicated by *, and statistical differences in relative weight between the DON group, the KA+DON group, and the DON group are indicated by a (ap < 0.05, *p < 0.05).

[0009] Figure 2 Figure 1 shows the results of kynurenic acid improving mouse behavior. In the figure, A shows the mouse's movement trajectory; B shows the distance the mouse traveled in the open field; C shows the distance the mouse traveled in the center of the open field; and D shows the time the mouse spent in the center of the open field (**p < 0.01).

[0010] Figure 3 Figure 1 shows the results of kynurenic acid alleviating vomitoxin toxicity. Figure A shows HE and AB-PAS staining of mouse intestines (section scale, 200 μm); B shows villus length of the jejunum and ileum; C shows gastrointestinal motility (TGIT); D shows the number of feces excreted within 2 hours; E shows the expression level of the inflammatory factor TNF-α in mouse serum; and F shows HE staining of mouse liver tissue (section scale, 100 μm). (*p < 0.05, **p < 0.01, ***p < 0.001).

[0011] Figure 4 Figure 1 shows the results of kynurenic acid modulating the intestinal microbiota in mice. Figure A shows the relative abundance of stress-tolerant bacteria in the mouse cecum; B shows the relative abundance of bacteria at the family level in the mouse cecum; and C shows the characteristic bacterial structure of mice (LDA value > 3) analyzed by LEfse. DETAILED DESCRIPTION

[0012] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0013] 1. Experimental Methods

[0014] 1.1 Experimental Animals Twenty-one 4-week-old, SPF-grade, healthy male C57BL / 6 mice weighing 15 ± 2g were selected as experimental animals. They were housed in an experimental animal room according to standard animal husbandry procedures with a 12-h light cycle and an indoor temperature of 22°C ± 2°C. The mouse feed, bedding, and drinking water were all autoclaved, and the animals had free access to water and food.

[0015] 1.2 Main Reagents KA was purchased from MedChemExpress, USA; carmine, methylcellulose, and DON were purchased from Sigma; and ELISA kits were purchased from Jianglai Biotechnology, China.

[0016] 1.3 Trial Grouping Mice were divided into three groups (n = 7): control group (Control), DON-treated group (DON), and KA + DON-treated group (KA + DON). Before the experiment, mice were grouped and acclimated for 3 days. Following the experiment, mice were treated daily. The KA + DON group received 10 mg / kg KA via gavage for 7 days. The CON and DON groups received the same dose of sterile water daily. After pretreatment, the DON group received 4 mg / kg DON via gavage, and the KA + DON group received both DON and KA via gavage for 21 days. Body weights were recorded regularly during this period, and intestinal motility tests and open-field tests were performed before the end of the experiment. After the experiment, blood was collected from the eyeballs of the mice, and the mice were sacrificed by cervical dislocation. Cecal contents were collected for 16S rRNA sequencing of the intestinal microbiota (performed by Hangzhou Lianchuan Biotechnology Co., Ltd.). Intestinal tissues and other tissues were collected for subsequent experiments.

[0017] 1.4 Measurement indicators and methods

[0018] 1.4.1 Intestinal motility function test Prepare a carmine-cellulose mixture and administer 200 μL to each mouse via gavage. Record the gavage time and place the mouse individually in a clean, transparent bowl. Provide jelly as food and water. Observe the mice for defecation approximately 2 hours later. Record the time of the first red stool for each mouse and the number of fecal particles within 2 hours of gavage.

[0019] 1.4.2 Observation of mouse behavior Mice were placed in an open field reaction chamber, and the instrument recorded their movements in the open field for 5 minutes. The software analyzed the movement trajectory and movement time of the mice over the 5-minute period. The software also analyzed the total distance traveled, the distance traveled in the central area, and the time the mice spent exploring the central area.

[0020] 1.4.3 Observation of mouse tissue morphology and structure Mouse intestinal and liver tissues were excised and immersed in 4% paraformaldehyde. After fixation for at least 24 hours, tissue samples were trimmed, paraffin-impregnated, embedded, and sectioned. Paraffin sections were dehydrated and then stained using a HE staining kit and an AB-PAS staining kit, respectively, according to the kit instructions.

[0021] 1.4.4 Serum TNF-α ELISA After collecting blood, centrifuge at 2000 rpm for 10 minutes. Quickly and carefully separate the serum and red blood cells on ice. Mix all required reagents before beginning the experiment. Repeat three times for each standard and test sample well. Dilute the specimen 1:1 with specimen diluent and add 50 μl to the reaction well. Dilute the test serum sample by half and add 50 μl to the reaction well. Immediately add 50 μL of biotinylated antibody. Apply a film to the plate, gently shake the plate to mix the sample, and incubate at 37°C for 1 hour. Pour off the liquid in the well, pat dry on absorbent paper, add 200 μL of wash solution to each well, gently shake to mix, pour off the liquid in the well, and pat dry with absorbent paper. Repeat this process three times. Add 80 μL of streptavidin-HRP to each well, gently shake the plate to mix the sample solution, and incubate at 37°C for 30 minutes. Pour off the liquid in the wells and pat dry on absorbent paper. Add 200 μL of wash buffer to each well, gently vortex to mix, pour off the liquid in the wells, and pat dry with absorbent paper. Repeat this process three times. Add 50 μL each of substrates A and B to each well, gently vortex to mix, and incubate at 37°C for 10 minutes. Shield the wells with tin foil to prevent light from affecting the experiment. Remove the microplate and quickly add 50 μL of stop solution. Measure the results immediately after adding the stop solution. Measure the OD value of each well at a wavelength of 450 nm in a microplate reader. Calculate the TNF-α concentration in the serum sample to be tested based on the standard curve.

[0022] 1.5 Data Analysis All data are expressed as mean ± SEM. GraphPad Prism 8 software was used to create the graphs. Statistical analysis was performed using ANOVA. *p < 0.05 indicates a significant difference, **p < 0.01, and ***p < 0.001 indicate a very significant difference.

[0023] 2. Results

[0024] DON inhibited the weight gain of mice, while kynurenic acid (KA+DON group) could effectively restore the weight gain of mice ( Figure 1 ).

[0025] The open field test showed that the motor ability of mice in the DON group was significantly decreased ( Figure 2 A), The total distance traveled by mice in the DON group within 5 min decreased ( Figure 2 B), the distance traveled in the center of the open field decreased ( Figure 2 C) and decreased open-field central movement time ( Figure 2 D), indicating that DON affects the autonomous movement ability and exploratory behavior of mice, while kynurenic acid can effectively promote the movement ability and exploratory behavior of mice.

[0026] Intestinal pathological sections showed that the length of villi in the jejunum and ileum of mice in the DON group was significantly reduced, inflammatory cells were infiltrated in the jejunum, and the number of colonic goblet cells decreased. Kynurenic acid can effectively improve the intestinal toxicity of DON in mice, mainly manifested by increasing the length of intestinal villi and the number of colonic goblet cells ( Figure 3 AB), and effectively restored the intestinal motility function of mice and promoted fecal excretion ( Figure 3 CD). In addition, kynurenic acid effectively inhibited the expression level of inflammatory factor TNF-α ( Figure 3 E). Mouse liver pathological sections showed that kynurenic acid could effectively inhibit DON-induced inflammatory cell infiltration in mouse liver tissue ( Figure 3 F).

[0027] By detecting and analyzing the 16S rRNA of the intestinal flora in the cecum of mice, the results showed that kynurenic acid can promote the abundance of bacteria with stress tolerance in the cecum of mice ( Figure 4 A). At the family level, DON increased the relative abundance of pro-inflammatory Helicobacteraceae bacteria and reduced the relative abundance of anti-inflammatory Oxalobacteraceae and Muribaculaceae bacteria in the mouse intestine. Kynurenic acid effectively modulated the intestinal flora, reducing the relative abundance of pro-inflammatory microorganisms and increasing the relative abundance of anti-inflammatory microorganisms ( Figure 4 B). In addition, kynurenic acid can also promote the abundance of beneficial bacteria such as Akkermansia ( Figure 4 C).

[0028] 3. Conclusion

[0029] Kynurenic acid can effectively alleviate the toxic effects induced by vomitoxin, mainly by effectively promoting animal weight gain, improving intestinal function, inhibiting the release of inflammatory factors and regulating intestinal flora.

Claims

1. The use of kynurenic acid in products for the prevention and treatment of vomitoxin toxicity.

2. The use according to claim 1, characterized in that: The product uses kynurenic acid as an active ingredient and is prepared into a pharmaceutical preparation for preventing and treating the toxicity of vomitoxin.

3. The use according to claim 2, characterized in that: The pharmaceutical preparation comprises one of an injection, a powder, a granule and a tablet.

4. The use according to claim 1, characterized in that: The product uses kynurenic acid as an active ingredient and is prepared into an animal health product, enteral nutrition agent, and feed additive for preventing and treating the toxicity of vomitoxin.

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