Use of beta-aminoisobutyric acid in the preparation of a preparation for the treatment of mastitis in animals

Abstract

The application relates to the field of biological medicine, and particularly relates to application of beta-amino isobutyric acid in preparation of an animal mastitis treatment preparation. The application relates to application of beta-amino isobutyric acid in preparation of an animal mastitis treatment preparation. The beta-amino isobutyric acid is obtained from the hindgut contents of a health Sanneng goat with the lowest number of somatic cells in milk through large population screening. The beta-amino isobutyric acid can significantly promote the proliferation efficiency of cow mammary epithelial cells, and can promote the expression of related proteins involved in the endoplasmic reticulum protein quality control pathway in the LPS-induced cow mammary epithelial cell inflammation model, thereby enhancing the folding efficiency of proteins in the inflammation process, accelerating the clearance of misfolded and unfolded proteins, finally reducing the concentration of pro-inflammatory cytokines, increasing the concentration of anti-inflammatory cytokines, and relieving the inflammation phenotype.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of β-aminoisobutyric acid in the preparation of formulations for treating mastitis in animals. Background Technology

[0002] Mastitis is one of the most challenging diseases in the dairy industry, causing significant economic losses globally by reducing milk quality and leading to the culling of breeding animals. Mastitis is mainly divided into acute clinical mastitis and subclinical mastitis, which is difficult to detect. Both types of diseases are characterized by elevated somatic cell counts (SCC) in milk and a systemic inflammatory phenotype. Traditional treatments for mastitis rely on isolating specific pathogens from milk to identify the source of infection and then treating them with antibiotics. However, this approach leads to antibiotic residues in milk, affecting the value of raw milk and its byproducts, and can also induce increased drug resistance in opportunistic pathogens associated with mastitis. Therefore, there is an urgent need to develop safer and more comprehensive treatments for mastitis in ruminants.

[0003] The gut microbiota is a complex ecosystem composed of highly diverse symbiotic microorganisms, forming a dynamically balanced microecological environment to maintain the host's immune defense function. Imbalances in its homeostasis may exacerbate the pathological process of mastitis through the "gut-mammary" axis regulatory mechanism, while reconstructing the gut microbiota through microbial transplantation can effectively alleviate mammary inflammation. Further research has 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. Given the technical bottlenecks and biosafety risks of current in vitro culture systems for probiotics, systematically screening their functional metabolites and verifying their therapeutic efficacy against mastitis in ruminants has become a more translational research direction.

[0004] β-Aminoisobutyric acid (BAIBA) is a small molecule derived from branched-chain amino acids and is considered a novel endogenous protective actin. BAIBA plays an important role in many life processes, particularly inflammation. Previous studies have shown that BAIBA can stimulate phosphorylation of adenosine monophosphate-activated protein kinase in mouse adipocytes in a dose-dependent manner and inhibit the secretion of pro-inflammatory cytokines TNF-α and MCP-1. Furthermore, BAIBA has been shown to inhibit TNF-α-induced apoptosis and cytoplasmic degradation of whole 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 this invention is to provide a functional metabolite, β-aminoisobutyric acid, that can alleviate the phenotype of mastitis in ruminants, reduce the number of somatic cells in milk, decrease 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 in this invention is derived from the hindgut contents of healthy Saanen dairy 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, enhance protein folding efficiency during inflammation by promoting the expression of proteins involved in the endoplasmic reticulum protein quality control pathway, including NEF, Hsp40, GPR94, EDEM, ERManI, PDIs, Hsp70, and Ubc6 / 7. This accelerates the clearance of misfolded and unfolded proteins, 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.

[0007] Furthermore, the treatment of dairy goats with clinical and subclinical mastitis by instilling β-aminoisobutyric acid into their milk ducts further confirmed that β-aminoisobutyric acid can significantly reduce the number of somatic cells in milk and inhibit the progression of mastitis.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides the application of β-aminoisobutyric acid in the preparation of drugs for treating mastitis in animals; the chemical formula of this functional metabolite is C4H9NO2; PubChem SID is 6143; CAS NO. is 4249-19-8.

[0010] Preferably, the animal is a ruminant.

[0011] Preferably, the ruminant is a cow 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 β-aminoisobutyric acid in the pharmaceutical preparation for treating mastitis in animals is 5-20 μM, and the feeding or administration interval is 12 h.

[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 reduce the concentration of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in cell homogenate, while increasing the concentration of anti-inflammatory cytokines IL-10.

[0016] Preferably, the drug further includes excipients; the excipients are one or more selected from sucrose, sorbitol, starch, dextrin, powdered sugar, sodium carboxymethyl cellulose, physiological saline, and sodium methyl cellulose.

[0017] Preferably, the dosage form of the drug is tablets, capsules, granules, injections, or creams.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The β-aminoisobutyric acid provided by this invention can promote the proliferation of mammary epithelial cells in ruminants.

[0020] The β-aminoisobutyric acid provided by this invention has the function of significantly reducing the concentrations of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in an LPS-induced bovine mammary epithelial cell inflammation model, and increasing the concentration of anti-inflammatory cytokines IL-10.

[0021] The β-aminoisobutyric acid provided by this invention can also enhance the protein folding efficiency 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 concentration of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and increasing the concentration of anti-inflammatory cytokine IL-10, so as to alleviate the inflammatory phenotype.

[0022] The β-aminoisobutyric acid provided by this invention can also alleviate the disease phenotype of ruminants suffering from clinical and subclinical mastitis and significantly reduce the number of cells in their milk. Attached Figure Description

[0023] Figure 1 This invention relates to Embodiment 1, which involves the determination of systemic inflammatory factor concentrations and identification of hindgut marker microbial species in healthy and mastitis-affected goats determined through large-group screening. A represents the screening of mastitis-affected and healthy goats from a large Saanen dairy goat lactating population; B represents the lactation phenotypes of healthy and mastitis-affected dairy goats; C represents the determination of pro-inflammatory cytokine concentrations in the milk of healthy and mastitis-affected dairy goats; D represents the determination of pro-inflammatory cytokine concentrations in the serum of healthy and mastitis-affected dairy goats; E represents the identification of hindgut marker microbial species in healthy and mastitis-affected dairy goats; and F represents the correlation analysis between hindgut marker microbial species and inflammatory phenotypes in healthy and mastitis-affected dairy goats.

[0024] Figure 2 In Example 2 of this invention, β-aminoisobutyric acid was identified as a potential functional metabolite with therapeutic effects on mastitis based on the analysis of functional genes and metabolites of hindgut microorganisms in two groups of dairy goats. Figure 2 A shows the correlation network between two groups of hindgut microbial species in dairy goats; Figure 2 B shows that Clostridiabacterium, a biomarker for identifying the hindgut microbiota of healthy sheep, is the core species that mediates quorum sensing in the microbial community. Figure 2 C represents the enrichment analysis of differentially expressed functional genes in the hindgut of dairy goats; Figure 2 D represents the functional enrichment analysis of differential metabolites in the hindgut of dairy goats; Figure 2 E represents the determination of β-aminoisobutyric acid enrichment in the hindgut of healthy sheep by analyzing differentially functional genes and metabolites in microorganisms. Figure 2 F represents the correlation analysis between β-aminoisobutyric acid and inflammatory markers in dairy goats with mastitis.

[0025] Figure 3 This invention relates to the therapeutic effect of β-aminoisobutyric acid on the inflammatory phenotype of an LPS-induced bovine mammary epithelial cell inflammation model in Example 3 of this invention; A and B represent the exploration of LPS concentration and treatment time in the bovine mammary epithelial cell inflammation model; C represents the effect of β-aminoisobutyric acid on the proliferation and cytotoxicity of bovine mammary epithelial cells; and D-G represent the effects of β-aminoisobutyric acid on the concentrations of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, as well as the anti-inflammatory cytokine IL-10, in the LPS-induced bovine mammary epithelial cell inflammation model.

[0026] Figure 4 This is Example 4 of the present invention, which explores the inflammatory phenotype mechanism of a bovine mammary epithelial cell inflammation model induced by LPS treatment with β-aminoisobutyric acid (β-aminoisobutyric acid) based on DIA proteomics. A shows the principal coordinate analysis of protein expression profiles based on the MAC group, LPS group, and β-aminoisobutyric acid group. B shows the pathways enriched with proteins that were significantly different after LPS treatment compared to the MAC group. C shows the pathways enriched with proteins that were significantly downregulated after LPS treatment but whose expression was restored after β-aminoisobutyric acid treatment. D~K quantify 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.

[0027] Figure 5This describes the effect of β-aminoisobutyric acid treatment on the somatic cell count in the milk of dairy goats suffering from subclinical and clinical mastitis, as described in Example 5 of this invention. A indicates that β-aminoisobutyric acid significantly reduces the somatic cell count in the milk of dairy goats with subclinical mastitis, and B indicates that β-aminoisobutyric acid significantly reduces the somatic cell count in the milk of dairy goats with clinical mastitis. Detailed Implementation

[0028] The technical solutions provided by the present invention will be 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.

[0029] All reagents required for the solution preparations in the following examples were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dairy goats suffering from subclinical and clinical mastitis were provided by Shaanxi Bofule Guanzhong Dairy Goat Breeding Co., Ltd. Quantitative reagent kits for the concentration of inflammatory factors in serum, milk, and cell suspensions were purchased from Shanghai Kexing Trading Co., Ltd.

[0030] Example 1: Metagenomic Analysis of Characteristic Hippic Gut Microbiota and Quorum Sensing Core Species in Healthy and Mastitis-Induced Dairy Goats

[0031] This invention selected 167 Saanen dairy goats in mid-lactation (parity: 2.17±0.40, lactation days: 121.47±7.95). All goats were housed in the same pen with stable temperature and good ventilation. They were milked at 5:00 AM and 5:00 PM daily, and fed immediately after milking. The average milk yield of the goats was calculated by averaging the daily milk yield collected in the first and fourth weeks of August 2022. On the last day of milk yield data collection, milk samples from the two milking sites were mixed at a 3:2 ratio, divided into two portions, and then analyzed for milk composition and quantified for somatic cell count in the milk.

[0032] The following day, the highest somatic cell count was selected (Mastitis, n = 6) and minimum (Health, n =6) Fresh feces were collected from the anus of sheep using abdominal massage, immediately flash-frozen in liquid nitrogen, and then stored at -80°C for metagenomic and untargeted metabolomics sequencing. Serum samples were collected from Mastitis and Health group sheep using jugular vein blood collection and frozen at -20°C for biochemical parameter analysis and quantification of inflammatory markers.

[0033] For metagenomic sequencing and data analysis, DNA was extracted from fecal samples from the Mastitis and Health groups using a micro-bead milling technique. 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 identified by 1% agarose gel electrophoresis. Paired-end libraries were constructed from the fragmented DNA using NEXTFLEX RapidDNA-Seq. Subsequently, metagenomic sequencing was performed using the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. A total of 142.11 Gb of raw sequencing data was generated from six fecal samples derived from dairy goats, with an average of 11.84 Gb per sample. The raw data generated from Illumina platform sequencing was processed using fastp (v0.20.0) to remove adapters and low-quality reads. To remove contamination from host DNA in the reads, BWA-MEM (v0.7.17) was used to align the 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%.

[0034] Subsequently, Megahit (v1.2.9) was used to assemble contigs from the high-quality sequences, yielding an average of 777,409.50 contigs per sample, with an average N50 of 639.69 bp. The assembled contigs were then used to predict ORFs using Prodigal (v2.6.3), ultimately yielding 15.78 million ORFs with an average length of 444.96 bp. After removing ORFs shorter than 100 bp, 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 NR predicted gene sequences into NR predicted protein sequences. Subsequently, SOAP2 (v2.21) was used to map the high-quality reads of each sample to the NR gene set to obtain the predicted NR gene abundance for each sample. DIAMOND (v2.1.8.162) was used to compare with the NCBI-NR database at an e-cutoff of 1e-5 to obtain species annotation information at each taxonomic level. LefSe analysis was used to identify species with significantly different abundances in the hindgut microbiota between Mastitis and Health sheep, serving as microbial biomarkers for each group.

[0035] Figure 1 A shows the screening process and quantitative results of somatic cell count in the milk of sheep in the Mastitis and Health groups; Figure 1 B shows the differences in lactation phenotypes between sheep in the Mastitis and Health groups; Figure 1 C shows the difference in the concentration of inflammatory cytokines in the milk of sheep in the Mastitis and Health groups; Figure 1 D shows the difference in serum inflammatory cytokine concentrations in sheep from the Mastitis and Health groups; Figure 1 E demonstrates 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 phenotypes in the Mastitis and Health groups.

[0036] The results showed that, compared with the healthy group, dairy goats infected with mastitis exhibited the following characteristics: a significantly increased somatic cell count in milk ( P < 0.001, milk production decreased significantly ( P < 0.05%, accompanied by a decrease in lactose content ( P < 0.05) and an increase in milk protein content ( P < 0.05). Notably, the concentrations of pro-inflammatory factors MPO, IL-6, IL-1β, and TNF-α in the breast milk of the diseased group were significantly increased ( P < 0.05), while pro-inflammatory cytokines LPS, IL-6, IL-1β and TNF-α were also significantly enriched in serum ( P <0.01). The above results fully confirm that mastitis not only leads to a decline in lactation performance, but also triggers a systemic immune metabolic imbalance through a pro-inflammatory cascade reaction.

[0037] Subsequently, based on metagenomic sequencing and using LefSe analysis, we identified the marker microbial species in the hindgut of sheep in the Mastitis and Health groups, respectively, confirming... Clostridia bacterium Several species have not obtained accurate taxonomic assignments at the species level. Clostridia spp. are enriched in the Health group, while Lachnospiraceae bacterium It was then identified as a microbial marker in the Mastitis group (LDA > 2, P < 0.05). Spearman correlation analysis revealed that the above unclassified multiple Clostridia spp. showed a significant negative correlation with the inflammatory phenotype, while marker microorganisms in the Mastitis group showed a significant positive correlation with pro-inflammatory parameters (|R| > 0.5). P < 0.05).

[0038] Example 2: Combined analysis of functional genes and metabolomics in the hindgut microbiota of dairy goats confirmed that β-aminoisobutyric acid is a potential functional metabolite that can treat mastitis.

[0039] KO annotation was performed on the NR predicted genes obtained from the metagenomic analysis in Example 1 using KOfamScan, and HMMER was used to align with the KOfam database (a hidden Markov database designed specifically for KO). For KEGG enrichment analysis, Z -score is a criterion used to assess whether a particular pathway is enriched in different groups.

[0040] For non-targeted metabolomics sequencing and analysis, fecal samples from Mastitis and Health sheep were thawed on ice. Each sample (50 mg ± 1 mg) was homogenized with 500 μL of ice-cold methanol / water (70%, v / v). The samples were vortexed for 3 min, sonicated in an ice-water bath for 10 min, and then vortexed again for 1 min. Subsequently, the samples were centrifuged at 12,000 rpm for 10 min. After centrifugation, 200 μL of the supernatant was incubated at -20°C for 30 min, followed by centrifugation again at 12,000 rpm for 30 min at 4°C. Then, 350 μL of the supernatant was transferred to a vacuum concentrator for evaporation drying. The dried residue was reconstituted with 150 μL of 70% methanol aqueous solution, vortexed for 3 min, and then sonicated in an ice-water bath for 10 min. Finally, centrifuge at 12,000 rpm for 3 minutes at 4°C, and take 120 μL of supernatant for analysis using the UHPLC-ESI-MS / MS system.

[0041] The ultra-high performance liquid chromatography (UHPLC) conditions were set as follows: Waters ACQUITY UPLC HSS T3 C18 column (1.8µm, 2.1 mm × 100 mm); mobile phase: ultrapure water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid; column temperature: 40°C; flow rate: 0.4 mL / min; injection volume: 2 μL. Mass spectrometry (MS) was performed using both positive and negative ion modes. Raw LC-MS / MS data were processed using Analyst 1.6.3 software. To broaden 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). Based on VIP > 1 and P A threshold of < 0.05 (Wilcoxon rank-sum test) was used to screen for differentially expressed metabolites. After mapping the identified metabolites to KEGG pathways, significantly enriched metabolic pathways were determined using hypergeometric tests.

[0042] Figure 2A shows the correlation network between two groups of hindgut microbial species in dairy goats; Figure 2 B demonstrates the identification of hindgut microbial markers in healthy sheep. Clostridia bacterium As a core species mediating quorum sensing in microbial communities; Figure 2 C represents the enrichment analysis of differentially expressed functional genes in the hindgut of dairy goats; Figure 2 D represents the functional enrichment analysis of differential metabolites in the hindgut of dairy goats; Figure 2 E represents the determination of β-aminoisobutyric acid enrichment in the hindgut of healthy sheep by analyzing differentially functional genes and metabolites in microorganisms. Figure 2 F represents the correlation analysis between β-aminoisobutyric acid and inflammatory markers in dairy goats with mastitis.

[0043] The results show that Clostridia bacterium As the core of microbial interactions in the hindgut of healthy dairy goats (|R|>0.5), P < 0.05), which can encode a wide range of genes related to microbial quorum sensing. IsrA , IsrB , IsrC , IsrD , IsrR This regulates the metabolic function of the gut microbiota. Furthermore, functional enrichment analysis based on differentially expressed microbial functional genes and metabolites showed that Valine, leucine, and isoleucine degradation were significantly enriched metabolic pathways. Therefore, we further investigated the differentially expressed microbial functional genes and metabolites involved in this pathway, discovering several such... AvtA , vorA , vorB , vorD and ACADM Microbial functional genes were expressed at increased levels in the Health group, and the concentration of L-valine, a precursor for β-aminoisobutyric acid synthesis, was also significantly increased in the Health group. P < 0.05, ultimately leading to a significant increase in the concentration of β-aminoisobutyric acid in the Health group ( P < 0.01). Subsequently, Spearman correlation analysis revealed that β-aminoisobutyric acid was significantly negatively correlated with multiple mastitis-related inflammatory markers (|R|>0.5, P < 0.05). Due to the excellent stability of the circulatory system, β-aminoisobutyric acid is expected to exert an anti-inflammatory effect on mastitis through the "gut-mammary" axis.

[0044] Example 3: Therapeutic effect of β-aminoisobutyric acid on LPS-induced inflammatory phenotype in bovine mammary epithelial cells

[0045] For the culture conditions of bovine mammary epithelial cells, DMEM medium supplemented with 10% fetal bovine serum was used to culture the cells at 37°C and 5% CO2. The bovine mammary epithelial cells were then stored in a continuously humidified cell culture incubator under standard culture conditions.

[0046] Bovine mammary epithelial cells were cultured for 24 hours after resuscitation, and their cell density and growth status were observed under a microscope. Cells were passaged when the cell density reached approximately 90%. Subsequently, bovine mammary epithelial 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 an LPS-induced inflammatory cell model.

[0047] To investigate the effect of β-aminoisobutyric acid (β-aminobutyric acid) on the proliferation efficiency of bovine mammary epithelial cells, cells that had been revived, cultured, and passaged were seeded at a density of 5 × 10³ cells / well in 96-well plates. After culturing in DMEM medium containing 10% fetal bovine serum for 24 hours to form a monolayer, β-aminoisobutyric acid at final concentrations of 5 μM, 10 μM, 15 μM, and 20 μM was added and incubated for 6 h, 12 h, 18 h, and 24 h, respectively. Immediately after incubation, 10 μL of CCK-8 reagent was added to each well, and the cells were 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.

[0048] Furthermore, to investigate the therapeutic effect of β-aminoisobutyric acid on the inflammatory phenotype of an LPS-induced bovine mammary epithelial cell inflammation model, cells that had been resuscitated, cultured, and passaged were used at a concentration of 5 × 10⁻⁶ cells / mL. 7 Cells were seeded at a density of 50 μg / mL in 6-well plates and treated with LPS for 12 h according to a pre-determined inflammatory model induction condition to induce an inflammatory response.

[0049] Subsequently, β-aminoisobutyric acid (β-aminobutyric acid) at final concentrations of 5 μM, 10 μM, 15 μM, and 20 μM was used to incubate inflammatory breast epithelial cells for 6 h, 12 h, 18 h, and 24 h, respectively, to investigate the optimal drug concentration and treatment time for β-aminoisobutyric acid to exert a therapeutic effect on breast inflammation. The therapeutic effect of β-aminoisobutyric acid was reflected by the concentrations of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and the anti-inflammatory cytokine IL-10.

[0050] Figure 3 A and B show the effects of LPS concentration gradient and treatment time on the concentration of pro-inflammatory factors in bovine mammary epithelial cell suspension. Figure 3C demonstrates the dose-time dependent effect of β-aminoisobutyric acid concentration-time bivariate on cell proliferation activity and toxic response; Figure 3 D~G demonstrates the antagonistic effect of β-aminoisobutyric acid on the inflammatory phenotype of an LPS-induced bovine mammary epithelial cell inflammation model under different concentration gradients and treatment sequences.

[0051] The results showed that treatment of bovine mammary epithelial cells with 50 μg / mL LPS for 12 h significantly increased the concentrations of pro-inflammatory cytokines TNF-α and IL-1β in the cell suspension. P < 0.001), this condition was established as the standardized inflammation induction protocol. Safety assessment of β-aminoisobutyric acid using the CCK-8 assay revealed that all concentrations (5–20 μM) and treatment times (6–24 h) had no significant inhibitory effect on cell proliferation activity. P > 0.05), of which 10 μM β-AIBA pretreatment for 12 h significantly increased cell proliferation rate ( P < 0.05). Based on the above inflammation model, cytokine levels were measured after incubation with 5, 10, 15, and 20 μM β-aminoisobutyric acid for 6, 12, 18, and 24 h, respectively. The results showed that during the 12 h treatment cycle, 5–20 μM β-aminoisobutyric acid significantly reduced the concentrations of pro-inflammatory 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). The above data indicate that β-aminoisobutyric acid can effectively improve LPS-induced inflammatory response in mammary epithelial cells.

[0052] Example 4: β-Aminoisobutyric acid regulates the clearance of misfolded proteins and alleviates inflammatory response through the endoplasmic reticulum unfolded protein response.

[0053] To elucidate the mechanism of β-aminoisobutyric acid (β-aminoisobutyric acid) in treating breast epithelial cell inflammation, we collected cell samples from the control group (MAC group), LPS-treated group (LPS group), and β-aminoisobutyric acid (BAIBA group) after washing them three times with PBS and centrifuging them at 4°C and 1500× 10⁻⁶. g Centrifuge for 5 min and discard the supernatant. Then, add the appropriate RIPA lysis buffer (0.1% protease inhibitor) according to the cell volume and lyse on ice for 30 min. Incubate the lysis buffer at 4°C and 12000× gCentrifuge for 15 min under the specified conditions, collect the supernatant, and determine the protein concentration using the Omni-Easy™ Instant BCA Protein Quantification Kit based on the BCA method. Store the collected protein samples at -80℃. For protein digestion and peptide extraction, 100 μg of protein sample was equilibrated with 100 mM triethylammonium bicarbonate (TEAB) buffer, and then subjected to reduction (10 mM tris(2-carboxyethyl)phosphine [TCEP], 37℃ for 60 min), alkylation (40 mM Iodoacetamide, at room temperature in the dark for 40 min), and acetone precipitation (-20℃ for 4 h, 10,000×). g (Centrifugation). Subsequently, the precipitate was redissolved in 100 µL of 100 mM TEAB and Trypsin was added at a mass ratio of 1:50 (enzyme:protein). The mixture was incubated overnight at 37°C. The enzymatic digest was dissolved in 0.1% trifluoroacetic acid (TFA), desalted using an HLB solid-phase extraction column, concentrated under vacuum, and then quantified into peptides using a NanoDrop One UV spectrophotometer.

[0054] For DIA proteomics sequencing and data analysis, samples were digested with trypsin and treated with an HLB desalting column. Equal volumes of peptides were then dissolved in mass spectrometry buffer. A Vanquish Neo ultra-high performance liquid chromatography system was used with a uPAC high-throughput column (75 μm × 5.5 cm). Gradient elution (180 SPD) was performed using 2% acetonitrile aqueous solution containing 0.1% formic acid (mobile phase A) and 80% acetonitrile aqueous solution (mobile phase B). Peptides were analyzed using an 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). Raw data were analyzed using Spectronaut™ 18 software with strict screening criteria: false detection rate (FDR) ≤1%, peptide confidence ≥99%, excluding shared and modified peptides. Quantification was performed using a peak area integration strategy for hexapeptides / proteins (three characteristic ions / peptides). Significance was determined using SPSS (v27). P value, and simultaneously incorporate the difference factor (FC) criterion: P Proteins with < 0.05 and FC > 1.2 were considered upregulated, while P Proteins with < 0.05 and FC < 0.83 were considered downregulated. DIAMOND (v2.1.8.162) was used to align protein sequences with various protein databases to identify the biological function and pathway of each differentially expressed protein.

[0055] Figure 4A shows the principal coordinate analysis (PCoA) based on the expression profiles of the MAC group, LPS group, and β-aminoisobutyric acid histones. Figure 4 B shows the pathways with significantly different protein enrichment after LPS treatment compared to the MAC group; Figure 4 C shows the pathway enriched by 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 these proteins.

[0056] Proteomics 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, R² = 0.463). Enrichment analysis of differentially expressed proteins based on the LPS group showed that multiple pro-inflammatory metabolic pathways were significantly activated ( P < 0.05), confirming the successful construction of the inflammation model. Further cluster analysis of proteins downregulated after LPS induction but restored by β-aminoisobutyric acid expression revealed that they were significantly enriched in the endoplasmic reticulum protein quality control pathway (GIP). P adj < 0.05). Quantitative analysis showed that β-aminoisobutyric acid (β-aminoisobutyric acid) could significantly restore the expression levels of key proteins in this pathway, including unfolded protein recognition factors (NEF, GPR94, Hsp40), misfolded protein labeling enzymes (EDEM, ERManI), anti-polymerization regulators (PDIs), and proteasome presentation-related factors (Ubc6 / 7, Hsp70). P < 0.05). The above experimental data confirm that β-aminoisobutyric acid promotes the recognition, labeling, and degradation of unfolded and misfolded proteins by activating the endoplasmic reticulum protein quality control pathway, thereby alleviating the inflammatory response of mammary epithelial cells.

[0057] Example 5: Therapeutic effect of β-aminoisobutyric acid on clinical and subclinical mastitis in dairy goats.

[0058] The experiment selected mid-lactation fetuses (parity = 2, DIM = 135.70 ± 3.82, mean ± SD) and veterinarily diagnosed with clinical type ( n = 4) and subclinical ( n= 5) Guanzhong dairy goats with mastitis were raised in separate pens in Fuping County, Shaanxi Province. They were administered 10 μM β-aminoisobutyric acid saline solution (2 mL / time) via milk perfusion at 08:00 AM and 20:00 PM daily for 7 consecutive days. Somatic cell counts in milk were measured before (CON) and after (BAIBA) treatment using a Combi 300 milk analysis system to evaluate its therapeutic effect on different types of mastitis.

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

[0060] The results showed that β-aminoisobutyric acid significantly reduced the somatic cell count in milk in dairy goats diagnosed with clinical or subclinical mastitis within a one-week treatment period. P < 0.05), relieving mastitis symptoms. These results fully demonstrate that β-aminoisobutyric acid is a highly effective functional metabolite for the treatment of mastitis.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of β-aminoisobutyric acid in the preparation of drugs for treating mastitis in animals, characterized in that, The animal in question is a dairy goat.

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

3. The application according to claim 1, characterized in that: The effective concentration of β-aminoisobutyric acid in the pharmaceutical preparation for treating mastitis in animals is 5-20 μM.

4. The application according to claim 1, characterized in that: The mastitis referred to is clinical mastitis or subclinical mastitis.

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

6. The application according to claim 1, characterized in that: The drug also includes excipients, which are one or more of sucrose, sorbitol, starch, dextrin, powdered sugar, sodium carboxymethyl cellulose, physiological saline, and sodium methyl cellulose.

7. The application according to claim 1, characterized in that: The dosage form of the drug is tablets, capsules, granules, injections, or creams.

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