Effect of Collagen on Vitamin E Alleviating the Damage of Vomitoxin on Bovine Ruminal Epithelial Cells

Abstract

The application relates to the field of biotechnology and discloses a role of Collagen in relieving damage of vomitoxin to bovine rumen epithelial cells, wherein the Collagen is COL1A1 protein, COL2A1 protein and COL5A1 protein. It is found in the application that in a DON damage model, the contents of the COL1A1 protein, the COL2A1 protein and the COL5A1 protein are obviously reduced, the contents of the COL1A1 protein, the COL2A1 protein and the COL5A1 protein are increased after adding vitamin E, the damage of vomitoxin to bovine rumen epithelial cells is reduced, and it is indicated that the expression amount of the COL1A1 protein, the COL2A1 protein and the COL5A1 protein is related to the damage of vomitoxin; then, the COL1A1 protein, the COL2A1 protein and the COL5A1 protein are overexpressed in bovine rumen epithelial cells, and it is found that the damage of vomitoxin to bovine rumen epithelial cells is reduced, which indicates that by up-regulating the expression of the COL1A1 protein, the COL2A1 protein and the COL5A1 protein, the damage of vomitoxin to bovine rumen epithelial cells can be relieved, and a new target for preparing a medicine for relieving the damage of vomitoxin is provided.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the role of Collagen in alleviating the damage of vomiting toxin to bovine rumen epithelial cells by vitamin E. Background Technology

[0002] Vomitoxin, also known as deoxynivalenol (DON), is a trichothecene B compound produced by fungi such as *Fusarium graminearum*. It is widely detected in corn, wheat, legumes, and corn silage, making it one of the most common types of mycotoxins. Due to its widespread distribution in feed and persistent toxicity, DON can enter the food chain and harm human and animal health. Studies have shown that DON can affect communication between rumen microorganisms. Increased DON concentration in the diet significantly reduces the biosynthesis of pantothenic acid and CoA in rumen microorganisms, leading to reduced microbial energy metabolism, inhibiting VFA synthesis, and resulting in decreased milk production in dairy cows. The absorption of nutrients by the rumen epithelium is affected by acidosis and rumenitis. Therefore, finding effective ways to mitigate DON toxicity and reduce its harm to humans and animals is of great significance.

[0003] Since DON contamination is unavoidable, mitigating and reducing its toxic effects on livestock and poultry has attracted widespread attention from scholars. However, traditional detoxification methods suffer from unstable effects, reduced nutritional value, and the generation of residual harmful substances, making large-scale production difficult. Vitamin E, as a natural antioxidant, has a wide range of biological functions with positive effects on oxidation, immunity, and reproduction. Furthermore, it ensures that vitamin E plays a broad and important role in promoting the growth and development of various livestock and poultry. Summary of the Invention

[0004] The purpose of this invention is to provide the effect of Collagen in alleviating the damage of vomitoxin to bovine rumen epithelial cells by vitamin E, thus providing a new target for the preparation of drugs that alleviate vomitoxin damage.

[0005] To achieve the above objectives, this invention provides the application of Collagen as a target in the preparation of drugs that alleviate the damage of vomitoxin to bovine rumen epithelial cells. Collagen is COL1A1 protein, COL2A1 protein, and COL5A1 protein. The amino acid sequence of the COL1A1 protein is shown in SEQ ID NO.19; The amino acid sequence of the COL2A1 protein is shown in SEQ ID NO.20; The amino acid sequence of the COL5A1 protein is shown in SEQ ID NO.21.

[0006] Furthermore, the application is to promote the expression of COL1A1, COL2A1, and COL5A1 proteins, promote ECM receptor function, protect bovine rumen epithelial cells, and alleviate vomitoxin damage.

[0007] To achieve the above objectives, the present invention also provides the application of Collagen as a target in screening drugs to alleviate the damage of vomitoxin to bovine rumen epithelial cells. Collagen is COL1A1 protein, COL2A1 protein, and COL5A1 protein. The amino acid sequence of the COL1A1 protein is shown in SEQ ID NO.19; The amino acid sequence of the COL2A1 protein is shown in SEQ ID NO.20; The amino acid sequence of the COL5A1 protein is shown in SEQ ID NO.21.

[0008] Furthermore, in application, COL1A1, COL2A1, and COL5A1 proteins are used as targets to detect their expression levels and screen for drugs that can alleviate bovine rumen epithelial cell damage caused by vomitoxin.

[0009] To achieve the above objectives, the present invention also provides the role of a formulation that promotes Collagen expression in the preparation of a drug that alleviates the damage of vomitoxin to bovine rumen epithelial cells, wherein Collagen is COL1A1 protein, COL2A1 protein, or COL5A1 protein; The amino acid sequence of the COL1A1 protein is shown in SEQ ID NO.19; The amino acid sequence of the COL2A1 protein is shown in SEQ ID NO.20; The amino acid sequence of the COL5A1 protein is shown in SEQ ID NO.21.

[0010] Furthermore, formulations that promote Collagen expression include small molecule compounds, antibody drugs, proteins, nucleic acid molecules, peptides, lipids, carbohydrates, or combinations thereof.

[0011] To achieve the above objectives, the present invention also provides a drug for alleviating the damage of vomitoxin to bovine rumen epithelial cells. The drug targets COL1A1 protein, COL2A1 protein, and COL5A1 protein, promotes the expression of COL1A1 protein, COL2A1 protein, and COL5A1 protein, and alleviates the damage of vomitoxin to bovine rumen epithelial cells.

[0012] Furthermore, the active ingredient of the drug includes small molecule compounds that promote Collagen expression, antibody drugs, proteins, nucleic acid molecules, peptides, lipids, carbohydrates, or combinations thereof.

[0013] The advantages and positive effects of the Collagen described in this invention in alleviating the damage of vomiting toxin to bovine rumen epithelial cells by vitamin E are as follows: This invention found that in the DON 0.4 μg / mL injury model, the levels of COL1A1, COL2A1, and COL5A1 proteins were significantly reduced. After adding vitamin E 50 μM / L, the levels of COL1A1, COL2A1, and COL5A1 proteins rebounded, and the damage to bovine rumen epithelial cells caused by vomitoxin was reduced, suggesting that the expression levels of COL1A1, COL2A1, and COL5A1 proteins are related to vomitoxin damage. Furthermore, overexpression of COL1A1, COL2A1, and COL5A1 proteins in bovine rumen epithelial cells reduced the damage to these cells caused by vomitoxin. This indicates that upregulating the expression of COL1A1, COL2A1, and COL5A1 proteins can alleviate the damage to bovine rumen epithelial cells caused by vomitoxin, providing a new target for the preparation of drugs to alleviate vomitoxin damage.

[0014] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0015] Figure 1 In the table, A represents the cell viability of bovine rumen epithelial cells treated with different DON concentrations at different culture times, B represents the total antioxidant status of bovine rumen epithelial cells cultured for 12 hours at different DON concentrations, C represents the total oxidative status of bovine rumen epithelial cells cultured for 12 hours at different DON concentrations, and D represents the oxidative stress index of bovine rumen epithelial cells cultured for 12 hours at different DON concentrations. Figure 2 In Figure A, cell viability is shown in different concentrations of vitamin E treatment groups; in Figure B, cell viability is shown in different concentrations of vitamin E combined with DON. Figure 3 In the table, A represents the intracellular malondialdehyde level in the vitamin E treatment group and the vitamin E and DON combined treatment group; B represents the total oxidative state; C represents the total antioxidant state; and D represents the oxidative stress index. Figure 4 In the figure, A represents the flow cytometry analysis results of the CON, D0.4, V50, and D0.4V50 groups, and B represents the apoptosis rate. Figure 5In the table, A represents the mRNA level of Occludin, B represents the mRNA level of Claudin-1, C represents the mRNA level of Zonulaoccludens-1, D represents the mRNA level of NF-Kappa b, E represents the mRNA level of IL-6, F represents the mRNA level of IL-1β, G represents the mRNA level of TNF-α, and H represents the mRNA level of IL-10. Figure 6 The results of SDS-PAGE electrophoresis of the sample proteins; Figure 7 This is a statistical graph of the mass spectrometry data of the sample proteins; Figure 8 This is a distribution map of peptide lengths in the sample protein. Figure 9 A heatmap of PCC analysis in sample repeatability testing; Figure 10 Volcano plots showing the differential expression levels of proteins upregulated or downregulated among the Control, DON0.4, VE50, and D0.4V50 groups. Specifically, A compares the Control and DON0.4 groups; B compares the DON0.4 and D0.4V50 groups; C compares the DON0.4 and VE50 groups; D compares the VE50 and Control groups; E compares the VE50 and D0.4V50 groups; F compares the Control and D0.4V50 groups; and G is a comprehensive statistical plot of upregulated or downregulated proteins in AF. Figure 11 In the middle section, A is a heatmap of cluster analysis of biological processes in GO enrichment, B is a heatmap of cluster analysis of cellular components, and C is a heatmap of cluster analysis of molecular functions. Figure 12 In the image, A represents the subcellular structure annotation of differentially expressed proteins in the DON / Control group, and B represents the subcellular structure annotation of differentially expressed proteins in the DON / DON-VE group. Figure 13 In the middle section, A represents the KEGG pathway enrichment analysis of the DON / Control group, and B represents the KEGG pathway enrichment analysis of the DON / DON-VE group. Figure 14 In Figure A, the relative quantitative expression analysis of COL1A1, COL2A1, COL6A1 and HSPG2 proteins significantly enriched in the ECM receptor pathway in the DON / Control group is shown. In Figure B, the relative quantitative expression analysis of COL1A1, COL2A1, COL6A1 and HSPG2 proteins significantly enriched in the ECM receptor pathway in the DON / DON-VE group is shown. Figure 15In Figure A, the relative quantitative expression analysis of COL1A1, COL2A1, COL5A1, and COL6A1 proteins significantly enriched in the digestive and absorption pathways of the DON / Control group is presented. In Figure B, the relative quantitative expression analysis of COL1A1, COL2A1, COL5A1, and COL6A1 proteins significantly enriched in the digestive and absorption pathways of the DON / DON-VE group is presented. Figure 16 In the middle section, A represents the DON / Control group enriched by ECM receptor interaction through the KEGG pathway, and the relative quantitative expression analysis of the enriched protein was further validated by PRM. In the middle section, B represents the DON / DON-VE group enriched by ECM receptor interaction through the KEGG pathway, and the relative quantitative expression analysis of the enriched protein was further validated by PRM. Figure 17 In Figure A, the digestion and absorption of proteins enriched through the KEGG pathway in the DON / Control group were analyzed, and the relative quantitative expression of the enriched proteins was further validated by PRM. In Figure B, the digestion and absorption of proteins enriched through the KEGG pathway in the DON / DON-VE group were analyzed, and the relative quantitative expression of the enriched proteins was further validated by PRM. Figure 18 The relative expression levels of mRNA of COL1A1, COL2A1, COL5A, and HSPG2 in the embodiments of the present invention; Figure 19 This invention illustrates the effects of DON and VE on cell viability and apoptosis rate after overexpression of COL1A1, COL2A1, and COL5A in this embodiment, where A represents cell viability and B represents cell apoptosis rate. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0017] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0018] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0019] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0020] Example 1 The following steps were taken to establish an in vitro model of bovine rumen epithelial cell injury: Bovine rumen epithelial cells were cultured in vitro and treated with 0, 0.4, 0.8, 1.6, 2.4, and 4.8 μg / ml DON for 0, 12, 24, and 48 h, respectively. Cell viability was determined using the CCK-8 assay. The results are as follows: Figure 1 As shown in Figure A, the culture time was determined to be 12 hours. Total oxidative state (TOS) and total antioxidant state (TAS) were measured using a kit, and the oxidative stress index (OSI) was calculated by dividing TOS / TAS. The results are shown below. Figure 1 As shown in Figures B, C, and D, the DON treatment concentrations were determined to be 0.4 and 0.8 μg / ml.

[0021] Example 2 Vitamin E alleviates DON-induced rumen epithelial cell damage in dairy cows: (1) Regarding cell viability: 100 μL of BREC cell suspension (5 × 10⁻⁶) 4 Cells were seeded at 70-80% (number of cells / mL) in 96-well cell culture plates. After reaching a confluence of 70-80%, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. The control group was treated with complete culture medium, while the vitamin E treatment groups required different concentrations (0, 12.5, 50, 200, 800, 3200 μM / L) of working vitamin E solution and cultured for 12 hours. After the treatment, the liquid in the wells was discarded, and each well was washed twice with DMEM / F12 medium. 100 μL of complete culture medium and 10 μL of CCK-8 solution were added to each well, and the cells were incubated at 37°C for 4 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was calculated. Results are as follows: Figure 2 As shown in Figure A. Cell viability was determined by combining 50 and 200 μM / L vitamin E with 0.4 and 0.8 μg / mL DON, and the results are shown below. Figure 2 As shown in B.

[0022] (2) Oxidative stress level: After cell passage culture, cell state and density were observed. Cells were collected by trypsin digestion to prepare a rumen epithelial cell suspension. The suspension was then cultured at 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 2 mL / ml in 6-well plates. Cell density and growth were observed. When the density reached 70-80%, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. The control group was treated with complete culture medium, while the experimental groups were cultured for 12 hours with different treatment media. Cells were then collected according to the kit instructions, and the intracellular MDA, TOS, TAS, and OSI levels were measured. Results are as follows: Figure 3As shown, the concentration of DON was determined to be 0.4 μg / ml, and the concentration of vitamin E was determined to be 50 μM / L.

[0023] (3) Determination of apoptosis rate: After cell passage culture, cell state and density were observed. Cells were collected by trypsin digestion to prepare a rumen epithelial cell suspension. The suspension was then cultured at 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 2 cells / ml in 6-well plates. Cell density and growth were observed. When the density reached 70%-80%, the cells were cultured for 12 hours. The original culture medium was removed, and the cells were washed twice with pre-chilled PBS. 500 μL of EDTA-free trypsin was added for approximately 5 minutes of digestion. After complete digestion, 2 mL of complete culture medium was added to stop the digestion. The entire liquid was transferred to a centrifuge tube and centrifuged at 2000 rpm for 10 minutes at room temperature. The cells were collected. The supernatant was carefully removed, and the cells were resuspended in 1 mL of pre-chilled PBS. The cells were transferred to a 1.5 mL centrifuge tube and centrifuged at 2000 rpm for 10 minutes. The cells were washed, and this process was repeated twice, discarding as much supernatant as possible. Dilute the 10×Binding Buffer in the kit to 1×Binding Buffer with deionized water beforehand. Add 200 μL of 1×Binding Buffer to a centrifuge tube to suspend the cells. Add 5 μL of Annexin V-FITC, mix well, and incubate at room temperature for 15 min in the dark. Five minutes before flow cytometry analysis, add 5 μL of PI staining solution and 200 μL of 1×Binding Buffer. Results are as follows: Figure 4 As shown, the combination treatment with VE and DON significantly reduced the DON-induced apoptosis rate (P<0.05).

[0024] (4) Expression of mRNAs of genes related to cell permeability, immunity, and inflammation: I. Total RNA Extraction and Quantitative PCR: ① BREC cell sample collection and total RNA extraction: After cell passage, cell state and density were observed. Cells were collected by trypsin digestion and liver cell suspension (16th generation adherent BREC cells) was prepared. The suspension was then cultured at 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 10 cells / mL in 6-well plates. 2 mL of solution was used per well. Cell density and growth were observed. When the density reached 70%-80%, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. Drug treatment was then administered, resulting in four treatment groups: control group (complete culture medium), vomitoxin group (0.4 μg / mL), vitamin E treatment group (50 μM / L), and vomitoxin + vitamin E combined treatment group (0.2 μg / mL DON + 50 μM / L VE). Cells were incubated for 12 h. 1) Remove the culture medium on ice, wash three times with PBS to remove serum and enzymes, add 1 ml of Trizol to each well, scrape the cells off with a cell scraper, collect them into 1.5 ml EP tubes, and lyse on ice for 20 min; 2) Add 200 μl of chloroform to the EP tube, shake vigorously up and down, let stand on ice for 10 min, pre-cool centrifuge, centrifuge at 4℃ and 1200 rpm for 20 min; 3) Transfer 200-300 μl of the supernatant into a new EP tube (do not aspirate the white membrane), add an equal volume of isopropanol, gently invert 5 times, let stand on ice for 10 min, and centrifuge at 1200 rpm for 10 min at 4℃. 4) Discard the supernatant, add 1 ml of 75% ethanol, tap the white precipitate to remove it, and centrifuge at 8000 rpm for 5 min at 4°C. 5) Discard the supernatant, add 500 μl of anhydrous ethanol, centrifuge at 8000 rpm for 3 min at 4 °C, discard the supernatant, and air dry for 5-10 min (the RNA precipitate will become transparent after drying).

[0025] 6) Dissolve 10-20 μl of DEPC in water, depending on the amount of RNA precipitate. 7) Measure RNA concentration. Sample RNA concentration > 500 ng / ml, A260 / A280 between 1.8 and 2.0, and A230 / A260 between 1.9 and 2.3. The obtained RNA solution can be reverse transcribed immediately or stored at -80°C.

[0026] ②cDNA synthesis: Reverse transcription was performed using the Tiangen Fast King cDNA first-strand synthesis kit. The specific procedure is as follows: 1) Thaw the template RNA and each system in the kit on an ice box. After thawing, shake to mix and then centrifuge briefly before use.

[0027] 2) Prepare the gDNA removal reaction system: 2 μL 5×gDNA Buffer + 5 μL template RNA solution, and add RNase-Free ddH2O to a final volume of 10 μL.

[0028] 3) Briefly centrifuge the prepared gDNA removal reaction system, place it in a PCR amplification instrument, incubate at 42℃ for 3 minutes, and place it on an ice box.

[0029] 4) Prepare the reverse transcription reaction system: 2μL 10×Fast RT Buffer + 1μL RT Enzyme Mix + 2μL FQ-RT Primer Mix, and add RNase-Free ddH2O to make up to 10μL.

[0030] 5) After mixing the prepared reverse transcription reaction system, briefly centrifuge it, add it to the incubated gDNA removal reaction system, place it in a PCR amplification instrument, incubate at 42℃ for 15 min, incubate at 95℃ for 3 min, and then place it on an ice box. The obtained cDNA is used for subsequent experiments.

[0031] ③ Real-time PCR The Tiangen qPCR Pre Mix (SYBR Green) kit was used for real-time quantitative PCR in this experiment. -ΔΔCt The method calculates the relative expression level of mRNA.

[0032] 1) Primer sequences for real-time PCR are shown in Table 1. β-actin was selected as the internal reference gene. Dyes and other materials were thawed on ice. 2) Prepare a 20 μL real-time PCR system as shown in Table 2 and mix well. 3) Select suitable reaction conditions for PCR reaction. This experiment used a two-step reaction method. The specific conditions are shown in Table 3.

[0033] Table 1. Primer sequences for real-time PCR

[0034] Table 2. Real-time PCR reaction system

[0035] Table 3. Quantitative Fluorescence Reaction Procedure

[0036] like Figure 5 As shown, the mRNA levels of tight junction proteins were significantly reduced when cells were in a damaged state (P<0.001). Compared with the DON group, the vitamin E group and the DON combination treatment group did not show a significant increase in ZO-1 mRNA levels, while the expression levels of Occludin and Claudin-1 mRNA were significantly increased (P<0.05). This indicates that vitamin E alleviated the DON-induced damage to the BREC cell barrier function.

[0037] Compared with the control group, the relative expression levels of NF-Kappab in the DON-treated groups were significantly decreased (P<0.0001). Figure 5 The presence of DON indicates that DON induces a strong immune response in bovine rumen epithelial cells. In contrast, the vitamin E and DON combination treatment group (D<0.4V50) showed a highly significant upregulation of relative NF-Kappab expression compared to the DON-treated group (P<0.0001), suggesting that vitamin E alleviated the DON-induced immune response.

[0038] Compared with the control group, the relative expression level of the pro-inflammatory gene IL-6 mRNA was significantly upregulated in the DON treatment group (P<0.05). Figure 5 In the middle E), the relative expression levels of IL-10 and TNF-α mRNA were significantly upregulated (P<0.001). Figure 5 In the F and G groups, the relative expression level of the anti-inflammatory gene IL-10 mRNA was significantly upregulated in the DON treatment group (P<0.001). Figure 5 The presence of H indicates that vomitoxin induces a strong inflammatory response in the rumen epithelial cells of dairy cows. Compared with the vomitoxin-treated group, the vitamin E combined with vomitoxin treatment group (D<0.4V50) showed no significant downregulation of the relative expression of the pro-inflammatory gene IL-6, while the relative expression of IL-10 and TNF were both significantly downregulated (P<0.001); the relative expression of the anti-inflammatory gene IL-10 mRNA was also significantly downregulated (P<0.001), indicating that vitamin E alleviated the inflammatory response induced by vomitoxin.

[0039] Example 3 4D Label-Free Quantitative Proteomics Analysis: BREC cells in good growth condition were passaged to 25cm. 2 Cells were cultured in culture flasks and treated according to experimental groups (Control, D0.4, V50, D0.4V50). The culture medium was discarded, and the cells were washed three times with pre-chilled PBS. Cells were then scraped off and collected into centrifuge tubes, centrifuged at 1000 rpm for 5 min at 4°C, the supernatant was discarded, the tubes were sealed with sealing film, flash-frozen in liquid nitrogen for 10 min, and then stored at -80°C for later use. For detection, protein extraction and quality control were performed first, followed by trypsin digestion. Then, analysis and data processing were performed using liquid chromatography-mass spectrometry (LC-MS). Finally, bioinformatics analysis was conducted on the differentially expressed proteins, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, to screen for enriched pathways.

[0040] 1. Protein content determination in samples: Protein concentration results are shown in Table 4, and SDS-PAGE results are as follows: Figure 6 The protein bands are clear, the distribution is normal, there is no protein degradation, the electrophoretic behavior between groups is not significantly different, and there is a certain degree of high-abundance protein. Quality control is qualified.

[0041] Table 4. Protein concentration determination results

[0042] 2. Mass spectrometry quality control analysis: Experimental DIA data were analyzed using the DIA-NN (v1.8) search engine and the database Bos_taurus_9913_PR_20240325.fasta (37501 sequences). A theoretical spectral library was constructed using deep learning algorithms, and a reverse library was added to calculate the false positive rate (FDR) caused by random matching. In this protein mass spectrometry analysis, 72353 peptides were identified, 62186 unique peptides were identified, the total number of identified proteins was 8562, and 8553 proteins were quantified. Figure 7 ).

[0043] After the database search is completed, quality control evaluation results are performed. Figure 8 Most peptides are distributed in the range of 7-20 amino acids, which conforms to the general rules based on enzymatic digestion and mass spectrometry fragmentation; most proteins correspond to more than two peptides; and they meet the proteomics quality control standards.

[0044] 3. Sample repeatability test: Based on the intensity values ​​of all samples, the Pearson correlation coefficient between each pair of samples was calculated, and a visual heatmap was plotted. Figure 9 This coefficient is used to measure the correlation between two sets of data.

[0045] 4. Analysis of differentially expressed proteins of vitamin E after DON-induced BHEC cell damage: BREC cell proteomic data such as Figure 10 As shown. Proteomics analysis of each group of samples was performed using liquid chromatography-mass spectrometry (LC-MS). During the proteomics analysis, the relative quantitative values ​​of each protein in each comparison group were subjected to a t-test, with P < 0.05 indicating a significant difference. Before the test, the relative quantitative values ​​of proteins were log2 transformed to ensure the test data conformed to the normal distribution required for the t-test. Through the above difference analysis, when the P value < 0.05, a change in expression level exceeding 1.5 was used as the threshold for significant upregulation, and a change less than 1 / 1.5 was used as the threshold for significant downregulation. In the data analysis, volcano plots were drawn using FC and P values ​​to represent the significant differences in sample data. A total of 1220 quantifiable DE proteins were identified in this experiment. From the volcano plot (red, upregulation; green, downregulation), the study on the mechanism by which vitamin E alleviates DON-induced damage to BRECs shows that: in the DON / Control group, 184 proteins were upregulated and 135 proteins were downregulated; DON... - The VE / Control group upregulated 114 proteins and downregulated 76 proteins; the DON / DON group... - In the VE group, 125 proteins were upregulated and 107 proteins were downregulated. To more intuitively and visually compare the proteomic differences among the groups, the results are as follows: Figure 10 As shown in G.

[0046] 5. Functional classification of differentially expressed proteins after vitamin E-induced DON-induced damage to BHEC cells: To understand the potential biological functions of differentially expressed proteins in the process of vitamin E alleviating DON-induced BHEC cell damage, functional analysis of differentially expressed proteins among different treatment groups was conducted using gene ontology (GO) and subcellular structural localization functional classification.

[0047] (1) GO secondary classification of differentially expressed proteins: Differential proteins were classified into three main categories using GO enrichment analysis based on bioinformatics: biological processes, cellular components, and molecular functions, thus elucidating the attributes of genes and gene products in organisms from different perspectives. Differential protein clustering analysis was then performed on each of the three GO categories. The results showed ( Figure 11 In the DON / Control group (A, B, C), when vitamin E alleviated DON-induced cell damage, the differentially regulated proteins were significantly downregulated. In the bioengineering classification, these differentially regulated proteins primarily involved proteins, amyloid, glycoproteins, endopeptidase activity, diterpenes, lipids, triglycerides, phospholipids, cholesterol, inflammatory responses, and cell-matrix adhesion. In the cellular component classification, they were mainly distributed in the extracellular space, the matrix containing collagen, the endoplasmic reticulum, collagen trimers, collagen trimer complexes, ribbon-like collagen fibers, fibrillary collagen trimers, and the basement membrane. In the molecular function classification, they were mainly concentrated in platelet-derived growth factor binding, extracellular matrix structural components, proteases, integrins, and amyloid-β binding. (DON / DON) - The proteins significantly upregulated in the VE group are mainly involved in the following biological processes: protein-lipid complex remodeling, retinoic acid metabolism, diterpene metabolism, lipid catabolism, phospholipid efflux, and SRP-dependent co-translation of proteins targeting membrane co-translation proteins. In the cellular component category, they are primarily distributed in secretory vesicles, blood microparticles, very low-density lipoprotein particles, high-density lipoprotein particles, triglyceride-rich plasma lipoprotein particles, chylomicrons, lipoprotein microparticles, plasma lipoprotein particles, protein-lipid complexes, globular high-density lipoprotein particles, endoplasmic reticulum, cytoplasmic ribosomes, ribosomal subunits, ribosomes, and cytoplasmic macroribosomal subunits. In the molecular function category, they are mainly involved in binding vitamin K-epoxide reductase (warfarin-sensitive), oxidoreductase activity, acting on the CH-OH group of the donor, acting as a receptor for apolipoprotein receptors and high-density lipoprotein particle receptors, ribosome structure, phosphatidylcholine-sterol O-acyltransferase activator, and lipase inhibitor activity.

[0048] (2) Subcellular localization of differentially expressed proteins: Mature proteins must reside within specific subcellular organelles to perform their stable biological functions. Subcellular organelles are essential components of the cell, consisting of micro-organs with specific morphologies and functions. To accurately understand the function of a protein, in addition to analyzing its structure, it is usually necessary to determine its subcellular location to identify the possible sites of its function. Subcellular localization of proteins is crucial for studying protein function. It allows researchers to pinpoint specific locations within the cell, such as the nucleus, various organelles, and the plasma membrane, thus providing insights into the mechanisms of gene action. Figure 12 As shown, the subcellular structure annotation results of differentially expressed proteins in each group reveal that, in order to further explore the regulatory mechanism by which vitamins alleviate DON-induced BREC cell damage, further analysis was conducted through subcellular division. The DON / Control group showed significant downregulation mainly in the extracellular space (31.85% of total), plasma membrane (17.78% of total), nucleus (17.04% of total), cytoplasm (14.07% of total), mitochondria (8.89% of total), cytoplasm and nucleus (2.96% of total), endoplasmic reticulum (4 cases, 2.9% of total), peroxisomes (4 cases, 2.96% of total), and other areas (2 cases, 1.48% of total). DON / DON - The VE group showed significant upregulation mainly in the cytoplasm (24.8% of the total), nucleus (20.8% of the total), extracellular fluid (16.8% of the total), plasma membrane (15.2% of the total), mitochondria (13.6% of the total), cytoplasm and nucleus (4% of the total), peroxisomes (2.4% of the total), cytoskeleton (1.6% of the total), and other (0.8% of the total).

[0049] (3) KEGG enrichment analysis of vitamin E on differentially expressed proteins after DON-induced BHEC cell injury: To further explore the regulatory pathways by which vitamins alleviate DON-induced BREC cell damage, KEGG pathway enrichment analysis was performed on differentially expressed proteins using bioinformatics. Figure 13 As can be seen from A and B, the DON / Control group and DON / DON -The vitamin E group was commonly enriched in the following pathways: map04512 ECM-receptor interaction, map04974 Protein digestion and absorption, map04614 Renin-angiotensin system, map00982 Drug metabolism - cytochrome P450, and map04979 Cholesterol metabolism. Furthermore, through... Figure 13 B shows the DON / Control group and DON / DON - The VE group was co-enriched in map04512 ECM-receptor interaction, map04151 PI3K-Akt signaling pathway, and map04974 Protein digestion and absorption. Specifically, the DON / Control and DON / DON groups were enriched. - The VE group showed significant enrichment of COL1A1, COL2A1, and HSPG2 proteins in the ECM receptor pathway, and their relative quantitative expression was as follows: Figure 14 As shown. DON / Control group and DON / DON - The VE group also showed significant enrichment of COL1A1, COL2A1, and COL5A1 proteins in the protein digestion and absorption pathways, with their relative quantitative expression as follows: Figure 15 As shown.

[0050] 6. PRM Verification: By significantly enriching and screening proteins and their pathways in the (KEGG pathway), 18 target proteins, as shown in Table 5, were obtained and subjected to PRM verification.

[0051] Table 5. Effects of differentially enriched proteins in vitamin E-induced DON-induced BREC cell damage on ECM receptor interactions, protein digestion, and absorption.

[0052] The proteins COL1A1, COL2A1, COL6A1, HSPG2, LAMC1, and ITGA5, which are significantly enriched in ECM receptor function, were summarized, and their relative quantitative expression results are as follows: Figure 16As shown in the figure. The results showed that COL1A1, COL2A1, and HSPG2 proteins exhibited similar trends in PRM validation and proteomics KEGG pathway analysis. The proteins COL1A1, COL2A1, and COL5A1, which were significantly enriched in protein digestion and absorption, were summarized, and their relative quantitative expression results are shown in the figure. Figure 17 As shown in the figure. The results show that COL1A1, COL2A1 and COL5A1 proteins also exhibited similar trends in PRM validation and proteomics KEGG pathway analysis, further demonstrating the rationality and reliability of proteomics analysis.

[0053] To further demonstrate that the addition of vitamin E 50 μM / L can alleviate bovine rumen epithelial cell damage induced by DON 0.4 μg / mL by upregulating COL1A1, COL2A1, and HSPG2 proteins in the ECM receptor pathway and COL1A1, COL2A1, and COL5A1 proteins in the protein digestion and absorption pathway, the relative expression levels of COL1A1, COL2A1, COL5A1, and HSPG2 mRNA were detected. The results are as follows: Figure 18 As shown in the figure. The results showed that the relative expression levels of COL1A1, COL2A1, COL5A, and HSPG2 mRNAs also exhibited similar trends in the PRM validation and proteomics KEGG pathway analysis results, further demonstrating the rationality and reliability of the proteomics analysis and PRM validation results.

[0054] The amino acid sequence of COL1A1 is shown in SEQ ID NO.19; the amino acid sequence of COL2A1 is shown in SEQ ID NO.20; and the amino acid sequence of COL5A1 is shown in SEQ ID NO.21.

[0055] In summary, at the molecular level, DON leads to oxidative damage, ribosome degradation, ribotoxic stress, and inhibition of DNA and protein synthesis, resulting in increased expression of inflammation-related genes and inducing apoptosis and autophagy. Our results indicate that the addition of VE 50 μmol / L effectively alleviated BREC barrier dysfunction caused by DON exposure of 0.4 μg / mL, reduced its permeability, and maintained the integrity of BREC mucosal structure and barrier function. Type I collagen α1 chains (COL1A1) are widely distributed in the stroma of parenchymal organs and connective tissues throughout the body. They are not only members of the collagen family but also important components of type I collagen, participating in the epithelial-mesenchymal transition closely related to malignant tumor development. Excessive accumulation of collagen in the extracellular matrix can lead to various diseases in animals, with type I collagen α1 chains (COL1A1) playing a crucial role in maintaining tissue development and homeostasis. Type II collagen (COL2A1) is mainly distributed in cartilage, accounting for about 95% of collagen and 60% of cartilage dry weight. It is a fibrous collagen. Most type II collagen diseases are caused by pathogenic mutations in COL2A1, leading to the substitution of glycine for serine in the triple-helix collagen domain, thereby disrupting normal protein folding and function, ultimately resulting in collagen synthesis defects. Type V collagen is a minor fibrous collagen subtype that can be separated by pepsin hydrolysis and plays a key role in regulating the formation of procollagen fibers in connective tissues expressing type I collagen. Therefore, the addition of vitamin E 50 μM / L can alleviate bovine rumen epithelial cell damage caused by DON 0.4 μg / mL by upregulating COL1A1, COL2A1, and HSPG2 proteins in the ECM receptor pathway and COL1A1, COL2A1, and COL5A1 proteins in the protein digestion and absorption pathway.

[0056] The results showed that in the DON 0.4 μg / mL injury model, the levels of COL1A1, COL2A1, and COL5A1 proteins were significantly reduced. After the addition of vitamin E 50 μM / L, the levels of COL1A1, COL2A1, and COL5A1 proteins rebounded. At the same time, the damage of vomitoxin to bovine rumen epithelial cells was reduced, suggesting that the expression levels of COL1A1, COL2A1, and COL5A1 proteins are related to vomitoxin damage.

[0057] Example 4 In vitro culture of bovine rumen epithelial cells → upregulation of COL1A1, COL2A1, and COL5A1 gene expression in bovine rumen epithelial cells → treatment with a certain concentration of DON and vitamin E → determination of cell viability (or apoptosis rate) by CCK-8 assay to verify that upregulation of COL1A1, COL2A1, and COL5A1 protein levels can protect bovine rumen epithelial cells, while vitamin E can alleviate the damage of DON to bovine rumen epithelial cells.

[0058] The specific steps are as follows: Bovine rumen epithelial cells were cultured in vitro and treated with 5 ng / mL recombinant bovine TGF-β1 cytokine for 48 h. A control group (without TGF-β1 treatment) was also included. Cells treated with and without TGF-β1 were gently washed once with PBS, then replaced with fresh medium containing 0.4 μg / ml DON or 0.4 μg / ml DON + 50 μM / L vitamin E. Cells were cultured for another 24 h. Cell viability was measured using the CCK-8 assay, and apoptosis rate was determined by flow cytometry. The results are as follows: Figure 19 As shown.

[0059] Depend on Figure 19 It was found that, compared with cells that did not overexpress DON, cells that overexpressed DON showed significantly increased cell viability and significantly decreased apoptosis rate, indicating that overexpression of COL1A1, COL2A1, and COL5A1 genes can reduce DON-induced cell damage. Compared with cells that overexpressed DON, cells that overexpressed DON and VE showed further increased cell viability and further decreased apoptosis rate, indicating that VE can alleviate DON-induced cell damage. This further demonstrates that by upregulating the expression of COL1A1, COL2A1, and COL5A1 proteins and adding appropriate VE, the damage of vomitoxin to bovine rumen epithelial cells can be alleviated, providing a new target for the preparation of drugs to alleviate vomitoxin-induced damage.

[0060] In summary, this study found that in the DON 0.4 μg / mL injury model, the levels of COL1A1, COL2A1, and COL5A1 proteins were significantly reduced. After adding vitamin E 50 μM / L, the levels of COL1A1, COL2A1, and COL5A1 proteins rebounded, and the damage to bovine rumen epithelial cells caused by vomitoxin was reduced, suggesting that the expression levels of COL1A1, COL2A1, and COL5A1 proteins are related to vomitoxin damage. Furthermore, overexpression of COL1A1, COL2A1, and COL5A1 proteins in bovine rumen epithelial cells reduced the damage to these cells caused by vomitoxin. This indicates that upregulating the expression of COL1A1, COL2A1, and COL5A1 proteins can alleviate the damage to bovine rumen epithelial cells caused by vomitoxin, providing a new target for the preparation of drugs to alleviate vomitoxin damage.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of vitamin E in the preparation of drugs for non-therapeutic purposes to alleviate the damage of vomitoxin to bovine rumen epithelial cells, characterized in that: The vitamin E works by regulating Collagen; The Collagen is COL1A1 protein, COL2A1 protein, and COL5A1 protein; The amino acid sequence of the COL1A1 protein is shown in SEQ ID NO.19; The amino acid sequence of the COL2A1 protein is shown in SEQ ID NO.20; The amino acid sequence of the COL5A1 protein is shown in SEQ ID NO.21; The vitamin E alleviates the damage of vomitoxin to bovine rumen epithelial cells by increasing the expression levels of COL1A1, COL2A1, and COL5A1 proteins in a vomitoxin injury model.

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