Effect of Collagen in relieving damage of vomitoxin to bovine rumen epithelial cells by vitamin E

Abstract

The invention discloses an effect of Collagen in relieving vomitoxin injury of bovine rumen epithelial cells by vitamin E, and belongs to the technical field of genetic engineering, the Collagen is COL1A1 protein, COL2A1 protein and COL5A1 protein; the amino acid sequence of the COL1A1 protein is as shown in SEQ ID NO. 19; the amino acid sequence of the COL2A1 protein is as shown in SEQ ID NO. 20; the amino acid sequence of the COL5A1 protein is as shown in SEQ ID NO. 21. According to the effect of Collagen in relieving bovine rumen epithelial cells damaged by vomitoxin through vitamin E, it is found that vitamin E can effectively protect the cell membrane integrity of the bovine rumen epithelial cells and can relieve cell viability decline, oxidative stress, immune and inflammatory reactions and the cell apoptosis rate caused by DON; vitamin E can promote ECM receptor effect and protein digestion and absorption ways by adjusting Collagen protein so as to protect rumen epithelial cells of dairy cows.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to the role of collagen in vitamin E alleviating the damage of bovine rumen epithelial cells by vomitoxin. Background Art

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a Class B trichothecenes produced by fungi such as Fusarium graminearum. It has been detected in large quantities in substances such as corn, wheat, beans and corn silage. It is the most common type of fungal toxin.

[0003] Due to its widespread distribution in feed and its persistent toxicity, DON can enter the food chain and pose a health hazard to humans and animals.

[0004] Studies have shown that DON can affect the information transmission between rumen microorganisms. Increased DON concentration in the diet will significantly reduce the biosynthesis of pantothenic acid and CoA by rumen microorganisms, leading to reduced microbial energy metabolism, inhibiting VFA synthesis, and causing a decrease in milk production in dairy cows. The absorption of nutrients by the rumen epithelium is affected by acidosis conditions and the occurrence of rumenitis. Therefore, it is of great significance to find effective ways to alleviate the toxicity of DON and reduce its harm to humans and animals.

[0005] Since DON pollution is unavoidable, how to alleviate and reduce the toxic effects of DON on livestock and poultry has also attracted widespread attention from scholars, but traditional detoxification methods have problems such as unstable effects, reduced nutritional value, and the generation of residual harmful substances. Vitamin E, as a natural antioxidant, has a wide range of biological functions and has positive effects on anti-oxidation, immunity and reproduction. At the same time, it can also ensure that vitamin E plays a wide and important role in promoting the growth and development of a variety of livestock and poultry. Summary of the invention

[0006] The purpose of the present invention is to provide the role of collagen in alleviating the damage of bovine rumen epithelial cells by vomitoxin, and to analyze the role of vitamin E in alleviating the damage of bovine rumen epithelial cells by vomitoxin using technical means such as 4D-label-free quantitative proteomics, so as to improve the antioxidant state of the cells, reduce a series of adverse effects such as DON enhancing the oxidative stress of the cells and increasing the apoptosis rate.

[0007] To achieve the above object, the present invention provides the effect of collagen in vitamin E alleviating the damage of bovine rumen epithelial cells by vomitoxin, wherein the collagen is COL1A1 protein, COL2A1 protein, 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 effect is that vitamin E promotes ECM receptor function and protein digestion and absorption pathways by regulating collagen protein.

[0008] Therefore, the effect of the collagen of the present invention on vitamin E in alleviating the damage of bovine rumen epithelial cells by vomitoxin has the following beneficial effects: (1) By detecting the effects of DON on the activity, oxidation status, apoptosis rate and other indicators of dairy cow rumen epithelial cells (BREC), an in vitro research model of DON-induced BREC cell damage was successfully established.

[0009] (2) Vitamin E can effectively protect the cell membrane integrity of rumen epithelial cells in dairy cows and alleviate the oxidative stress, immune, inflammatory response and cell apoptosis rate caused by vomitoxin.

[0010] (3) Vitamin E protects the rumen epithelial cells of dairy cows by regulating the levels of collagen proteins COL1A1, COL2A1, and COL5A1, thereby regulating protein digestion and absorption pathways.

[0011] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Part A shows the cell viability of rumen epithelial cells of dairy cows treated with different DON concentrations at different culture times, Part B shows the total antioxidant status of rumen epithelial cells of dairy cows cultured at different DON concentrations for 12 hours, Part C shows the total oxidation status of rumen epithelial cells of dairy cows cultured at different DON concentrations for 12 hours, and Part D shows the oxidative stress index of rumen epithelial cells of dairy cows cultured at different DON concentrations for 12 hours. Figure 2 A in the middle is the cell viability of the group treated with different concentrations of vitamin E, and B is the cell viability of the group treated with different concentrations of vitamin E combined with DON; Figure 3 A is the intracellular malondialdehyde level in the vitamin E treatment group and the vitamin E and DON combined treatment group, B is the total oxidation state, C is the total antioxidant state, and D is the oxidative stress index; Figure 4 A in the figure shows the flow cytometry analysis results of the CON, D0.4, V50, and D0.4V50 groups, and B shows the cell apoptosis rate; Figure 5A is the mRNA level of Occludin, B is the mRNA level of Claudin-1, C is the mRNA level of Zonulaoccludens-1, D is the mRNA level of NF-Kappa b, E is the mRNA level of IL-6, F is the mRNA level of IL-1β, G is the mRNA level of TNF-α, and H is the mRNA level of IL-10; Figure 6 It is the SDS-PAGE electrophoresis test result of sample protein; Figure 7 It is a statistical diagram of mass spectrometry data of sample protein; Figure 8 It is the peptide length distribution diagram of the sample protein; Fig. 9 This is the heat map of PCC analysis in sample repeatability test; Fig.10 Volcano plots of up-regulated or down-regulated proteins compared with the Control, DON0.4, VE50, and D0.4V50 groups, where A is the comparison between the Control and DON0.4 groups, B is the comparison between the DON0.4 and D0.4V50 groups, C is the comparison between the DON0.4 and VE50 groups, D is the comparison between VE50 and Control, E is the comparison between VE50 and D0.4V50, and F is the comparison between the Control and D0.4V50 groups; G is a comprehensive statistical diagram of up-regulated or down-regulated proteins in AF; Fig.11 A is the heat map of cluster analysis of biological processes in GO enrichment, B is the heat map of cluster analysis of cellular components, and C is the heat map of cluster analysis of molecular functions; Fig.12 A in the middle is the annotation of the subcellular structure of differentially expressed proteins in the DON / Control group, and B is the annotation of the subcellular structure of differentially expressed proteins in the DON / DON-VE group; Fig.13 A in the middle is the KEGG pathway enrichment analysis of the DON / Control group, and B is the KEGG pathway enrichment analysis of the DON / DON-VE group; Fig.14 A in the middle is the relative quantitative expression analysis of COL1A1, COL2A1, COL6A1 and HSPG2 proteins significantly enriched in the ECM receptor action pathway in the DON / Control group, and B is the relative quantitative expression analysis of COL1A1, COL2A1, COL6A1 and HSPG2 proteins significantly enriched in the ECM receptor action pathway in the DON / DON-VE group; Fig.15A in the middle is the relative quantitative expression analysis of COL1A1, COL2A1, COL5A1 and COL6A1 proteins significantly enriched in the protein digestion and absorption pathway in the DON / Control group, and B is the relative quantitative expression analysis of COL1A1, COL2A1, COL5A1 and COL6A1 proteins significantly enriched in the protein digestion and absorption pathway in the DON / DON-VE group; Fig.16 A in the middle is the relative quantitative expression analysis of the enriched proteins in the DON / Control group after enrichment to ECM receptor interaction through KEGG pathway and further verification by PRM, and B is the relative quantitative expression analysis of the enriched proteins in the DON / DON-VE group after enrichment to ECM receptor interaction through KEGG pathway and further verification by PRM; Fig.17 A in the middle shows the relative quantitative expression analysis of protein digestion and absorption enriched through the KEGG pathway in the DON / Control group and further verified by PRM; B shows the relative quantitative expression analysis of protein digestion and absorption enriched through the KEGG pathway in the DON / DON-VE group and further verified by PRM. DETAILED DESCRIPTION

[0013] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0014] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0015] Embodiment 1 The in vitro model of dairy cow rumen epithelial cell injury was established as follows: Cow 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. The cell viability was determined using the CCK-8 method. The results are as follows: Figure 1 As shown in A, the culture time was determined to be 12 h. The difference between different treatments was indicated by ac.

[0016] The total oxidation state (TOS) and total antioxidant state (TAS) were determined using the kit, and the oxidative stress index (OSI) was calculated by TOS / TAS. The results were as follows: Figure 1 As shown in B, C, and D, the DON treatment concentrations were determined to be 0.4 and 0.8 μg / mL. The difference between different treatments was indicated by ae.

[0017] Embodiment 2 Vitamin E alleviates DON-induced rumen epithelial cell damage in dairy cows in the following steps: (1) About cell viability Add 100 μL of BREC cell suspension Inoculate in a 96-well cell culture plate. When the cell density reaches 70% to 80%, discard the culture medium, wash twice with pre-cooled PBS, replace with complete culture medium for the control group, and replace with vitamin E working solution of different concentrations (0, 12.5, 50, 200, 800, 3200 μM / L) for 12 hours for the vitamin E treatment group. After the experimental treatment, discard the liquid in the well, wash each well twice with DMEM / F12 culture medium, add 100 μL complete culture medium and 10 μL CCK-8 solution to each well, incubate in a 37°C cell culture incubator for 4 hours, and use an enzyme reader to measure the absorbance (OD value) at a wavelength of 450 nm to calculate the cell viability. The results are as follows: Figure 2 As shown in A. As the concentration of vitamin E treatment increases, cell viability increases; and when the concentration increases to 3200 μM / L, cell viability decreases. The difference between different treatments is indicated by ad.

[0018] 50 and 200 μM / L vitamin E were used in combination with 0.4 and 0.8 μg / mL DON to measure cell viability. The results are as follows Figure 2 As shown in B. Compared with the DON group, under the same vitamin E treatment conditions, the difference between 0.4μg / mL DON and vitamin E was extremely significant. However, there was no significant effect on cell viability between 0.4μg / mL DON and 50 and 200μM / L VE. The difference between different treatments is indicated by af.

[0019] (2) Oxidative stress level After cell subculture, the cell status and density were observed, and the cells were collected by trypsin digestion to prepare rumen epithelial cell suspension. 5 Cells were inoculated at a density of 100 cells / mL in a 6-well culture plate. 2 mL of solution was added to each well. The cell density and growth were observed. When the density reached 70% to 80%, the culture medium was discarded and the cells were washed twice with pre-cooled PBS. The control group was replaced with complete culture medium, and the experimental group was cultured with different treatment solutions for 12 hours. Then the cells were collected according to the requirements of the kit, and the intracellular malondialdehyde, total oxidation state, total antioxidant state, and oxidative stress index content were determined.

[0020] The results are as follows Figure 3As shown, af indicates the significant difference between different treatments. The combined effect of vitamin E and DON showed that the MDA level in BREC cells was significantly decreased by using 50μmol / L and 200μmol / L VE to alleviate 0.4μg / mL DON, compared with the use of 50μmol / L and 200μmol / L VE to alleviate 0.8μg / mL DON. The MDA content in cells treated with 50μmol / L VE and 0.4μg / mL DON showed a significant increase compared with the MDA content in 0.4μg / mL DON ( P ﹤0.0001). The intracellular TAS content and TOS content showed an opposite trend of change. The intracellular TAS and TOS contents under the combined action of 50μmol / L VE and 0.4μg / mL DON showed a significant increase compared with 0.4μg / mL DON ( P ﹤0.0001). The DON treatment concentration was determined to be 0.4 μg / mL and the vitamin E concentration was determined to be 50 μM / L.

[0021] (3) Determination of cell apoptosis rate After cell subculture (the treatment groups were: Control group, DON 0.4 μg / mL group, VE 50 μmol / L group, and DON 0.4 μg / mL + VE 50 μmol / L group), the cell state and density were observed, and the cells were collected by trypsin digestion to prepare rumen epithelial cell suspension. 5 Inoculate the cells at a density of 100 / mL in a 6-well culture plate. 2 mL of solution per well. Observe the cell density and growth. When the density reaches 70% to 80%, remove the original culture medium after 12 hours of culture and wash twice with pre-cooled PBS. Add 500 μL of EDTA-free trypsin to digest for about 5 minutes. After observing that the cells are completely digested, add 2 mL of complete culture medium to terminate the digestion. Transfer all the liquid to a centrifuge tube and centrifuge at 2000 rpm for 10 minutes at room temperature to collect the cells.

[0022] Carefully remove the upper liquid, add 1 mL of pre-cooled PBS to resuspend the cells, transfer to a 1.5 mL centrifuge tube, centrifuge at 2000 rpm for 10 min, wash the cells, repeat the operation twice, and discard the supernatant as much as possible.

[0023] Dilute the 10×Binding Buffer in the kit to 1×Binding Buffer with deionized water in advance, add 200μL of 1×Binding Buffer to the centrifuge tube to suspend the cells, add 5μL of Annexin V-FITC and mix well, incubate at room temperature for 15min in dark, add 5μL of PI staining and 200μL of 1×Binding Buffer 5min before loading, and perform flow cytometry analysis immediately.

[0024] The results are as follows Figure 4 As shown, compared with the control group, the rate of apoptotic cells in BREC cells treated with 0.4 μg / mL DON for 12 h increased significantly ( P ﹤0.05). Adding 50μmol / L VE alleviated the apoptotic cell ratio of BREC cells treated with 0.4μg / mL DON for 12h and significantly reduced the rate of apoptotic cells ( P ﹤0.001). This indicates that the combined treatment of VE and DON significantly reduced the cell apoptosis rate induced by DON ( P <0.05). The significance of the difference between different treatments was indicated by ac.

[0025] (4) mRNA expression of cell permeability, immunity, and inflammation-related genes Total RNA extraction and fluorescence quantitative PCR, including: ①BREC cell sample collection and total RNA extraction After cell subculture, the cell status and density were observed, and the cells were collected by trypsin digestion to prepare liver cell suspension (BREC cells grown on the wall at the 16th generation). 5 The cells were inoculated at a density of 100 / mL in a 6-well culture plate, with 2 mL of solution in each well.

[0026] The cell density and growth were observed. When the density reached 70% to 80%, the culture medium was discarded, the cells were washed twice with pre-cooled PBS, and drug treatment was performed. The cells were divided into 4 treatment groups, namely: 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). The cells were cultured in an incubator for 12 h.

[0027] 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 with a cell scraper, collect them into a 1.5 mL EP tube, and lyse them on ice for 20 min.

[0028] 2) Add 200 μL of chloroform to the EP tube, shake vigorously up and down, let it stand on ice for 10 minutes, and centrifuge in a precooled centrifuge at 4°C, 1200 rpm for 20 minutes.

[0029] 3) Pipette 200 μL of supernatant into a new EP tube (do not suck up the white membrane), add an equal volume of isopropanol, gently invert 5 times, let stand on ice for 10 minutes, and centrifuge at 1200 rpm for 10 minutes at 4°C.

[0030] 4) Pour off the supernatant, add 1 mL of 75% ethanol, knock up the white precipitate, and centrifuge at 8000 rpm at 4°C for 5 min.

[0031] 5) Discard the supernatant, add 500 μL of anhydrous ethanol, centrifuge at 8000 rpm at 4°C for 3 min, discard the supernatant, and air dry for 10 min (the RNA precipitate will become transparent after drying).

[0032] 6) According to the amount of RNA precipitation, add 20 μL DEPC water to dissolve; 7) Measure RNA concentration. Sample RNA concentration>500ng / mL, A260 / A280 between 1.8~2.0, A230 / A260 between 1.9~2.3. The obtained RNA solution can be reverse transcribed immediately or stored in a -80℃ refrigerator.

[0033] ② Synthesis of cDNA This experiment uses Tiangen Fast King cDNA First Strand Synthesis Kit for reverse transcription. The specific operation method is as follows: 1) Thaw the template RNA and each system in the kit on ice, shake and mix well, centrifuge briefly and set aside.

[0034] 2) Prepare the gDNA removal reaction system: 2uL 5×gDNA Buffer + 5uL template RNA solution, and add RNase-Free ddH2O to make up to 10uL.

[0035] 3) Briefly centrifuge the prepared gDNA removal reaction system, place it in a PCR amplification instrument, incubate at 42°C for 3 minutes, and place it on an ice box.

[0036] 4) Prepare the reverse transcription reaction system: 2uL 10×Fast RT Buffer+1uL RT Enzyme Mix+2uL FQ-RT Primer Mix, add RNase-Free ddH2O to make up to 10uL.

[0037] 5) Mix the prepared reverse transcription reaction system and centrifuge briefly, add it to the incubated gDNA removal reaction system, put it into the PCR amplification instrument, incubate at 42℃ for 15min, incubate at 95℃ for 3min, and then put it on ice. The obtained cDNA is used for subsequent experiments.

[0038] ③ Fluorescence quantitative PCR Tiangen qPCR Pre Mix (SYBR Green) kit was used in the real-time fluorescence quantitative PCR of this experiment, and the 2−ΔΔCt method was used to calculate the relative expression level of mRNA.

[0039] 1) The primer sequences for fluorescent quantitative PCR are shown in Table 1. β-actin was selected as the internal reference gene and the dye was thawed on ice.

[0040] 2) Set up 20 μL of real-time PCR system as shown in Table 2 and mix well.

[0041] 3) Select appropriate reaction conditions for PCR reaction. This experiment adopted a two-step reaction method. The specific conditions are shown in Table 3.

[0042] Table 1 Primer sequences for fluorescent quantitative PCR ;

[0043] Table 2 Fluorescence quantitative PCR reaction system ;

[0044] Table 3 Fluorescence quantitative reaction procedure ;

[0045] like Figure 5 As shown in A, B, and C, when cells are in a damaged state, the mRNA level of the tight junction protein Zonula occludens-1 (ZO-1) is significantly reduced ( P <0.001). Compared with the DON group, the ZO-1 mRNA level in the vitamin E group and DON combination treatment group did not increase significantly, while the Occludin and Claudin-1 mRNA expression levels increased significantly ( P< 0.05). This indicates that vitamin E alleviates the barrier function of BREC cells damaged by DON. The difference between different treatments is indicated by ad.

[0046] Compared with the control group, the relative expression of NF-Kappa b in the DON treatment group was significantly decreased ( P< 0.0001, Figure 5 D in the middle), indicating that DON caused a strong immune response in the rumen epithelial cells of dairy cows. Compared with the DON treatment group, the relative expression of NF-Kappa b in the vitamin E and DON combination treatment group (D0.4V50) was significantly upregulated ( P< 0.0001), indicating that vitamin E alleviated the immune response induced by DON. The difference between different treatments is indicated by ad.

[0047] Compared with the control group, the relative mRNA expression of the pro-inflammatory gene IL-6 in the DON treatment group was significantly upregulated ( P< 0.05, Figure 5 E), while the relative mRNA expression of IL-1β and TNF-α was significantly upregulated (P< 0.001, Figure 5 Middle F, G); The relative mRNA expression of the anti-inflammatory gene IL-10 in the DON treatment group was significantly upregulated ( P< 0.001, Figure 5 H), indicating that vomitoxin causes a strong inflammatory response in the rumen epithelial cells of dairy cows. Compared with the vomitoxin treatment group, the relative expression of the pro-inflammatory gene IL-6 in the vitamin E and vomitoxin treatment group (D0.4V50) was not significantly downregulated, while the relative expression of IL-1β and TNF-α were extremely significantly downregulated ( P< 0.001); the relative expression of anti-inflammatory gene IL-10 mRNA was significantly downregulated ( P< 0.001), indicating that vitamin E alleviated the inflammatory response induced by vomitoxin. The difference between different treatments is indicated by ad.

[0048] (5) 4D label-free quantitative proteomics analysis Passage the BREC cells that are growing well to 25cm 2 Cultured in culture flasks, cells were treated according to the experimental groups (Control, D0.4, V50, D0.4V50), the culture medium was discarded, washed 3 times with pre-cooled PBS, the cells were scraped and collected in centrifuge tubes, centrifuged at 4°C and 1000rpm for 5 minutes, the supernatant was discarded, the tubes were sealed with sealing film, and the cells were quickly frozen in liquid nitrogen for 10 minutes and then transferred to a -80°C refrigerator for storage. During the detection, protein extraction quality control and trypsin hydrolysis were first performed, and then analysis and data processing were performed by liquid chromatography-mass spectrometry. Finally, bioinformatics analysis was performed through the screened differential protein expression, including GO analysis and KEGG pathway enrichment analysis, so as to screen the enriched pathways.

[0049] 1) Determination of sample protein quality The protein concentration results of the experimental groups (CON, D0.4, V50, D0.4V50) are shown in Table 4, and the SDS-PAGE results are shown in Figure 6 The protein bands were clear, the distribution of protein bands was normal, the protein was not degraded, the electrophoretic behavior of the groups was not significantly different, and there was a certain degree of high-abundance protein, and the quality control was qualified.

[0050] Table 4 Protein concentration determination results ;

[0051] 2) Mass spectrometry quality control analysis The experimental DIA data used the DIA-NN (v1.8) search engine, the database was Bos_taurus_9913_PR_20240325.fasta (37501 sequences), and a deep learning algorithm was used to construct a theoretical spectrum library. A reverse library was added to calculate the false positive rate (FDR) caused by random matching. Figure 7 As shown, the number of identified peptides was 72,353, the number of unique peptides identified was 62,186, the total number of identified proteins was 8,562, and the number of quantified proteins was 8,553.

[0052] After the database search is completed, the quality control evaluation results are as follows Figure 8 As shown, the results show that most peptides are distributed in 7-20 amino acids, which conforms to the general rules based on enzymatic hydrolysis and mass spectrometry fragmentation; most proteins correspond to more than two peptides; and they meet the proteomics quality control standards.

[0053] 3) Sample repeatability test The Pearson correlation coefficient between two samples is calculated based on the intensity values ​​of all samples, and a visual heat map is drawn. This coefficient is used to measure the degree of correlation between two sets of data. Fig. 9 As shown, the redder the color, the closer the Pearson correlation coefficient is to 1, and the stronger the correlation between the two samples.

[0054] 4) Analysis of differentially expressed proteins in BREC cells damaged by DON induced by vitamin E Liquid chromatography-mass spectrometry was used to perform proteomic analysis on each group of samples. During the proteomic analysis, the relative quantitative value of each protein in each comparison group sample was subjected to T test. P <0.05 indicated significant differences.

[0055] Before the test, the relative quantitative value of protein was transformed by Log2 to make the test data conform to the normal distribution required by the T test. P When the differential expression value was less than 0.05, the change of differential expression value exceeding 1.5 was regarded as the threshold of significant upregulation, and that less than 1 / 1.5 was regarded as the threshold of significant downregulation.

[0056] In data analysis, FC and P value are used to draw volcano plots, such as Fig.10As shown, it is used to express the significant difference of sample data. A total of 1220 quantifiable DE proteins were identified this time. From the volcano plot (red, upregulated; blue, downregulated), it can be seen from the study of the mechanism of vitamin E alleviating the damage of DON to BREC that: the DON / Control group had 184 upregulated proteins and 135 downregulated proteins; the DON-VE / Control group had 114 upregulated proteins and 76 downregulated proteins; the DON / DON-VE group had 125 upregulated proteins and 107 downregulated proteins. In order to more intuitively and visually compare the proteomic differences between the groups, the upregulated or downregulated proteins were statistically analyzed, and the results are shown as follows: Fig.10 As shown in G.

[0057] 5) Functional classification of differentially expressed proteins after DON-induced BREC cell damage by vitamin E In order to understand the possible biological functions of differentially expressed proteins in the process of vitamin E alleviating DON-induced BREC cell damage, the functional analysis of differentially expressed proteins between different treatment groups was performed using Gene Ontology (GO) and subcellular structure localization functional classification.

[0058] (1) GO secondary classification of differentially expressed proteins GO enrichment analysis of differentially expressed proteins was performed using bioinformatics and divided into three categories: biological processes, cellular components, and molecular functions, explaining the properties of genes and gene products in organisms from different perspectives.

[0059] Differential protein clustering analysis was performed on the three major categories in GO classification, such as Fig.11 As shown in the figure, the horizontal direction represents the enrichment test results of different groups, and the vertical direction represents the description of the differential protein enrichment related function. Different groups of differential proteins and colors correspond to functional descriptions, indicating the degree of enrichment, and the degree of enrichment is represented by * (the stronger the enrichment, the more *, and the stronger the color).

[0060] When vitamin E alleviated the damage of DON to cells, the DON / Control group significantly downregulated proteins. In the bioengineering classification, the differentially expressed proteins were mainly regulated in proteins, amyloid, glycoprotein, endopeptidase activity, diterpenes, lipids, triglycerides, phospholipids, cholesterol, inflammatory response and cell-matrix adhesion; in the cell component classification, they were mainly distributed in the extracellular space, matrix containing collagen, matrix endoplasmic reticulum, collagen trimer, collagen trimer complex, band collagen fibers, fibrillar collagen trimer and 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.

[0061] The proteins significantly upregulated in the DON / DON-VE group were mainly regulated in protein-lipid complex remodeling, retinoic acid metabolism, diterpenoid metabolism, lipid catabolism, phospholipid efflux and SRP-dependent co-translational protein targeting membrane co-translational protein in the biological process classification; in the cellular component classification, they were mainly 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, spherical high-density lipoprotein particles, endoplasmic reticulum, cytoplasmic ribosomes, ribosomal subunits, ribosomes and cytoplasmic large ribosomal subunits; in the molecular function classification, they were mainly concentrated in binding vitamin-K-epoxide reductase (warfarin sensitive), oxidoreductase activity, CH-OH groups acting on donors, disulfide as receptor apolipoprotein receptor, high-density lipoprotein particle receptor binding, structural composition of ribosomes, phosphatidylcholine-sterol O-acyltransferase activator and lipase inhibitor activity.

[0062] (2) Subcellular localization of differentially expressed proteins Mature proteins can only play a stable biological function in specific subcellular organelles. Subcellular organelles are important components of cells and are micro-organs with certain morphology and functions. In order to accurately understand the function of a certain protein, in addition to analyzing its protein structure, it is usually necessary to understand its subcellular localization to determine the possible place where it can play its function.

[0063] Protein subcellular localization is very important for studying protein functions. Subcellular localization can locate a protein or expression product in a specific location in the cell, such as the nucleus, various organelles, and the plasma membrane, thus providing a research direction for understanding the mechanism of gene action.

[0064] In order to further explore the mechanism regulation of vitamins in alleviating DON-induced BREC cell damage, further analysis was performed by subcellular division, and annotation maps of the subcellular structures of differentially expressed proteins were drawn, such as Fig.12 shown.

[0065] The significant downregulation in the DON / Control group was mainly manifested in the extracellular space (31.85% of the total), plasma membrane (17.78% of the total), nucleus (17.04% of the total), cytoplasm (14.07% of the total), mitochondria (8.89% of the total), cytoplasm and nucleus (2.96% of the total), endoplasmic reticulum in 4 cases (2.9% of the total), peroxisome in 4 cases (2.96% of the total), and others in 2 cases (1.48% of the total).

[0066] The significant upregulation in the DON / DON-VE group was mainly manifested in the cytoplasm (accounting for 24.8% of the total), nucleus (accounting for 20.8% of the total), extracellular (accounting for 16.8% of the total), plasma membrane (accounting for 15.2% of the total), mitochondria (accounting for 13.6% of the total), cytoplasm, nucleus (accounting for 4% of the total), peroxisomes, (accounting for 2.4% of the total), cytoskeleton (accounting for 1.6% of the total), and others (accounting for 0.8% of the total).

[0067] (3) KEGG enrichment analysis of differentially expressed proteins in BREC cells after DON-induced injury by vitamin E In order to further explore the regulatory pathways by which vitamins alleviate DON-induced BREC cell damage, KEGG pathway enrichment analysis of differentially expressed proteins was performed using bioinformatics.

[0068] pass Fig.13 As can be seen from A and B, the DON / Control group and the DON / DON-VE group were jointly enriched in the ECM receptor interaction, protein digestion and absorption, renin-angiotensin system, drug metabolism-cytochrome P450, and cholesterol metabolism pathways.

[0069] And through Fig.13 The DON / Control group and DON / DON-VE group shown in A and B were jointly enriched in ECM receptor interaction, PI3K-Akt signaling pathway, and protein digestion and absorption.

[0070] Among them, the DON / Control group and the DON / DON-VE group were significantly enriched in COL1A1, COL2A1, COL5A1 and HSPG2 proteins in the ECM receptor pathway. The relative quantitative expression of the proteins was as follows: Fig.14 The significant differences among different treatments are indicated by * and ns.

[0071] The DON / Control group and the DON / DON-VE group also significantly enriched the COL1A1, COL2A1, COL5A1, and COL6A1 proteins in the protein digestion and absorption pathways. The relative quantitative expression of the proteins is shown in Figure 2 Fig.15 The significant differences among different treatments are indicated by * and ns.

[0072] 6) PRM Verification By significantly enriching and screening proteins and their pathways in (KEGG pathway), 18 target proteins shown in the screened proteins in Table 5 were obtained for PRM verification.

[0073] Table 5 Effects of vitamin E on ECM receptor interaction, protein digestion and absorption of differentially enriched proteins in alleviating DON-induced BREC cell damage ;

[0074] Among them, the proteins COL1A1, COL2A1, COL6A1, HSPG2, LAMC1, and ITGA5 that were significantly enriched in ECM receptors were summarized, and the results of relative quantitative expression were as follows: Fig.16 The results showed that COL1A1, COL2A1 and HSPG2 proteins showed similar trends in PRM validation and proteomics KEGG pathway analysis results.

[0075] The proteins COL1A1, COL2A1 and COL5A1 that were significantly enriched in protein digestion and absorption were summarized, and the relative quantitative expression results were as follows Fig.17 The results showed that COL1A1, COL2A1 and COL5A1 proteins also showed similar trends in PRM validation and proteomics KEGG pathway analysis results, further proving the rationality and reliability of proteomics analysis.

[0076] 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.

[0077] Therefore, the present invention found that vitamin E can effectively protect the cell membrane integrity of cow rumen epithelial cells and alleviate the decline in cell viability, oxidative stress, immune and inflammatory response and cell apoptosis rate caused by DON. Vitamin E protects cow rumen epithelial cells by regulating collagen to promote ECM receptor action and protein digestion and absorption pathways.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. The effect of collagen in non-therapeutic vitamin E on alleviating the damage of bovine rumen epithelial cells to vomitoxin, characterized in that: The collagen is COL1A1 protein, COL2A1 protein, 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 effect is that vitamin E promotes ECM receptor function and protein digestion and absorption pathways by regulating collagen.

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