Mastitis metabolism marker and application thereof in preparation of kit for preventing, diagnosing or treating mastitis

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a mastitis metabolism marker and application thereof in preparation of a kit for preventing, diagnosing or treating mastitis. The invention relates to a marker for diagnosing and predicting mastitis, which is characterized in that the marker is at least one of IL-6 (interleukin-6), IL-1beta (interleukin-1 beta), MPO (Maximum Peroxidase), NAGase (Nicotinase), ZO-1 (Zin-1), Occludin (Occludin) and Claudin-3 (Claudin-3). In three samples, expression increase of metabolites related to bile acid, alcohol and subclass of derivatives of bile acid, alcohol and subclass of derivatives of bile acid, alcohol and subclass of derivatives of bile acid are observed, concentration of demethylcholic acid in breast tissue is increased, and it is indicated that microorganisms can influence the progress of mouse mastitis by adjusting secondary bile acid. Therefore, targeted regulation and control of bile acid metabolism may become a potential treatment strategy for mastitis treatment. According to the method, the metabolic state of mastitis can be known more comprehensively, and a new insight is provided for the influence of mastitis on the immune system.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a mastitis metabolic marker and its application in the preparation of a kit for preventing, diagnosing or treating mastitis. Background Art

[0002] Mastitis is a common disease in lactating women and female mammals, usually caused by microbial infections such as Staphylococcus aureus. During lactation or after childbirth, mastitis is often accompanied by long-term duct congestion, as well as clinical manifestations such as local erythema, severe pain, and swelling. Mastitis can hinder breastfeeding and lead to the transmission of pathogens from the mother to the baby. Once upon a time, antibiotic therapy was very common, but overuse can damage women's health and the ranch economy. In addition, there are also problems such as frequent recurrence and failure to recover in mastitis. Therefore, further clarifying the pathogenesis of mastitis is crucial for accurate diagnosis and effective control.

[0003] Studies have shown that the disorder of the gut microbiota plays an important role in the pathogenesis of inflammatory diseases such as mastitis. Transplanting the feces of mastitis cows into germ-free mice results in systemic inflammatory responses in the mouse serum, spleen, colon, and mammary gland tissues. The dysregulation of the gut microbiota is a promoting factor for the occurrence of mastitis, and regulating the gut microbiota to reduce systemic inflammatory responses can be used as a potential intervention strategy for mastitis.

[0004] The pathogenesis of mastitis involves metabolic and immune responses. Therefore, it is necessary to comprehensively understand its potential regulatory mechanisms. Gut microbes play a crucial role in regulating the production of short-chain fatty acids (such as butyrate) and secondary bile acids, thereby having a mitigating effect on mastitis. Somatic cell count (SCC) is a commonly used indicator for mammary gland infection. In cows with mastitis, the SCC index increases significantly, which is positively correlated with the contents of β-hydroxybutyric acid, isoleucine, and acetate in the host body. Therefore, the metabolic level is closely related to the mammary gland health status. However, the relationship between metabolism and inflammation in mastitis remains unclear. Summary of the Invention

[0005] The purpose of the present invention is to provide a mastitis metabolic marker and its application in the preparation of a kit for preventing, diagnosing or treating mastitis. This application helps to more comprehensively understand the metabolic state of mastitis and provides new insights into its impact on the immune system.

[0006] To achieve the above purpose, the mastitis metabolic marker of the present invention adopts the following technical solution: A mastitis metabolic marker, wherein the marker is at least one of IL-6, IL-1β, MPO, NAGase, ZO-1, Occludin, and Claudin-3.

[0007] Preferably, the expression levels of IL-6, IL-1β, MPO, and NAGase in mastitis tissues are higher than those in normal mammary tissues.

[0008] Preferably, the expression levels of ZO-1, Occludin, and Claudin-3 in mastitis tissues are lower than those in normal mammary tissues.

[0009] Preferably, the abundance of bacterial taxa in mastitis tissues is less than that in normal mammary tissues, while the diversity of bacterial taxa in mastitis tissues is greater than that in normal mammary tissues.

[0010] Preferably, the expression level of Proteobacteria in mastitis tissues is higher than that in normal mammary tissues; at the genus level, the expression levels of Roseburia, Parabacteroides, Butyricimonas, UBA1819, and Dubosiella in mastitis tissues are higher than those in normal mammary tissues.

[0011] Preferably, the carotenoid biosynthesis pathway, meiosis-yeast metabolism pathway, D-arginine and D-ornithine metabolism pathway in mastitis tissues are all higher than those in normal mammary tissues, while the polycyclic aromatic hydrocarbon degradation pathway and proteasome degradation pathway are all lower than those in normal mammary tissues.

[0012] Preferably, the targets of mastitis are Itgav, Itgb3, bile acid metabolism, or at least one of them.

[0013] To achieve the above object, the following technical solutions are adopted for the application of the mastitis metabolic markers of the present invention in the preparation of a kit for preventing, diagnosing, or treating mastitis: An application of a mastitis metabolic marker in the preparation of a kit for preventing, diagnosing, or treating mastitis, wherein the sample of the diagnostic kit is from human ex vivo serum.

[0014] An application of a mastitis metabolic marker in the preparation of a drug for preventing, diagnosing, or treating mastitis, wherein the drug includes nucleic acid drugs, antibody drugs, small molecule chemical drugs, or cell therapy drugs.

[0015] Beneficial effects: In this application, an increase in the expression of metabolites related to bile acids, alcohols, and their derivative subclasses was observed in three samples. The elevated concentration of demethylcholic acid in mammary tissues indicates that microorganisms can affect the progression of murine mastitis by regulating secondary bile acids. Therefore, targeted regulation of bile acid metabolism may become a potential treatment strategy for mastitis, and this pathway may play a role in inducing physiological and pathological changes in mastitis. Description of the Drawings

[0016] Figure 1For histopathological analysis of mastitis and differential expression of MPO, NAGase, IL-6, and IL-1β in blood and mammary tissues, A shows histopathology by H&E staining (scale bar: 20 μm, black arrows indicate tissue congestion); B shows the expression levels of MPO, NAGase, IL-6, and IL-1β in serum samples detected by ELISA (*P<0.05 and **P<0.01); C shows the expression levels of MPO, NAGase, IL-6, and IL-1β in mammary tissues detected by ELISA (*P<0.05 and **P<0.01); Figure 2A For α-diversity analysis; 2B’ and 2B’’ are distribution histograms at the phylum and genus levels respectively; 2C is a linear discriminant analysis with LDA = 2; 2D is a phylogenetic tree; 2E is a heatmap of KEGG analysis by PICRUSt2; Figure 3A -C are OPLS-DA score plots of metabolite profiles in colorectal contents, plasma, and mammary tissues of group C and group M respectively; 3D-3F are compound classifications of metabolites in colorectal contents, plasma, and mammary tissues respectively; Figure 4A Is a Venn diagram of metabolites in colorectal contents, plasma, and mammary tissues; 4B’, 4B’’, and 4B’’’ are the differences among the top 50 metabolites in colorectal contents, plasma, and mammary tissues respectively; 4C-4E are the peak intensities of key metabolites in colorectal contents, plasma, and mammary tissues respectively; Figure 5A Is the enrichment of the top 20 metabolites in colorectal contents in KEGG pathways; 5B and 5C are the analysis of plasma and mammary tissue metabolites using KEGG pathways respectively; Figure 6A Is a volcano plot, where the scatter points in the upper left quadrant represent downregulated proteins, the scatter points in the upper right quadrant represent upregulated proteins, and the scatter points in other regions represent non-significant proteins; 6B is a heatmap; 6C is an enrichment plot of the top 15 differential proteins; 6D is a bubble plot of downregulated pathway enrichment in KEGG (P<0.05), and the arrows indicate 6 pathways related to cell adhesion and cytoskeleton regulation; 6E is the partial gene regulation relationship of these 6 pathways; Figure 7A Is a heatmap of proteomics; 7B is the PCR verification of proteomics in mammary tissues (*P<0.05, **P<0.01, and ***P<0.001); 7C is a Western blot; 7D is the differential expression of 3 proteins (*P<0.05 and ***P<0.001); Figures 8A - 8CDifferentially expressed metabolites and proteins in colorectal content, plasma, and mammary gland tissue, respectively. □ represents pathways, △ represents proteins, ○ represents metabolites, and the degree represents the number of connections between proteins; 8D represents the calculation of the correlation between the reaction intensity data of proteins and differentially expressed metabolites in mammary gland tissue by Pearson correlation analysis. A significance level of P≤0.05 and a correlation ≥0.95 were used to construct the network diagram. Detailed implementation manners

[0017] The embodiments of the present invention will be described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present invention and should not be construed as a limitation to the present invention.

[0018] I. Materials and methods 1.1 Molecular identification Staphylococcus aureus was isolated and cultured from the milk of dairy cows with mastitis. The collected milk samples were stored in sterile glass flasks at 4°C. 5 mL of the milk sample was transferred to 50 mL of 7.5% sodium chloride broth and shaken on a shaker at 37°C and 170 r / min for 24 h. Using the streak plate isolation method, after inoculating and culturing on mannitol salt agar (MSA) for 18 h, yellow colonies were observed and presumed to be Staphylococcus aureus ( Int. J. Vet. Sci. Med. , 2018, 6 , 53). Multiple colonies were purified and cultured, and Staphylococcus aureus was sequenced.

[0019] 1.2 Animal model This experiment was approved by the Ethics Committee of a certain affiliated hospital in Zhengzhou (No. 2023-KY-0845) and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Lactating rats (Chengdu Dashuo) at 6 - 8 weeks of age and weighing 180 - 210 g were randomly divided into group C (control group) and group M (Staphylococcus aureus mastitis group), with 12 rats in each group.

[0020] Before the experiment, the lactating rats were anesthetized in a desiccator containing 10 mL of ether for 0.5 - 3 min. The fourth pair of mammary glands and the surrounding area were disinfected with 75% alcohol. Then, a sterile syringe was gently inserted into the nipple duct, and 0.1 mL of a Staphylococcus aureus suspension with a concentration of 5×10 8 CFU / mL was injected into the mammary gland twice a day for three consecutive days. Blood samples were collected, and the rats were euthanized by removing their necks. Mammary gland tissue and intestinal contents were obtained under sterile conditions and stored in a -80°C refrigerator for later use.

[0021] 1.3 Pathology and kit analysis The breast tissue samples were fixed with 4% paraformaldehyde fixative, dehydrated with ethanol, and embedded in paraffin. Hematoxylin and eosin staining was performed and observed under a microscope. The breast tissue samples were homogenized in ice-cold saline, and the supernatant was collected after centrifugation. Blood was centrifuged to obtain serum for subsequent analysis. Enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Hepai Biotechnology) were used to quantitatively test myeloperoxidase (MPO), N-acetyl-β-d-glucosaminidase (NAGase), interleukin (IL)-6, and IL-1β.

[0022] 1.4 16S rRNA Treatment with DADA2 in QIIME2 (2020.11) was performed, including denoising, chimera removal, and sequence duplicate data deletion ( Front Vet Sci. ,2023, 10 , 1219729). Alpha diversity was used to evaluate the species richness and evenness of the samples. Bray-Curtis and Jaccard similarity coefficients were used for beta diversity analysis. To evaluate the statistical significance of microbial differences, LEfSe analysis was performed in combination with linear discriminant analysis (LDA). PICRUSt2 software was used to determine the predicted functional composition of known microbial genes and quantify the functional differences between different samples.

[0023] 1.5 LC-MS / MS analysis Thirty milligrams of colorectal contents and 100 μL of homogenized breast tissue or plasma samples were collected separately and transferred to 1.5 mL centrifuge tubes. Analysis was performed using a liquid chromatography-mass spectrometry system consisting of a high-resolution mass spectrometer and a mass spectrometry system. The chromatographic column was UPLCHSST3 (100 mm × 2.1 mm, 1.8 μm), and the column temperature was 45°C. The mobile phase composition was as follows: mobile phase A was an aqueous solution containing 0.1% formic acid, mobile phase B was acetonitrile, and the flow rate was 0.35 mL / min. During the mass spectrometry signal acquisition process, positive ion scanning and negative ion scanning modes were used.

[0024] 1.6 TMT quantitative proteomics analysis Total protein was extracted from rat mammary gland tissues, and its concentration was determined by the BCA method and then detected by SDS-PAGE. Meanwhile, some samples were digested with trypsin and labeled with polypeptides. Equal amounts of labeled samples were combined for chromatographic separation. LC-MS / MS was used for analysis and processing. An Acclaim PepMap RSLC 75μm×50cm (RP-C18, ThermoFisher) column was used, and injection was performed at a flow rate of 300 nL / min for gradient elution. Proteome Discoverer 2.4.1.15 software (ThermoFisher Scientific) was used for data analysis. Multiple databases were used for functional annotation analysis of the identified proteins, and GO and pathway analyses were performed on the differentially expressed proteins.

[0025] 1.7 RT-qPCR analysis Total RNA was extracted from mammary gland tissues using Trizol (TaKaRa Biotech, China). RT-PCR experiments were performed using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, China) kit according to the instructions. The gene sequences of Fst, PGR, IFIH1, Tnfaip8l2, IRGM, IRF5, PAL2G4A, Prxl2b, and GAPDH were all from NCBI GenBank. Primer Premier 5.0 software (Table 1) was used for primer design. Referring to the GAPDH gene, the 2-ΔΔCT method was used to calculate the relative expression levels of each mRNA.

[0026] Table 1 Primer sequences for RT-qPCR 1.8 Western blot analysis Total protein was extracted from 100 mg mammary gland tissue samples, homogenized on ice with RIPA protein inhibitor, and centrifuged at 12,000 rpm for 10 min. The protein concentration in the supernatant was determined using a BCA protein detection kit. Protein samples (50 μg) were separated on a 12% SDS-PAGE gel, transferred to a PVDF membrane, and blocked with 5% bovine serum albumin (BSA, Sigma Aldrich, St. Louis, MO, USA) in TBST for 2 h. After incubating with the primary antibody solution overnight at 4°C, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. Protein expression was detected using an enhanced chemiluminescence detection system, and its optical density was measured using Image-Pro Plus 6.0 software for quantification; β-actin was used as an endogenous control.

[0027] 1.9 Statistical analysis Statistical analysis was performed using GraphPad Prism 8.0 software, and the data were expressed as mean ± standard deviation. The normality of the data was evaluated using the Shapiro-Wilk test, and the significant differences between two groups were analyzed using the t-test or Wilcoxon test. P < 0.05 was considered the significance level, indicating a statistically significant difference (*P < 0.05; **P < 0.01; ***P < 0.001).

[0028] II. Results and analysis 2.1 Staphylococcus aureus mastitis model As shown in Figure 1 A, histological examination by H&E staining showed obvious inflammatory infiltration in the mammary tissues of mastitis rats. As shown in Figure 1 B and 1C, compared with the control group, the expression levels of IL-6, IL-1β, MPO, and NAGase were significantly increased in the mammary tissues and blood samples of mastitis rats, which could be used as indicators of mammary epithelial cell injury. The results indicated that the mastitis rat model was successfully established.

[0029] 2.2 Composition and changes of the intestinal flora in mastitis rats A total of 49,587 - 60,627 effective units were generated from the colorectal contents, and 353 - 485 species were identified. Through 16S rRNA sequencing analysis, groups C and M showed similar α-diversity and β-diversity. Although the differences were not significant (P > 0.05), the abundance of the flora in group M decreased while the diversity increased, as Figure 2A shown, indicating that the structure and function of the intestinal microbiota might have changed. As shown in Figure 2B’ and 2B’’, among the 12 bacterial phyla in groups M and C, Bacteroidetes (mean 61.14%), Firmicutes (29.05%), and Proteobacteria (6.6%) were the most abundant. At the genus level, a total of 150 genera were identified, mainly including Bacteriaceae (25.17%), Prevotella (10.46%), Bacteroides (9.92%), Alloprevotella (9.31%), and UCG-005 (5.64%). As shown in Figure 2CIt can be seen that LEfSe analysis showed that Proteobacteria was significantly enriched in group M. In addition, at the genus level, Roseburia, Parabacteroides, Butyricimonas, UBA1819, and Dubosiella were enriched in group M, while Eubacterium mucigenum, Prevotellaceae NK3B31 group, Ruminococcus, and Lachnospiraceae were significantly enriched in group C. KEGG predicted based on 16S showed that the carotenoid biosynthesis pathway was significantly upregulated at level 3 in group M (P = 0.041), both the D-arginine and D-ornithine metabolism pathways were significantly upregulated (P = 0.015), and the meiosis-yeast metabolism pathway was also significantly upregulated (P = 0.012); while the polycyclic aromatic hydrocarbon degradation pathway was downregulated (P = 0.031), and the proteasome degradation pathway was downregulated (P = 0.039). The above results indicate that the alteration of the microbiota is related to the alteration of functions ( Figure 2D and 2E as shown).

[0030] 2.3 Metabolic profiles of colorectal contents, plasma, and mammary gland tissues of mastitis rats In samples of colorectal contents ( Figure 3A ), plasma ( Figure 3B ), and mammary gland tissues ( Figure 3C ), OPLS scores showed different metabolite distributions between group C and group M. A total of 198 metabolites were identified in colorectal contents, of which 79 and 119 were detected in positive and negative ion modes respectively, with an R2Y value of 0.999 and a Q2 value of 0.744. A total of 143 metabolites were identified in plasma analysis, of which 93 and 50 were detected in positive and negative ion modes respectively, with an R2Y value of 0.999 and a Q2 value of 0.844. A total of 480 metabolites were identified in mammary gland tissues, of which 255 and 225 were detected in positive and negative ion modes respectively, with an R2Y value of 0.994 and a Q2 value of 0.894. The Venn diagram shows the common metabolites among these three sample types. At the superclass level, lipids and lipid-like molecules (38.19%, 32.78%, and 31.02%), organic heterocyclic compounds (13.92%, 16.91%, and 16.84%), and organic acids and their derivatives (12.47%, 17.52%) were dominant in colorectal contents, plasma, and mammary gland tissue samples ( Figures 3D - 3F ). At the subclass level, amino acids, peptides, and analogs (9.38%) were the most abundant in the colorectum, followed by fatty acids and conjugates (4.37%), carbohydrates and carbohydrate conjugates (3.06%), and flavonoids (2.38%).

[0031] In plasma samples, amino acids, polypeptides and analogues (10.44%), fatty acids and conjugates (5.02%), carbohydrates and their conjugates (3.73%), while carbonyl compounds accounted for approximately 1.83%. In mammary tissue samples, amino acids, polypeptides and analogues (13.34%), followed by fatty acids and conjugates (4.48%). Carbohydrates and their conjugates accounted for approximately 4%, while eicosanoids accounted for 2.18%.

[0032] 2.4 Metabolites and metabolic pathways in mastitis rats To demonstrate the differential metabolites (P < 0.05 and VIP > 1.0) in different mastitis samples, Figure 4A showed 7 common metabolites between colorectal contents and mammary tissue, including pregnanetriol and pelargonic acid. There were 4 similar metabolites between colorectal contents and plasma, including 7-ketodeoxycholic acid and stearoyl carnitine. 5 similar metabolites were found between plasma and mammary tissue, including solerol and dihydroxy α-linolenic acid. In addition, 4-hydroxybenzaldehyde, serinol and 2-methylbenzoic acid were also observed. The results suggest that there may be metabolic similarities among the three different samples of mastitis rats. Hierarchical cluster analysis was performed on the top 50 metabolites with the highest abundances in colorectal contents, plasma and mammary tissue samples (Figure 4B’, 4B’’ and 4B’’’).

[0033] Compared with the control group, the concentrations of bile acids, alcohols and their derivatives (including isolithocholic acid, ursodeoxycholic acid and 7-ketodeoxycholic acid) increased among 11 metabolites in the colorectal contents of mastitis rats. In addition, the contents of 3 cholestanes and 6 sterols increased. Stearoylcarnitine also showed a higher concentration, but pregnanetriol decreased (Figure 4C). Compared with the control group, the levels of L-isoleucine, L-valine, L-tyrosine and stearoyl carnitine were upregulated in the plasma of mastitis rats. In addition, the concentrations of 7 bile acids, alcohols and their derivatives (including cholic acid and 7-ketodeoxycholic acid) were upregulated (Figure 4D).

[0034] Compared with the control group, the expression levels of L-arginine and L-cysteine were lower in the mammary tissue of mastitis rats, and the concentrations of various carbohydrates and their conjugates decreased. The expressions of 11 linoleic acid and its derivatives, as well as pregnanetriol, were also downregulated; in addition, 3 bile acids, alcohols and derivatives including demethylcholic acid were upregulated. In addition, 28 eicosanoids including prostaglandin F2a and prostaglandin D1 changed (Figure 4E).

[0035] It is known from Figure 5A that in colorectal contents, the upregulated metabolic pathways mainly include primary bile acid synthesis, steroid biosynthesis and steroid hormone biosynthesis. It is known from Figure 5BIt can be seen that in plasma, the metabolic pathways are mainly concentrated in the prolactin signaling pathway and the synthesis of aminoacyl-tRNA, and significant changes in plasma metabolites occurred (P<0.05). From Figure 5C It can be seen that for mammary gland tissue, the metabolic pathways are mainly concentrated in the synthesis of unsaturated fatty acids, linoleic acid metabolism, arachidonic acid metabolism, and the oxytocin signaling pathway, and significant changes in metabolites in the mammary gland tissue occurred (P<0.05).

[0036] 2.5 Interaction between proteomics and metabolic changes A total of 283 proteins were identified in the proteomics of mammary gland tissue, of which 135 were up-regulated and 148 were down-regulated. The volcano plot shows the differentially expressed proteins (DEPs) (FC>1.2 or FC<0.83) ( Figure 6A ). The heat map also shows the abundances of the top 10 up-regulated and down-regulated proteins, including Fst and Pgr ( Figure 6B ). GO analysis of the differentially expressed proteins showed significant enrichment of "cytosol" in the cellular component. In terms of molecular function, identical protein binding and calcium ion binding became prominent categories ( Figure 6C ). In the biological process section, it was found that "innate immune response" was up-regulated within group M, including genes such as Eif2ak2, Irgm, Ifih1, Irf5, Trim30d, Cyld, Oasl, Ipo7, and Tnfaip8l2, indicating that mastitis infection triggers a multi-faceted innate immune response. In the KEGG analysis, it was observed that the pathways related to cell adhesion and cytoskeleton regulation were significantly down-regulated, including pathways such as focal adhesion, EMC receptor interaction, Rap1 signaling pathway, regulation of actin cytoskeleton, adherens junction, and regulation of cell adhesion molecules (CAMs) ( Figure 6D ). Two integrin adhesion receptors, Itgav and Itgb3, are potential targets for intervening in the development of mastitis and may act as key regulators ( Figure 6E ).

[0037] In the ECM receptor interaction pathway, western blot analysis was performed to identify five enriched proteins, Itgav, Itgb3, Dag1, Lamb2, and Lamc1. In addition, impaired immune response and cell adhesion in the mammary gland may lead to damage to the blood-milk barrier. In western blot experiments, to evaluate the barrier damage in the mammary gland tissues of rats induced by mastitis, the expression levels of tight junction (TJ) proteins ZO-1, Occludin, and Claudin-3 were measured. The results showed that ZO-1, Occludin, and Claudin-3 were significantly downregulated in the mammary gland tissues of rats induced by mastitis, and the differential expression of these three proteins (*P<0.05 and ***P<0.001) indicated impaired integrity of the blood-milk barrier. To establish the correlation between more differential proteins and the occurrence of mastitis, eight target genes (Fst, PGR, IFIH1, Tnfaip8l2, IRGM, IRF5, PAL2G4A, and Prxl2b) were also selected for PCR verification. The verification results were all consistent with the proteomic analysis ( Figures 7A - 7D ).

[0038] Based on KGML network analysis, the regulatory relationships between differential metabolites in different biological samples and proteins in mammary gland tissues were determined. In these networks, most of the related proteins were downregulated. In the protein-colorectal content metabolite network, the necrosis pathway was mainly enriched, and the protein digestion and absorption pathway was the key pathway in plasma. Upregulated metabolites included L-isoleucine, L-tyrosine, and L-valine, and downregulated proteins included A0a0H2UHX5 (gene name Mme), D3ZN64 (gene name Col28a1), and F1M7S4 (gene name Cpa3), etc. A stronger correlation was observed between mammary gland tissue metabolites and proteins enriched in arachidonic acid metabolism, cancer pathways, and necrosis. The typical upregulated protein related to mastitis was A0A0G2KAA9 (gene name Pla2g4a), and the downregulated protein was M0R5U4 (gene name Adcy9). Arachidonic acid and prostaglandin F2a were continuously upregulated metabolites, while L-arginine and inosine monophosphate were continuously downregulated metabolites. In summary, Figures 8A - 8C the interaction between the proteome and metabolome was revealed. In addition, B0BNM2 (gene name Mbd2) was considered a representative protein in the network related to mammary gland metabolic proteins ( Figure 8D ).

[0039] This application studied the metabolic changes in colorectal contents, plasma, and mammary gland tissues. Cholestane steroids and secondary sterols showed an upward trend in colorectal contents. Steroids and their derivatives are synthesized by enzymatic metabolism of cholesterol. Elevated levels of cholestanol steroids and sterols indicate steroid hormone metabolic disorders in mastitis patients. Plasma analysis showed upregulated expression of L-isoleucine, L-valine, and L-tyrosine. Changes in 28 eicosanoids, including prostaglandin F2α and prostaglandin D1, were detected in mammary gland tissues. These are the most important active metabolites of the bioactive lipid arachidonic acid and play a key role in the inflammatory process. The upregulation of inflammatory mediators such as IL-1β, IL-6, and MPO in the mammary gland tissues of mastitis-induced rats may be related to this. Therefore, it is inferred that mastitis has an adverse effect on the body through multiple metabolic pathways such as steroid hormones, amino acids, and arachidonic acid.

[0040] This study clarified the significance of the composition and metabolic changes of the microbiota during the progression of mastitis, and also studied the gene expression patterns related to innate immunity and interaction with extracellular matrix receptors under this pathological condition. Significant differences in metabolites were observed between different samples, indicating a common pathway behind these changes. Specifically, this application proposes that the regulation of bile acids and arachidonic acid has the potential as a biomarker for mastitis.

[0041] In the present invention, terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0042] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A mastitis metabolic marker, wherein the marker is at least one of IL-6, IL-1β, MPO, NAGase, ZO-1, Occludin, and Claudin-3.

2. The mastitis metabolic marker according to claim 1, wherein, the expression levels of IL-6, IL-1β, MPO, and NAGase in mastitis tissues are higher than those in normal mammary tissues.

3. The mastitis metabolic marker according to claim 1, wherein, the expression levels of ZO-1, Occludin, and Claudin-3 in mastitis tissues are lower than those in normal mammary tissues.

4. The mastitis metabolic marker according to claim 1, wherein, the abundance of bacterial taxa in mastitis tissues is less than that in normal mammary tissues, while the diversity of bacterial taxa in mastitis tissues is greater than that in normal mammary tissues.

5. The mastitis metabolic marker according to claim 1, wherein, the expression level of Proteobacteria in mastitis tissues is higher than that in normal mammary tissues; at the genus level, the expression levels of Roseburia, Parabacteroides, Butyricimonas, UBA1819, and Dubosiella in mastitis tissues are higher than those in normal mammary tissues.

6. The mastitis metabolic marker according to claim 1, wherein, the carotenoid synthesis pathway, meiosis-yeast metabolism pathway, D-arginine and D-ornithine metabolism pathway in mastitis tissues are all higher than those in normal mammary tissues, while the polycyclic aromatic hydrocarbon degradation pathway and proteasome degradation pathway are both lower than those in normal mammary tissues.

7. The mastitis metabolic marker according to claim 1, wherein, the targets of mastitis are at least one of Itgav, Itgb3, and bile acid metabolism.

8. Use of a mastitis metabolic marker according to any one of claims 1 to 7 in the preparation of a kit for preventing, diagnosing, or treating mastitis, wherein the sample of the diagnostic kit is from human ex vivo serum.

9. Use of a mastitis metabolic marker according to any one of claims 1 to 7 in the preparation of a drug for preventing, diagnosing, or treating mastitis, wherein the drug includes nucleic acid drugs, antibody drugs, small molecule chemical drugs, or cell therapy drugs.

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