Application of beta-aminoisobutyric acid in preparation of preparation for treating animal mastitis

Abstract

The invention relates to the technical field of biological medicines, in particular to application of beta-aminoisobutyric acid in preparation of a preparation for treating animal mastitis. The invention discloses application of beta-aminoisobutyric acid in preparation of a preparation for treating animal mastitis. The beta-aminoisobutyric acid provided by the invention is derived from the posterior intestine content of the healthy saanen milk goat with the lowest somatic cell count in the milk obtained by large group screening. According to the present invention, the proliferation efficiency of dairy cow mammary epithelial cells can be significantly promoted, and the expression of the related proteins involved in the endoplasmic reticulum protein quality control pathway can be promoted in the LPS-induced dairy cow mammary epithelial cell inflammation model so as to enhance the folding efficiency of the proteins during the inflammation process; the removal of mistakenly folded and unfolded proteins is accelerated, and finally, the concentration of proinflammatory cytokines is reduced, and the concentration of anti-inflammatory cytokines is increased, so that the inflammatory phenotype is relieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of beta-aminoisobutyric acid in preparing a preparation for treating animal mastitis. Background Art

[0002] Mastitis is one of the most challenging diseases in the dairy industry, causing significant economic losses to the global dairy industry by reducing milk quality and leading to the culling of sows. Mastitis is mainly divided into acute clinical mastitis and difficult-to-detect subclinical mastitis. The course of both types of diseases is accompanied by an increase in milk somatic cell count (SCC) and a systemic inflammatory phenotype. Traditional diagnosis and treatment of mastitis relies on isolating specific pathogens from milk to identify the source of infection and targeted treatment with antibiotics. However, this treatment method will result in antibiotic residues in milk, thereby affecting the value of raw milk and its by-products, and will also induce increased resistance of opportunistic pathogens associated with mastitis. Therefore, there is an urgent need to develop safer and more comprehensive treatments for ruminant mastitis.

[0003] The intestinal flora is a complex ecosystem composed of highly diverse symbiotic microorganisms, forming a dynamically balanced microecological environment to maintain the host's immune defense function. Its homeostatic imbalance may aggravate the pathological process of mastitis through the "intestinal-mammary" axis regulation mechanism, and reconstructing the intestinal flora through microbial transplantation can effectively alleviate the mammary inflammatory response. Studies have further confirmed that specific probiotic strains can regulate the immune response of mammary tissue and maintain mammary barrier function by producing bioactive substances such as tryptophan derivatives and short-chain fatty acids through metabolism. In view of the technical bottlenecks and biosafety risks of existing in vitro culture systems of probiotics, systematic screening of their functional metabolites and verification of their therapeutic efficacy against ruminant mastitis has become a research direction with greater translational value.

[0004] β-Aminoisobutyric acid (BAIBA), a small molecule derived from branched-chain amino acids, is considered a novel endogenous protective actin. BAIBA plays an important role in numerous life processes, particularly inflammation. Previous studies have shown that BAIBA stimulates phosphorylation of AMP-activated protein kinase (AMP-activated protein kinase) in mouse adipocytes in a dose-dependent manner and inhibits the secretion of the proinflammatory cytokines TNF-α and MCP-1. Furthermore, BAIBA has been shown to inhibit TNF-α-induced apoptosis and cytoplasmic degradation of all neurons in the nucleus pulposus in an AMPK / NF-κB axis-dependent manner. However, no studies have reported the role of BAIBA in the treatment of mastitis in ruminants. Summary of the Invention

[0005] The purpose of the present invention is to provide a functional metabolite - β-aminoisobutyric acid, which can alleviate the disease phenotype of ruminant mastitis, reduce the somatic cell count in milk, reduce the concentration of pro-inflammatory cytokines in mammary epithelial cell homogenate, and increase the concentration of anti-inflammatory cytokines.

[0006] The β-aminoisobutyric acid provided by the present invention is derived from the hindgut contents of healthy Saanen goats with the lowest somatic cell count in their milk, obtained through large-scale population screening. It can significantly promote the proliferation efficiency of bovine mammary epithelial cells and, in an LPS-induced bovine mammary epithelial cell inflammation model, promote the expression of NEF, Hsp40, GPR94, EDEM, ERManI, PDIs, Hsp70, and Ubc6 / 7 proteins involved in the endoplasmic reticulum protein quality control pathway, thereby enhancing protein folding efficiency during inflammation and accelerating the clearance of misfolded and unfolded proteins. Ultimately, it reduces the concentrations of proinflammatory cytokines IL-1β, IL-6, and TNF-α, and increases the concentration of the anti-inflammatory cytokine IL-10, thereby alleviating the inflammatory phenotype.

[0007] In addition, teat perfusion treatment of dairy goats suffering from clinical and subclinical mastitis further confirmed that β-aminoisobutyric acid can significantly reduce the number of somatic cells in milk and inhibit the progression of mastitis.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides the use of beta-aminoisobutyric acid in preparing a preparation for treating animal mastitis; the functional metabolite has a chemical formula of C4H9NO2, a PubChem SID of 6143, and a CAS NO. of 4249-19-8.

[0010] Preferably, the animal is a ruminant.

[0011] Preferably, the ruminant is a cattle or a sheep.

[0012] Preferably, the mastitis is caused by infection with Escherichia coli, Pseudomonas, Klebsiella, Streptococcus agalactiae or Staphylococcus aureus.

[0013] Preferably, the effective concentration of the β-aminoisobutyric acid in the preparation for treating animal mastitis is 5 to 20 μM, and the feeding or administration interval is 12 hours.

[0014] Preferably, the mastitis is clinical mastitis or subclinical mastitis.

[0015] Preferably, the β-aminoisobutyric acid can increase the proliferation efficiency of animal mammary epithelial cells, and can reduce the concentrations of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in cell homogenate, and increase the concentration of anti-inflammatory cytokine IL-10.

[0016] Preferably, the preparation is a medicine, feed or feed additive.

[0017] Preferably, the drug further comprises excipients; the excipients are one or more of sucrose, sorbitol, starch, dextrin, powdered sugar, sodium hydroxymethyl cellulose, physiological saline and sodium methyl cellulose.

[0018] Preferably, the dosage form of the drug is tablets, capsules, granules, injections, creams or ointments.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The beta-aminoisobutyric acid provided by the invention can promote the proliferation of mammary epithelial cells of ruminants.

[0021] The β-aminoisobutyric acid provided by the present invention has the function of significantly reducing the concentrations of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in an LPS-induced cow mammary epithelial cell inflammation model, and increasing the concentration of the anti-inflammatory cytokine IL-10.

[0022] The β-aminoisobutyric acid provided by the present invention can also enhance the folding efficiency of proteins during inflammation by promoting the expression of NEF, Hsp40, GPR94, EDEM, ERManI, PDIs, Hsp70 and Ubc6 / 7 proteins involved in the protein quality control pathway in the endoplasmic reticulum, thereby accelerating the clearance of misfolded and unfolded proteins, and ultimately reducing the concentrations of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and increasing the concentration of the anti-inflammatory cytokine IL-10, thereby alleviating the inflammatory phenotype.

[0023] The beta-aminoisobutyric acid provided by the present invention can also alleviate the disease phenotype of ruminants suffering from clinical or subclinical mastitis and significantly reduce the number of cells in their milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is the determination of systemic inflammatory factor concentrations and identification of hindgut marker microbial species in healthy and mastitis goats determined based on large-scale population screening in Example 1 of the present invention; A is the screening of mastitis and healthy goats from a large lactating population of Saanen dairy goats, B is the lactation phenotype of healthy and mastitis goats, C is the determination of pro-inflammatory cytokine concentrations in the milk of healthy and mastitis goats, D is the determination of pro-inflammatory cytokine concentrations in the serum of healthy and mastitis goats, E is the identification of hindgut marker microbial species in healthy and mastitis goats, and F is the correlation analysis between hindgut marker microbial species and inflammatory phenotypes in healthy and mastitis goats.

[0025] Figure 2 In Example 2 of the present invention, β-aminoisobutyric acid was determined to be a functional metabolite with potential therapeutic effect for mastitis based on the analysis of functional genes and metabolites of hindgut microorganisms of two groups of dairy goats; Figure 2 A shows the correlation network of hindgut microbial species in two groups of dairy goats; Figure 2 B shows the identification of Clostridiabacterium, a microbial marker in the hindgut of healthy sheep, as a core species mediating quorum sensing of the microbial community; Figure 2 C is the enrichment analysis of differentially expressed microbial functional genes in the hindgut of dairy goats; Figure 2 D is the functional enrichment analysis of differential metabolites in the hindgut of dairy goats; Figure 2 E is the enrichment of β-aminoisobutyric acid in the hindgut of healthy sheep determined by analyzing differentially functional microbial genes and differential metabolites; Figure 2 F is the correlation analysis between β-aminoisobutyric acid and inflammatory markers in dairy goats with mastitis.

[0026] Figure 3 It is the therapeutic effect of β-aminoisobutyric acid on the inflammatory phenotype of the LPS-induced bovine mammary epithelial cell inflammation model in Example 3 of the present invention; A and B are explorations of the concentration and treatment time of the LPS-induced bovine mammary epithelial inflammatory cell model, C is the effect of β-aminoisobutyric acid on the proliferation and cytotoxicity of bovine mammary epithelial cells, and D to G are the effects of β-aminoisobutyric acid on the concentrations of proinflammatory cytokines IL-1β, IL-6 and TNF-α and the anti-inflammatory cytokine IL-10 in the LPS-induced bovine mammary epithelial cell inflammation model.

[0027] Figure 4This is an exploration of the inflammatory phenotype mechanism of the LPS-induced bovine mammary epithelial cell inflammation model treated with β-aminoisobutyric acid based on DIA proteomics in Example 4 of the present invention; A shows the principal coordinate analysis based on the protein expression profiles of the MAC group, LPS group and β-aminoisobutyric acid group, B is the pathway with significantly differential protein enrichment after LPS treatment compared with the MAC group, C is the pathway enriched for proteins that were significantly downregulated after LPS treatment but whose expression was restored after β-aminoisobutyric acid treatment, and D~K quantified the differences in the expression levels of NEF, Hsp40, GPR94, EDEM, ERManI, PDIs, Hsp70 and Ubc6 / 7 proteins involved in the endoplasmic reticulum protein quality control pathway.

[0028] Figure 5 This is the effect of β-aminoisobutyric acid treatment on the somatic cell count in the milk of goats with subclinical and clinical mastitis in Example 5 of the present invention. A represents that β-aminoisobutyric acid can significantly reduce the somatic cell count in the milk of goats with subclinical mastitis, and B represents that β-aminoisobutyric acid can significantly reduce the somatic cell count in the milk of goats with clinical mastitis. DETAILED DESCRIPTION

[0029] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] The reagents required for solution preparation in the following examples were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dairy goats with subclinical and clinical mastitis were provided by Shaanxi Bofule Guanzhong Dairy Goat Breeding Co., Ltd. Kits for quantifying inflammatory cytokine concentrations in serum, milk, and cell suspensions were purchased from Shanghai Kexing Trading Co., Ltd.

[0031] Example 1: Analysis of characteristic microorganisms and quorum sensing core species in the hindgut of healthy and mastitis dairy goats based on metagenomics

[0032] The present invention selected 167 Saanen dairy goats in the middle stage of lactation (parity: 2.17±0.40, lactation days: 121.47±7.95). All goats were raised in the same pen with stable temperature and good ventilation. They were milked at 5:00 am and 5:00 pm every day and fed immediately after milking. The average milk production of the goats was obtained by collecting the daily milk production in the first and fourth weeks of August 2022 to obtain the average value as their final average milk production. On the last day of milk production data collection, milk samples from the two milking points were mixed in a ratio of 3:2 and divided into two replicates for milk component analysis and milk somatic cell count quantification.

[0033] The following day, fresh feces were collected from the anus using abdominal massage from sheep with the highest (Mastitis, n = 6) and lowest (Healthy, n = 6) somatic cell counts. These feces were immediately snap-frozen in liquid nitrogen and subsequently stored at -80°C for metagenomic and non-targeted metabolomics sequencing. Serum samples were collected from both the Mastitis and Healthy groups by venous blood sampling and stored at -20°C for analysis of biochemical parameters and quantification of inflammatory markers.

[0034] For metagenomic sequencing and data analysis, DNA was extracted from fecal samples from the Mastitis and Healthy groups using a micro-bead milling protocol. The concentration and purity of the extracted DNA were assessed using a NanoDrop 2000 UV-Vis spectrophotometer, and the quality of the extracted DNA samples was verified by 1% agarose gel electrophoresis. Paired-end libraries were constructed from the fragmented DNA using NEXTFLEX Rapid DNA-Seq. Metagenomic sequencing was then performed using the Illumina NovaSeq 6000 platform, generating 150-bp paired-end reads. Six fecal samples from dairy goats generated a total of 142.11 Gb of raw sequencing data, with an average of 11.84 Gb per sample. The raw data generated by Illumina sequencing were stripped of adapters and low-quality reads using fastp (v0.20.0). To remove contamination from host DNA in reads, BWA-MEM (v0.7.17) was used to align reads to the goat reference genome to obtain high-quality sequences. A total of 104.14 Gb of high-quality sequences were obtained, with an average of 8.68 Gb per sample and an average host contamination rate of 26.76%.

[0035] Subsequently, Megahit (v1.2.9) was used to assemble high-quality sequences into contigs, yielding an average of 777,409.50 contigs per sample with an average N50 of 639.69 bp. The assembled contigs were further analyzed using Prodigal (v2.6.3) for ORF prediction, ultimately yielding 15.78 million ORFs with an average length of 444.96 bp. After removing ORFs less than 100 bp in length, CD-HIT (v4.8.1) was used to cluster the ORFs, resulting in a goat hindgut microbial NR gene set with an average length of 518.74 bp and containing 4,399,515 non-redundant (NR) predicted genes. For species and functional annotation, EMBOSS Transeq (v6.6.0.0) was used to translate the predicted NR gene sequences into predicted NR protein sequences. High-quality reads from each sample were then mapped to the NR gene set using SOAP2 (v2.21) to obtain predicted NR gene abundance for each sample. DIAMOND (v2.1.8.162) was used to compare against the NCBI-NR database with an e-cutoff of 1e-5 to obtain species annotation information at each taxonomic level. LefSe analysis was used to identify species with significant differences in hindgut microbiome abundance between Mastitis and Healthy sheep, which served as microbial biomarkers for both groups.

[0036] Figure 1 A shows the screening process of sheep in the Mastitis and Health groups and the quantitative results of somatic cell counts in milk; Figure 1 B shows the differences in lactation phenotypes between sheep in the Mastitis and Health groups; Figure 1 C shows the difference in inflammatory cytokine concentrations in milk of sheep in the Mastitis and Health groups; Figure 1 D shows the differences in inflammatory cytokine concentrations in the serum of sheep in the Mastitis and Health groups; Figure 1 E shows the identification of marker microbial species in the hindgut of sheep in the Mastitis and Health groups; Figure 1 F shows the correlation analysis between hindgut markers and mastitis inflammatory phenotype in Mastitis and Health groups.

[0037] Results showed that compared with the healthy group, dairy goats infected with mastitis exhibited the following characteristics: a significant increase in milk somatic cell count (P<0.001), a significant decrease in milk production (P<0.05), a decrease in lactose content (P<0.05), and an increase in milk protein content (P<0.05). Notably, milk concentrations of the proinflammatory cytokines MPO, IL-6, IL-1β, and TNF-α were significantly increased in the diseased group (P<0.05). Furthermore, proinflammatory cytokines LPS, IL-6, IL-1β, and TNF-α were significantly enriched in the serum (P<0.01). These results confirm that mastitis not only leads to a decline in lactation performance but also triggers systemic immune metabolic imbalances through a cascade of proinflammatory cytokines.

[0038] Subsequently, we used metagenomic sequencing and LefSe analysis to identify signature microbial species in the hindgut of sheep in the Mastitis and Healthy groups. We confirmed that several Clostridia spp., including Clostridia bacterium, which had not been accurately taxonomically assigned at the species level, were enriched in the Healthy group, while Lachnospiraceae bacterium was identified as a microbial marker in the Mastitis group (LDA>2, P<0.05). Spearman correlation analysis revealed that the unclassified Clostridia spp. were significantly negatively correlated with the inflammatory phenotype, while the signature microorganisms in the Mastitis group were significantly positively correlated with pro-inflammatory parameters (|R|>0.5, P<0.05).

[0039] Example 2 Combined analysis of functional genes and metabolomics of hindgut microorganisms in dairy goats confirmed that β-aminoisobutyric acid is a functional metabolite that can potentially treat mastitis

[0040] KO annotation was performed on the NR predicted genes obtained from the metagenomic analysis of Example 1 based on KofamScan, and HMMER was used to compare them against the Kofam database (a hidden Markov database designed specifically for KO). For KEGG enrichment analysis, Z-score was used as a criterion to assess whether a specific pathway was enriched in each group.

[0041] For untargeted metabolomics sequencing and analysis, fecal samples from Mastitis and Health sheep were thawed on ice, and 50 mg (±1 mg) of each sample was homogenized with 500 μL of ice-cold methanol / water (70%, v / v). The samples were vortexed for 3 minutes, sonicated in an ice-water bath for 10 minutes, and then vortexed again for 1 minute. After this, the samples were centrifuged at 12,000 rpm for 10 minutes. After centrifugation, 200 μL of the supernatant was incubated at -20°C for 30 minutes and then centrifuged again at 12,000 rpm for 30 minutes at 4°C. 350 μL of the supernatant was then transferred to a vacuum concentrator and evaporated to dryness. The dry residue was reconstituted with 150 μL of 70% methanol / water solution, vortexed for 3 minutes, and sonicated in an ice-water bath for 10 minutes. Finally, the supernatant was centrifuged at 12,000 rpm for 3 minutes at 4°C, and 120 μL of the supernatant was analyzed by UHPLC-ESI-MS / MS.

[0042] Ultra-high-performance liquid chromatography (UPLC) conditions were as follows: a Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 2.1 mm × 100 mm); a mobile phase consisting of ultrapure water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid; a column temperature of 40°C; a flow rate of 0.4 mL / min; and an injection volume of 2 μL. Mass spectrometry was acquired in both positive and negative ion modes. LC-MS / MS raw data were processed using the Analyst 1.6.3 software package. To expand the scope of metabolite identification, metabolite annotation was performed based on the self-built MWDB (METWARE database) and public databases (Metlin, HMDB, KEGG, and MoNA). Differential metabolites were screened using a threshold of VIP > 1 and P < 0.05 (Wilcoxon rank-sum test). Identified metabolites were mapped to KEGG pathways, and significantly enriched metabolic pathways were identified using a hypergeometric test.

[0043] Figure 2 A shows the correlation network of hindgut microbial species in two groups of dairy goats; Figure 2 B shows the identification of Clostridia bacterium, a microbial marker in the hindgut of healthy sheep, as a core species mediating quorum sensing of the microbial community; Figure 2 C is the enrichment analysis of differentially expressed microbial functional genes in the hindgut of dairy goats; Figure 2 D is the functional enrichment analysis of differential metabolites in the hindgut of dairy goats; Figure 2 E is the enrichment of β-aminoisobutyric acid in the hindgut of healthy sheep determined by analyzing differentially functional microbial genes and differential metabolites; Figure 2 F is the correlation analysis between β-aminoisobutyric acid and inflammatory markers in dairy goats with mastitis.

[0044] Results showed that Clostridia bacteria, a core microbial interaction in the hindgut of healthy dairy goats (|R|>0.5, P<0.05), extensively encode the quorum sensing-related genes IsrA, IsrB, IsrC, IsrD, and IsrR, thereby regulating the metabolic functions of the intestinal microbiota. Furthermore, functional enrichment analysis based on differentially expressed microbial functional genes and metabolites revealed that valine, leucine, and isoleucine degradation were significantly enriched metabolic pathways. Therefore, we further investigated the differentially expressed microbial functional knockout (KOs) and metabolites involved in this pathway and found that the expression of several microbial functional genes, such as AvtA, vorA, vorB, vorD, and ACADM, was increased in the healthy group. Furthermore, the concentration of L-valine, a precursor for β-aminoisobutyric acid synthesis, was significantly increased in the healthy group (P<0.05), ultimately leading to a significant increase in β-aminoisobutyric acid concentration in the healthy group (P<0.01). Subsequently, Spearman correlation analysis revealed a significant negative correlation between β-aminoisobutyric acid and multiple mastitis-related inflammatory markers (|R|>0.5, P<0.05). Because β-aminoisobutyric acid has excellent circulatory stability, it is expected to exert its anti-inflammatory effects on mastitis through the "gut-mammary gland" axis.

[0045] Example 3 Therapeutic Effect of β-aminoisobutyric Acid on LPS-Induced Inflammatory Phenotype of Cow Mammary Epithelial Cells

[0046] Regarding the culture conditions of bovine mammary epithelial cells, the bovine mammary epithelial cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37° C. and 5% CO 2 . The bovine mammary epithelial cells were stored in a continuously humidified cell culture incubator under standard culture conditions.

[0047] Bovine mammary epithelial cells were cultured for 24 hours after resuscitation. Cell density and growth were observed under a microscope and passaged when the cell density reached approximately 90%. Subsequently, the cells were treated with LPS at concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL for 12 hours, 24 hours, and 48 hours, respectively, to explore the optimal conditions for the LPS-induced inflammatory cell model.

[0048] In order to explore the effect of β-aminoisobutyric acid on the proliferation efficiency of bovine mammary epithelial cells, cells that had been revived, cultured, and passaged were cultured at a rate of 5×10 3Cells were seeded in a 96-well plate at a density of 10 cells / well. After culturing for 24 hours in DMEM medium containing 10% fetal bovine serum to form a monolayer, β-aminoisobutyric acid was added at final concentrations of 5 μM, 10 μM, 15 μM, and 20 μM, respectively, and incubated for 6 h, 12 h, 18 h, and 24 h, respectively. Immediately after the incubation, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C in the dark for 2 hours. The absorbance at 450 nm (reference 650 nm) was immediately measured using a microplate reader, and the cell proliferation efficiency corresponding to each treatment concentration and treatment time was calculated.

[0049] Furthermore, in order to explore the therapeutic effect of β-aminoisobutyric acid on the inflammatory phenotype of the LPS-induced bovine mammary epithelial cell inflammation model, the cells that had been revived, cultured, and passaged were plated at 5×10 7 The cells were seeded in a 6-well plate at a density of cells / well, and the cells were treated with LPS at a concentration of 50 μg / mL for 12 h according to the predetermined inflammatory model induction conditions to induce an inflammatory response.

[0050] Subsequently, mammary epithelial cells in inflammatory state were incubated with β-aminoisobutyric acid at final concentrations of 5μM, 10μM, 15μM, and 20μM for 6h, 12h, 18h, and 24h, respectively, to explore the optimal drug concentration and treatment time for the therapeutic effect of β-aminoisobutyric acid on mammary inflammation. The therapeutic effect of β-aminoisobutyric acid was reflected by the concentrations of proinflammatory cytokines IL-1β, IL-6, and TNF-α and anti-inflammatory cytokine IL-10.

[0051] Figure 3 A, B show the effects of LPS concentration gradient and treatment time on the concentration of proinflammatory factors in bovine mammary epithelial cell suspension; Figure 3 C shows the dose-time dependent effects of β-aminoisobutyric acid concentration-time bivariate on cell proliferation activity and toxicity; Figure 3 D to G show the antagonistic effect of β-aminoisobutyric acid on the inflammatory phenotype of the LPS-induced bovine mammary epithelial cell inflammation model at different concentration gradients and treatment time sequences.

[0052] Results showed that treatment of bovine mammary epithelial cells with 50 μg / mL LPS for 12 hours significantly increased the concentrations of proinflammatory cytokines TNF-α and IL-1β in the cell suspension (P<0.001), establishing this condition as a standardized inflammation induction protocol. The safety of β-aminoisobutyric acid (β-AIBA) was assessed using the CCK-8 assay, revealing that no significant inhibitory effect on cell proliferation was observed at any concentration (5-20 μM) or treatment time (6-24 hours) (P>0.05). Pretreatment with 10 μM β-AIBA for 12 hours significantly increased cell proliferation (P<0.05). Based on the above inflammatory model, cytokine levels were measured after incubation with 5, 10, 15, and 20 μM β-aminoisobutyric acid for 6, 12, 18, and 24 hours. The results showed that during the 12-hour treatment period, 5-20 μM β-aminoisobutyric acid significantly reduced the concentrations of the proinflammatory cytokines IL-1β, IL-6, and TNF-α (P < 0.01), while significantly increasing the concentration of the anti-inflammatory cytokine IL-10 (P < 0.001). These data indicate that β-aminoisobutyric acid can effectively ameliorate LPS-induced inflammatory responses in mammary epithelial cells.

[0053] Example 4: β-aminoisobutyric acid regulates the clearance of misfolded proteins and alleviates inflammatory responses through the endoplasmic reticulum unfolded protein response

[0054] To reveal the mechanism of β-aminoisobutyric acid in treating mammary epithelial cell inflammation, we washed the cell samples of the control group (MAC group), LPS (LPS group) and β-aminoisobutyric acid (BAIBA group) with PBS three times and collected them in a centrifuge tube. The cells were centrifuged at 1500 × g for 5 min at 4 ° C and the supernatant was discarded. Thereafter, the corresponding RIPA lysis buffer (0.1% protease inhibitor) was added according to the cell amount and lysed on ice for 30 min. The lysate was centrifuged at 12000 × g for 15 min at 4 ° C, the supernatant was collected and lysed using Omni-Easy TM The Instant BCA Protein Assay Kit uses the BCA method to determine protein concentration. Collected protein samples were stored at -80°C. For protein digestion and peptide extraction, 100 μg of protein sample was equilibrated in 100 mM triethylammonium bicarbonate (TEAB) buffer, followed by reduction (10 mM tris(2-carboxyethyl)phosphine [TCEP], 37°C for 60 min), alkylation (40 mM iodoacetamide, dark-protected, room temperature) and acetone precipitation (-20°C for 4 h, centrifugation at 10,000 × g). The precipitate was then reconstituted in 100 μL of 100 mM TEAB and digested overnight at 37°C with Trypsin at a mass ratio of 1:50 (enzyme:protein). The digest was then solubilized in 0.1% trifluoroacetic acid (TFA), desalted using an HLB solid-phase extraction cartridge, concentrated under vacuum, and subjected to peptide quantification using a NanoDropOne UV spectrophotometer.

[0055] For DIA proteomics sequencing and data analysis, samples were digested with trypsin and treated with HLB desalting columns. Equal amounts of peptides were dissolved in mass spectrometry buffer and eluted using a Vanquish Neo ultra-high performance liquid chromatography system combined with a uPAC high-throughput column (75 μm × 5.5 cm). Gradient elution (180 SPD) was performed with 2% acetonitrile aqueous solution containing 0.1% formic acid (mobile phase A) and 80% acetonitrile aqueous solution (mobile phase B). The peptides were analyzed by Orbitrap Astral mass spectrometer in DIA mode (positive ion mode, ion source voltage 1.5 kV; primary mass spectrometry scan range 380–980 m / z, secondary mass spectrometry 150–2000 m / z). The raw data were obtained by Spectronaut. TM 18 software was used for database search analysis, with strict screening criteria set: protein / peptide false discovery rate (FDR) ≤ 1%, peptide confidence ≥ 99%, exclusion of shared peptides and modified peptides, and quantification using a six-peptide / protein (three characteristic ions / peptide) peak area integration strategy. Significant P values ​​were determined using SPSS (v27), and the fold difference (FC) criterion was also included: proteins with P < 0.05 and FC > 1.2 were considered upregulated, while proteins with P < 0.05 and FC < 0.83 were considered downregulated. Protein sequences were aligned with various protein databases using DIAMOND (v2.1.8.162) to identify the biological functions of each differentially expressed protein and annotation of the pathway to which it belongs.

[0056] Figure 4 A shows the principal coordinate analysis (PCoA) based on the protein expression profiles of the MAC group, LPS group, and β-aminoisobutyric acid group; Figure 4 B shows the pathways with significantly differential protein enrichment after LPS treatment compared with the MAC group; Figure 4 C shows the pathways enriched for proteins that were significantly downregulated after LPS treatment but whose expression was restored after β-aminoisobutyric acid treatment; Figure 4 D~K quantified the expression levels of differentially expressed proteins involved in the endoplasmic reticulum protein quality control pathway and demonstrated the functions of the proteins.

[0057] Proteomic analysis showed that the protein expression profiles of the MAC group, LPS group and β-aminoisobutyric acid treatment group were significantly separated (P = 0.001, R 2=0.463). Enrichment analysis of differentially expressed proteins in the LPS group showed that multiple pro-inflammatory metabolic pathways were significantly activated (P < 0.05), confirming the successful establishment of the inflammation model. Further cluster analysis of proteins that were downregulated after LPS induction and restored by β-aminoisobutyric acid revealed that they were significantly enriched in the endoplasmic reticulum protein quality control pathway (Protein processing in endoplasmic reticulum: GIP; P adj <0.05). Quantitative analysis showed that β-aminoisobutyric acid significantly restored the expression levels of key proteins in this pathway, including unfolded protein recognition factors (NEF, GPR94, Hsp40), misfolded protein marker enzymes (EDEM, ERManI), anti-aggregation regulatory factors (PDIs), and proteasome presentation-related factors (Ubc6 / 7, Hsp70) (P < 0.05). These experimental data confirm that β-aminoisobutyric acid promotes the recognition, marking, and degradation of unfolded and misfolded proteins by activating the endoplasmic reticulum protein quality control pathway, thereby alleviating the inflammatory response in mammary epithelial cells.

[0058] Example 5: Therapeutic Effect of β-aminoisobutyric acid on Clinical and Subclinical Mastitis in Dairy Goats. Guanzhong dairy goats in mid-lactation (parity = 2, DIM = 135.70 ± 3.82, mean ± SD) diagnosed by veterinarians with clinical (n = 4) and subclinical (n = 5) mastitis were selected and housed in separate pens in Fuping County, Shaanxi Province. 10 μM β-aminoisobutyric acid in saline (2 mL / time) was administered via teat perfusion at 8:00 AM and 8:00 PM daily for 7 consecutive days. Milk somatic cell counts were measured before (CON) and after (BAIBA) treatment using a Combi 300 milk analysis system to evaluate the therapeutic efficacy against different types of mastitis.

[0059] Figure 5 A shows the therapeutic effect of β-aminoisobutyric acid on dairy goats suffering from subclinical mastitis; Figure 5 B shows the therapeutic effect of β-aminoisobutyric acid on dairy goats with clinical mastitis.

[0060] The results showed that for dairy goats diagnosed with clinical or subclinical mastitis, β-aminoisobutyric acid significantly reduced milk somatic cell counts (P<0.05) and alleviated mastitis symptoms within a one-week treatment period. These results fully confirm that β-aminoisobutyric acid is an extremely effective functional metabolite for the treatment of mastitis.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of β-aminoisobutyric acid in the preparation of preparations for treating animal mastitis.

2. The use according to claim 1, characterized in that The animal is a ruminant.

3. The use according to claim 2, characterized in that: The ruminant is a cattle or a sheep.

4. The use according to claim 1, characterized in that: The mastitis is caused by infection with Escherichia coli, Pseudomonas, Klebsiella, Streptococcus agalactiae or Staphylococcus aureus.

5. The use according to claim 1, characterized in that: The effective concentration of the beta-aminoisobutyric acid in the preparation for treating animal mastitis is 5-20 μM.

6. The use according to claim 1, characterized in that: The mastitis is clinical mastitis or subclinical mastitis.

7. The use according to claim 1, characterized in that: The β-aminoisobutyric acid can increase the proliferation efficiency of animal mammary epithelial cells, reduce the concentrations of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in cell homogenate, and increase the concentration of anti-inflammatory cytokine IL-10.

8. The use according to claim 1, characterized in that: The preparation is a medicine, feed or feed additive.

9. The use according to claim 8, characterized in that: The medicine further comprises excipients; the excipients are one or more of sucrose, sorbitol, starch, dextrin, powdered sugar, sodium hydroxymethyl cellulose, physiological saline and sodium methyl cellulose.

10. The use according to claim 8, characterized in that: The dosage form of the medicine is tablet, capsule, granule, injection, cream or ointment.

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