Use of sodium beta-hydroxybutyrate in improving intestinal mucosal immunity of pigs

Abstract

The application provides application of sodium beta-hydroxybutyrate in improving pig intestinal mucosal immunity, and sodium beta-hydroxybutyrate is used for preparing a regulator or / and biological product for improving pig intestinal mucosal immunity. The sodium beta-hydroxybutyrate can effectively reduce the secretion level of pro-inflammatory cytokines in pig serum, promote the proliferation and activation of pig intestinal mucosal lymphocytes, and especially can specifically activate a TCR signal pathway and start the immune activation of intestinal T cells, so as to improve the intestinal mucosal immune function of pigs. The application value of the sodium beta-hydroxybutyrate as a precise regulator of pig intestinal mucosal immunity is clarified, the limitation of traditional indirect regulation modes depending on antibiotics, probiotics and the like is broken through, and a targeted regulation scheme directly acting on intestinal immune cells is provided.

Description

Technical Field

[0001] This invention belongs to the field of animal bioengineering and animal immune regulation technology, specifically relating to the application of sodium β-hydroxybutyrate in enhancing the immunity of the intestinal mucosa in pigs. Background Technology

[0002] The porcine intestinal mucosal immune system comprises components such as gut-associated lymphoid tissue, intraepithelial lymphocytes, lamina propria lymphocytes, and secretory immunoglobulins. This system plays a crucial role in maintaining intestinal homeostasis, resisting pathogen invasion, and regulating the body's inflammatory response. During weaning, piglets' intestinal tissue is not fully developed, and their immune function is not yet fully established. Coupled with the multiple stimuli brought about by weaning stress, these animals are highly susceptible to intestinal diarrhea, causing significant economic losses to the pig farming industry. Therefore, developing technologies to enhance the intestinal mucosal immunity of pigs is a key research direction in the current animal husbandry and veterinary industry.

[0003] Currently, the technical solutions used to improve the intestinal immune status of pigs can be mainly divided into the following categories: (1) Antibiotic growth promoters: These substances can indirectly improve intestinal health and enhance animal production performance by inhibiting the proliferation of harmful intestinal microorganisms and alleviating the body's inflammatory response. However, long-term irrational use of antibiotics can induce a series of problems such as bacterial resistance, drug residues, and intestinal microecological system disorder.

[0004] (2) Probiotic microecological preparations: Probiotics mainly exert their immune-regulating effects indirectly by reshaping the composition of the intestinal flora and optimizing the intestinal microecological environment; prebiotics, on the other hand, indirectly enhance the level of intestinal immunity by promoting the proliferation of beneficial microorganisms in the body's intestines. However, probiotic preparations have shortcomings such as weak survival ability of live bacteria in vitro, poor tolerance in gastric acid and bile salt environments, and low intestinal colonization efficiency; at the same time, product storage conditions and individual animal differences can interfere with their effects, resulting in large fluctuations in actual effects and poor predictability.

[0005] (3) Organic acids and their salts (such as sodium butyrate): Sodium butyrate belongs to short-chain fatty acid salts and is now widely used in livestock and poultry farming. Existing studies have confirmed that this type of substance can improve the morphology of animal intestinal tissue, strengthen the intestinal barrier function, and play a certain regulatory role in the intestinal flora. However, sodium butyrate is difficult to act directly on intestinal mucosal immune cells, and the relevant pathways and molecular mechanisms are still unclear, with obvious limitations in its target and regulatory scope.

[0006] (4) Plant extracts, plant essential oils and antimicrobial peptides: Plant extracts such as carvacrol and cinnamaldehyde, as well as antimicrobial peptides, have been used in research related to pig intestinal health. Most of these substances only exhibit antibacterial and antioxidant activities, and there is a lack of systematic research on their targeted regulation of the intestinal mucosal immune system; at the same time, they generally suffer from poor stability of active components, unclear target points, and significant fluctuations in actual application effects. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing an application of sodium β-hydroxybutyrate in improving the immunity of the intestinal mucosa in pigs. Sodium β-hydroxybutyrate enhances the immune function of the intestinal mucosa in pigs by activating the TCR signaling pathway and initiating T cell immune activation.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the application of sodium β-hydroxybutyrate in improving the immunity of porcine intestinal mucosa, wherein the sodium β-hydroxybutyrate is used to prepare a modulator and / or biological product for improving the immunity of porcine intestinal mucosa.

[0009] Preferably, the application includes one or more of the following applications: The sodium β-hydroxybutyrate is used to promote the proliferation and activation of lymphocytes in the jejunum and ileum; The sodium β-hydroxybutyrate is used to reduce serum levels of IL-1β, IL-4, IL-6 and IL-10; The sodium β-hydroxybutyrate is used to increase the relative expression levels of GZMA and PRF1 in T cells.

[0010] Preferably, the sodium β-hydroxybutyrate enhances the immune function of the porcine intestinal mucosa by activating the TCR signaling pathway, initiating T cell immune activation.

[0011] Preferably, the breed of pig is the Bama miniature pig.

[0012] Compared with the prior art, the present invention has the following advantages: 1. The present invention has controllable cost and is easy to promote: sodium β-hydroxybutyrate is widely available and the preparation process is mature.

[0013] 2. This invention clarifies for the first time the application value of sodium β-hydroxybutyrate as a precise regulator of porcine intestinal mucosal immunity, breaking through the limitations of traditional indirect regulation methods such as antibiotics and probiotics, and providing a targeted regulation scheme that directly acts on intestinal immune cells.

[0014] 3. The sodium β-hydroxybutyrate of the present invention can effectively reduce the secretion level of pro-inflammatory cytokines in porcine serum, significantly promote the proliferation and activation of porcine intestinal mucosal lymphocytes, and in particular, can specifically activate the TCR signaling pathway, initiate the immune activation of intestinal T cells, and enhance the activity of cytotoxic T cells.

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 This is a graph showing the blood ketone monitoring results of pigs in the control group and experimental group in Example 1 of the present invention.

[0017] Figure 2 These are HE staining images of the jejunum, ileum, and mesenteric lymph nodes of pigs in the control and experimental groups in Example 1 of this invention.

[0018] Figure 3 This is a proteomic analysis diagram of jejunal tissue from the control group and experimental group in Example 1 of the present invention.

[0019] Figure 4 This is a graph showing the detection of cytokines in the blood of the experimental group and the control group in Example 1 of the present invention.

[0020] Figure 5 This is a graph showing the relative expression levels of GZMA and PRF1 in T cells of the experimental and control groups in Example 1 of the present invention; wherein, Figure A represents GZMA; and Figure B represents PRF1. Detailed Implementation

[0021] Example 1

[0022] This example demonstrates the application of sodium β-hydroxybutyrate in enhancing the immune response of the porcine intestinal mucosa.

[0023] Sodium β-hydroxybutyrate in this embodiment was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with product number S832832-500g and chemical formula C4H7NaO3.

[0024] Twelve 2-month-old Bama miniature pigs (8kg-10kg body weight) were randomly selected for a feeding experiment and divided into two groups. The experimental group was fed sodium β-hydroxybutyrate once a day at a fixed time (3mmol / kg body weight, dissolved in drinking water). The control group was fed sodium bicarbonate orally (3mmol / kg body weight, dissolved in drinking water) to eliminate the effect of sodium ions on the experimental pigs. The feeding was carried out for a total of 3 days. (1) Blood ketone monitoring in experimental pigs: Blood samples were collected daily to monitor blood ketone levels (sampling was performed at 0h, 1.5h, 8h, and 24h after feeding on the first day; at 8h and 24h after feeding on the second day; and at 8h and 24h after feeding on the third day). Results were as follows: Figure 1 As shown, the concentration of β-hydroxybutyrate (β-HB) in the blood of pigs reached its highest value 8 hours after oral administration of sodium β-hydroxybutyrate, and decreased 24 hours after oral administration.

[0025] (2) Oral administration of sodium β-hydroxybutyrate promotes the proliferation and activation of intestinal lymphocytes: After feeding for 3 days, the pigs were euthanized, and tissue samples (jejunum, ileum, and mesenteric lymph nodes) were collected for HE staining analysis. The results are as follows: Figure 2 As shown in the figure (scale bar: 200 μm), compared with the control group, the number of lymphocytes in the jejunum and ileum lamina propria of pigs in the oral β-hydroxybutyrate group (experimental group) showed an increased trend. Specifically, lymphocytes were densely distributed in the lamina propria, forming small clusters in some areas, and their proportion with surrounding epithelial cells and stromal cells was significantly increased. Furthermore, the lymphocyte morphology was predominantly activated (increased nucleocytoplasmic ratio, clear cytoplasm), indicating that these cells were in a functionally active state. Simultaneously, the mesenteric lymph nodes in the experimental group showed enlarged lymphoid follicles and increased lymphocyte density, suggesting that oral β-hydroxybutyrate can promote the proliferation and activation of lymphocytes in the porcine mesenteric lymph nodes.

[0026] (3) Sodium β-hydroxybutyrate initiates immune activation of intestinal T cells by specifically activating the TCR signaling pathway: Jejunal tissue was selected for proteomics analysis, and the results are as follows: Figure 3 As shown, the differentially expressed proteins were significantly enriched in the T cell receptor signaling pathway, indicating that sodium β-hydroxybutyrate initiates the immune activation program of intestinal T cells by specifically activating the TCR signaling pathway.

[0027] (4) Oral administration of sodium β-hydroxybutyrate to remodel the immune response in pigs: Blood and serum were collected for cytokine antibody microarray analysis [IL-1β (interleukin-1β), IL-4 (interleukin-4), IL-6 (interleukin-6), IL-8 (interleukin-8), IL-10 (interleukin-10), IL-12p40p70, GM-CSF (granulocyte-macrophage colony-stimulating factor), IFNg (gene encoding interferon-γ), TGFb1 (transforming growth factor β1), TNFa (tumor necrosis factor-α)] to comprehensively evaluate the regulatory effect of sodium β-hydroxybutyrate on the immune response of porcine intestinal mucosa.

[0028] The results are as follows Figure 4 As shown, compared with the control group, the serum secretion of inflammatory cytokines IL-1β, IL-4, IL-6, and IL-10 was significantly reduced and IFNg was significantly increased in pigs that were orally administered sodium β-hydroxybutyrate (experimental group). This result indicates that oral administration of sodium β-hydroxybutyrate can remodel the immune response in pigs.

[0029] (5) Sodium β-hydroxybutyrate can activate cytotoxic T cell immune responses: CD3 of healthy adult pigs was sorted using magnetic beads. + T cells in ImmunoCult TM XF T cells were cultured in vitro in serum-free medium (STEM CELL, Canada) and stimulated with anti-CD3e IgG (2 μg / mL), sodium β-hydroxybutyrate (5 mM), and treated with PU139 (a pan-histone acetyltransferase inhibitor, 10 μM). The relative expression levels of GZMA (granzyme A) and PRF1 (perforin 1) were then detected by RT-qPCR. The nucleotide sequences of the GZMA upstream primer and downstream primer are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

[0030] The results are as follows Figure 5 As shown in A and B, sodium β-hydroxybutyrate can further upregulate the expression of anti-CD3e IgG-induced GZMA and PRF1, indicating that sodium β-hydroxybutyrate can activate porcine cytotoxic T cell responses in a histone acetylation-dependent manner.

[0031] In summary, addressing the shortcomings of existing porcine intestinal mucosal immune regulation technologies, such as unclear immune regulation mechanisms and a lack of precise targeted regulation methods, as well as the current situation of insufficient application and limited research on the mechanism of action of sodium β-hydroxybutyrate in the field of porcine intestinal immunity, this invention, for the first time, clearly demonstrates the application value of sodium β-hydroxybutyrate as a precise regulator of porcine intestinal mucosal immunity. It breaks through the limitations of traditional indirect regulation methods relying on antibiotics and probiotics, providing a targeted regulation scheme that directly acts on intestinal immune cells. Furthermore, it reveals that sodium β-hydroxybutyrate enhances porcine intestinal mucosal immune function by activating the TCR signaling pathway and initiating T cell immune activation, thus resolving the problems of unclear immune regulation mechanisms and unstable effects in existing technologies.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. An application of sodium β-hydroxybutyrate in enhancing porcine intestinal mucosal immunity, characterized in that, The sodium β-hydroxybutyrate is used to prepare modulators and / or biological products that enhance the immunity of the porcine intestinal mucosa.

2. The application of sodium β-hydroxybutyrate according to claim 1 in enhancing porcine intestinal mucosal immunity, characterized in that, The application includes one or more of the following: The sodium β-hydroxybutyrate is used to promote the proliferation and activation of lymphocytes in the jejunum and ileum; The sodium β-hydroxybutyrate is used to reduce serum levels of IL-1β, IL-4, IL-6 and IL-10; The sodium β-hydroxybutyrate is used to increase the relative expression levels of GZMA and PRF1 in T cells.

3. The application of sodium β-hydroxybutyrate according to claim 1 in enhancing porcine intestinal mucosal immunity, characterized in that, The sodium β-hydroxybutyrate enhances the immune function of the pig intestinal mucosa by activating the TCR signaling pathway, initiating T cell immune activation.

4. The application of sodium β-hydroxybutyrate according to claim 1 in enhancing porcine intestinal mucosal immunity, characterized in that, The breed of pig is the Bama miniature pig.

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