Effect of APOA gene in relieving vomitoxin injury of bovine liver cells by vitamin E

Abstract

The invention discloses an effect of an APOA gene in relieving vomitoxin injury of bovine liver cells by vitamin E, and relates to the technical field of the effect of vitamin E in relieving vomitoxin injury of bovine liver cells. The APOA gene refers to an APOA1 gene, an APOA2 gene and an APOA4 gene; the CDS sequence of the APOA1 gene is as shown in SEQ ID NO. 19; the CDS sequence of the APOA2 gene is as shown in SEQ ID NO. 20; the CDS sequence of the APOA4 gene is as shown in SEQ ID NO. 21; the effect is that the vitamin E regulates an APOA gene to be enriched to a cholesterol metabolic pathway. According to the effect of the APOA gene in relieving vomitoxin-caused cattle liver cells, it is found that the vitamin E can effectively protect the cell membrane integrity of the cattle liver cells and can relieve cell viability decline, oxidative stress, inflammatory response and cell apoptosis caused by vomitoxin; vitamin E regulates the cholesterol metabolic pathway by regulating the APOA gene level so as to play a role in protecting liver cells of dairy cows.

Description

Technical Field

[0001] The present invention relates to the technical field of the role of vitamin E in alleviating bovine liver cells damaged by vomitoxin, and in particular to the role of APOA gene in the role of vitamin E in alleviating bovine liver cells damaged by vomitoxin. Background Art

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a secondary metabolite produced by Fusarium. In recent years, the detection rate and contamination rate in feed and feed raw materials have been very high. According to surveys, the contamination rate of DON in global feed raw material samples reached 64% during 2008-2017, which not only caused huge economic losses to the breeding industry, but also endangered the health of humans and livestock.

[0003] Long-term consumption of feed contaminated with DON will slow down the growth rate of livestock and poultry, reduce nutrient absorption rate, cause cytotoxicity, immunotoxicity and even death. The liver is the main metabolic organ in the body and the most important target organ of DON. It is involved in regulating many biological processes, such as metabolism, immunity, and protein synthesis. The toxin and its metabolites are very likely to be deposited in large quantities in the liver, changing the morphology of liver tissue and inducing inflammatory responses and oxidative stress. Therefore, it is of great significance to find effective ways to alleviate the toxicity of DON and reduce its harm to humans and animals.

[0004] 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. However, traditional detoxification methods have problems such as unstable effects, reduced nutritional value, and the generation of residual harmful substances, making them difficult to mass produce.

[0005] As a natural antioxidant, vitamin E has a wide range of biological functions, which can be summarized as follows: (1) It has an antioxidant effect; (2) It promotes the body's synthesis of certain biologically active substances; (3) It improves the reproductive capacity of animals; (4) It improves the immune capacity of animals; and (5) Vitamin E also has a detoxification effect. Summary of the invention

[0006] To solve the above problems, the present invention provides the role of APOA gene in vitamin E alleviating bovine liver cells damaged by DON, and uses proteomics and other technical means to analyze the role of vitamin E in alleviating bovine liver cells damaged by DON, so as to improve the antioxidant state of cells and reduce a series of adverse effects of DON such as enhanced oxidative stress on cells and increased apoptosis rate.

[0007] To achieve the above object, the present invention provides the role of APOA gene in vitamin E alleviating the damage of bovine liver cells to vomitoxin, wherein the APOA gene refers to APOA1 gene, APOA2 gene and APOA4 gene; The CDS sequence of the APOA1 gene is shown in SEQ ID NO.19; The CDS sequence of the APOA2 gene is shown in SEQ ID NO.20; The CDS sequence of the APOA4 gene is shown in SEQ ID NO.21; The effect is that vitamin E regulates the enrichment of APOA gene into the cholesterol metabolism pathway.

[0008] The APOA gene of the present invention has the following beneficial effects in that vitamin E alleviates the damage of bovine liver cells to vomitoxin: (1) By detecting the effects of DON on the activity, oxidation status, apoptosis rate and other indicators of bovine liver cells (BHEC), an in vitro research model of DON-induced BHEC cell damage was successfully established.

[0009] (2) Vitamin E can effectively protect the cell membrane integrity of dairy cow liver cells and alleviate the decreased cell viability, oxidative stress, inflammatory response and cell apoptosis caused by vomitoxin.

[0010] (3) Vitamin E protects cow liver cells by regulating APOA gene levels and thereby regulating cholesterol metabolism 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 A is the activity of cow liver cells under different DON concentrations and different treatment times, B is the malondialdehyde level of cow liver cells measured by the kit, C is the total oxidation state of cow liver cells measured by the kit, and D is the total antioxidant state of cow liver cells measured by the kit; 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 is the oxidative stress level of cells in the vitamin E treatment group and the vitamin E and DON combined treatment group, where A is the intracellular MDA content, B is the intracellular TOS content, and C is the intracellular TAS content; Figure 4 Determination of cell apoptosis rate in CON, D0.2, V50, and D0.2V50 groups, where A is the result of flow cytometry analysis, and B is the cell apoptosis rate; Figure 5are the results of fluorescence quantitative PCR detection, wherein A is the mRNA level of Zonula occludens-1, B is the mRNA level of Claudin-1, C is the mRNA level of Occludin, D is the mRNA level of IL-6, E is the mRNA level of IL-10, F is the mRNA level of TNF, G is the mRNA level of Bax, H is the mRNA level of Bcl-2, and I is the ratio of Bax / Bcl-2; 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 A is the peptide length distribution diagram of the sample protein, B is the peptide quantity distribution diagram, and C is the protein coverage distribution diagram; Fig. 9 A is the PCC analysis heat map in the sample repeatability test, B is the PCA analysis result map, and C is the RSD box plot; Fig.10 A is a volcano plot of up-regulated or down-regulated proteins in the CON, D0.2, V50, and D0.2V50 groups, B is a statistical plot of up-regulated or down-regulated proteins in different groups, and C is a Venn diagram of up-regulated or down-regulated proteins in different groups; Fig.11 GO analysis of differentially expressed proteins at 0.2D / CON; Fig.12 GO analysis of differentially expressed proteins at 50V / CON; Fig.13 GO analysis of differentially expressed proteins at 0.2D50V / CON; Fig.14 GO analysis of differentially expressed proteins at 0.2D50V / 0.2D; Fig.15 Annotate the subcellular structures of differentially expressed proteins; Fig.16 A is the COG / KOG functional annotation of differentially expressed proteins in the 0.2D / CON group, B is the COG / KOG functional annotation of differentially expressed proteins in the 50V / CON group, C is the COG / KOG functional annotation of differentially expressed proteins in the 0.2D50V / CON group, and D is the COG / KOG functional annotation of differentially expressed proteins in the 0.2D50V / 0.2D group; Fig.17 A is the GO enrichment analysis of differentially expressed proteins in the 0.2D / CON group, B is the GO enrichment analysis of differentially expressed proteins in the 50V / CON group, C is the GO enrichment analysis of differentially expressed proteins in the 0.2D50V / CON group, and D is the GO enrichment analysis of differentially expressed proteins in the 0.2D50V / 0.2D group; Fig.18 A in the middle shows the top 10 KEGG signaling pathways enriched in 0.2D / CON, and B shows the KEGG signaling pathways enriched in up- and down-regulation, respectively; Fig.19 is the PRM protein expression level of candidate proteins APOA1, APOA2 and APOA4. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0014] Embodiment 1 The in vitro model of dairy cow liver cell injury was established as follows: Cow liver cells were selected for in vitro culture and treated with 0, 0.05, 0.1, 0.2, 0.3, and 0.5 μg / mL DON, respectively, for 0, 9, 18, and 36 h.

[0015] The cell viability was determined using the CCK-8 method. Figure 1 As shown in A, the culture time was determined to be 18 h.

[0016] Malondialdehyde (MDA), total oxidative status (TOS), and total antioxidant status (TAS) were determined using the kit. The results were as follows: Figure 1 As shown in B, C, and D. The DON treatment concentration was determined to be 0.02 μg / mL.

[0017] Embodiment 2 Vitamin E alleviates DON-induced liver cell damage in dairy cows in the following steps: (1) About cell viability 100 μL of BHEC cell suspension (5 × 10 4 / mL) were inoculated in a 96-well cell culture plate. When the cell density reached 70-80%, 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 vitamin E treatment group was replaced with different concentrations (0, 12.5, 50, 200, 800, 3200 μM / L) of vitamin E working solution for 18 hours.

[0018] After the culture is completed, discard the liquid in the wells, wash each well twice with DMEM / F12 medium, add 100μL complete medium and 10μL CCK-8 solution to each well, incubate in a 37℃ cell culture incubator for 2-3h, and measure the absorbance (OD value) at a wavelength of 450nm with an enzyme marker. Calculate the cell viability, the results are as follows Figure 2 As shown in A.

[0019] When the VE concentration was 12.5-200 μM / L, the BHEC cell viability increased significantly ( P <0.01), and the cell viability was the highest at 50μM / L. When the concentration was 800μM / mL, there was no significant difference in cell viability ( P >0.05), when the concentration was 3200 μM / mL, the cell viability was significantly reduced ( P <0.001), so 12.5, 50, and 200 μM / L vitamin E and DON were selected for combined treatment. Different letters (ad) between treatments indicate significant differences ( P <0.05).

[0020] 12.5, 50, and 200 μM / L vitamin E were used in combination with 0.2 μg / mL DON to measure cell viability. The results are as follows: Figure 2 As shown in B.

[0021] The BHEC cell viability in the DON-treated group was significantly decreased compared with that in the control group ( P <0.001), while after combined treatment with vitamin E, cell viability tended to increase. When the VE concentration was 50 and 200 μM / L, the cell viability increased significantly compared with the DON treatment group ( P <0.01), and 50μM / L had the best effect.

[0022] (2) Oxidative stress level After cell subculture, the cell status and density were observed, and the cells were collected by trypsin digestion to prepare liver cell suspension (BHEC cells grown adherently at the 16th generation). 5 Inoculate cells at a density of 100 / ml in a 6-well culture plate. 2 mL of solution per well. Observe cell density and growth. When the density reaches 70-80%, discard the culture medium and wash twice with pre-cooled PBS.

[0023] The control group was replaced with complete culture medium, and the experimental group was cultured with different treatment medium for 18 hours. Then the cells were collected according to the requirements of the kit and the intracellular MDA, TOS, and TAS contents were determined. The results are as follows Figure 3 shown.

[0024] The MDA content in the VE group was significantly lower than that in the DON group ( P<0.001), the TAS content was higher than that of the DON group, but the difference was not significant; the TOS content was significantly lower than that of the DON group ( P <0.01).

[0025] In addition, when 50 and 200 μM / L vitamin E were added to treat cells, the total antioxidant status (TAS) was significantly higher than that of the control group ( P <0.001), indicating that vitamin E can alleviate the oxidative damage of cow liver cells induced by vomitoxin and effectively improve the antioxidant capacity of cow liver cells. Combined with the results of cell viability, 50μM / L vitamin E and 0.2μg / mL vomitoxin were finally selected for combined treatment for subsequent experiments.

[0026] (3) Determination of cell apoptosis rate After cell subculture, the cell status and density were observed, and the cells were collected by trypsin digestion to prepare liver cell suspension (BHEC cells grown adherently at the 16th generation). 5 The cells were inoculated at a density of 100 / 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%-80%, the original culture medium was removed after 18 hours of culture, and the cells were washed twice with pre-cooled PBS. 500 μL of EDTA-free trypsin was added for digestion for about 5 minutes.

[0027] After observing that the cells are completely digested, add 2 mL of complete medium to stop digestion, transfer all the liquid to a centrifuge tube, centrifuge at room temperature at 2000 rpm for 10 minutes to collect the cells. Carefully remove the upper layer of 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 minutes to wash the cells, repeat the operation twice, and discard the supernatant as much as possible.

[0028] Dilute the 10×Binding Buffer in the kit to 1×Binding Buffer with deionized water in advance, add 300μL 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 15 minutes in the dark, add 5μL of PI staining and 200μL of 1×Binding Buffer 5 minutes before the machine, and immediately perform flow cytometry analysis.

[0029] The results are as follows Figure 4 As shown in Figure 2, the relative expression of the pro-apoptotic gene Bax in the DON-treated group was significantly increased compared with the control group ( P <0.001, the relative expression of the anti-apoptotic gene Bcl-2 was significantly downregulated ( P <0.01), the ratio of Bax to Bcl-2 was significantly upregulated (P <0.001); compared with the DON treatment group, the relative expression of Bax in the DON and vitamin E treatment group was significantly downregulated ( P <0.001), and the relative expression of Bcl-2 was significantly upregulated ( P <0.01), the ratio of Bax to Bcl-2 decreased significantly ( P <0.001). The combined treatment of VE and DON significantly reduced the apoptosis rate induced by DON ( P <0.05).

[0030] Embodiment 3 Total RNA extraction and fluorescence quantitative PCR (1) BHEC 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 (BHEC cells grown adherently 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.

[0031] Observe the cell density and growth. When the density reaches 70%-80%, discard the culture medium, wash twice with pre-cooled PBS, and add drugs. There are 4 treatment groups, namely: control group (complete culture medium), vomitoxin group (0.2μg / mL), vitamin E treatment group (50μM / L), vomitoxin + vitamin E combined treatment group (0.2μg / mL DON + 50μM / LVE), and culture them in an incubator for 18h.

[0032] After pouring out the culture medium, wash twice with pre-cooled PBS, add 1 mL of RNAiso Plus, shake gently to fully lyse the cells, scrape them all off with a cell scraper and transfer them to a 1.5 mL centrifuge tube, and repeatedly pipette until there is no obvious precipitation in the lysate.

[0033] Let stand at room temperature for 5 minutes, add 200 μL of pre-cooled chloroform, mix until the solution turns milky white, let stand at room temperature for 5 minutes, centrifuge at 12000g for 15 minutes at 4℃. Carefully remove the centrifuge tube from the centrifuge. At this time, the homogenate is divided into 3 layers: colorless supernatant, middle white protein layer and lower organic phase.

[0034] Transfer 350 μL of supernatant to another new 1.5 mL centrifuge tube, add 1 mL of pre-cooled isopropanol, mix thoroughly by inverting, and let stand at room temperature for 10 min. Centrifuge at 12000g for 10 min at 4°C. Carefully discard the supernatant, add 1 mL of pre-cooled 75% ethanol, gently invert the tube to wash the wall of the centrifuge tube, centrifuge at 7500g for 5 min at 4°C, and carefully discard the supernatant. Open the centrifuge tube cap and dry the precipitate at room temperature for a few minutes. After the precipitate is dry, add an appropriate amount of RNase-free water to dissolve the precipitate.

[0035] The concentration and quality of total RNA were evaluated using a UV / visible fluorescence spectrophotometer. The A260 / A280 ratio must be in the range of 1.8-2.2 to obtain an RNA solution of qualified purity. The extracted RNA solution was stored in a -80°C refrigerator.

[0036] (2) Synthesis of cDNA Total RNA was reverse transcribed into cDNA using Tiangen FastKing cDNA First Strand Synthesis Kit. First, thaw the template RNA and reagents in the kit on ice. After thawing, vortex each solution to mix well and centrifuge briefly, and place on ice. Prepare the genomic DNA removal system solution, centrifuge briefly, incubate at 42°C for 3 minutes, and then place on ice. Prepare the reverse transcription reaction system mixture, add the mixture to the gDNA removal step reaction solution, mix well, incubate at 42°C for 15 minutes, incubate at 95°C for 3 minutes, and then place on ice to obtain the cDNA sample. Store at -20°C for further analysis.

[0037] (3) Fluorescence quantitative PCR The Tiangen Talent fluorescence quantitative detection kit was used for detection, and the 2-ΔΔCt method was used to calculate the relative expression of mRNA. The fluorescence quantitative PCR primer sequences are shown in Table 1. β -actin was used as an internal reference gene.

[0038] All reagents were melted on ice and mixed by inverting. Real-Time PCR reaction solution was prepared on ice to establish a 20 μL Real-Time PCR reaction system with 10 μL 2×Talent qPCR premix, 2 μL 50×ROXReference Dye, 4.8 μL RNase-Free ddH 2 O, 0.6 μL reverse primer (10 μM), 0.6 μL forward primer (10 μM) and 2 μL cDNA, and mix the system.

[0039] A two-step reaction procedure was used, which was divided into a pre-denaturation stage and a PCR reaction stage. The first step was pre-denaturation at 95°C for 3 min, for 1 cycle; the second step was denaturation at 95°C for 5 s, for 40 cycles; and the final step was annealing at 60°C for 15 s, for 40 cycles.

[0040] Table 1 Primer sequences for fluorescent quantitative PCR ;

[0041] like Figure 5 As shown, the mRNA level of tight junction protein Zonula occludens-1 (ZO-1) was significantly reduced when cells were in a damaged state ( P <0.05, Figure 5 Claudin-1 and Occludin mRNA levels were significantly decreased ( P <0.001, Figure 5 However, compared with the DON group, the mRNA content of ZO-1 in the vitamin E and DON group was significantly increased, but the difference was not significant. The mRNA expression levels of Occludin and Claudin-1 were extremely significantly increased ( P <0.05). This indicates that vitamin E alleviates the BHEC cell barrier function damaged by DON.

[0042] Compared with the control group, the relative expression levels of IL-6, IL-10 and TNF in the DON-treated group were significantly upregulated ( P <0.001, Figure 5 D, E, and F in the figure indicate that DON causes a strong inflammatory response in cow liver cells. Compared with the DON treatment group, the relative expression levels of IL-6, IL-10, and TNF in the vitamin E and DON treatment group (D0.2V50) were significantly downregulated (P<0.001), indicating that vitamin E alleviated the inflammatory response caused by DON.

[0043] The mRNA expression levels of apoptosis-related genes Bax and Bcl-2 were further detected. Figure 5 As shown in G, H, and I, the relative expression of Bax in the DON-treated group was significantly increased compared with the control group ( P <0.001), and the anti-apoptotic factor Bcl-2 was significantly downregulated compared with the control group ( P <0.01), the ratio of Bax to Bcl-2 was significantly upregulated ( P <0.001); compared with the DON treatment group, the relative expression of Bax in the DON and vitamin E treatment group was significantly downregulated ( P <0.001), and the anti-apoptotic factor Bcl-2 was significantly upregulated (P <0.01), the ratio of Bax to Bcl-2 decreased significantly ( P <0.001).

[0044] (5) 4D label-free quantitative proteomics analysis Passage the BHEC cells with good growth status to 25cm 2 Cultured in culture flasks, treated cells according to experimental groups (CON, D0.2, V50, D0.2V50), discarded culture medium, washed 3 times with pre-cooled PBS, scraped cells, and collected into centrifuge tubes. Centrifuged at 4°C, 1000 rpm for 5 min, removed supernatant, sealed with sealing film, quick-frozen in liquid nitrogen for 10 min, and stored in a -80°C refrigerator for later use.

[0045] During the detection, protein extraction quality control and trypsin hydrolysis are first performed, followed by analysis and data processing through liquid chromatography-mass spectrometry. Finally, bioinformatics analysis is performed on the screened differential protein expressions, including Gene Ontology analysis (GO analysis) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, to screen the enriched pathways.

[0046] ① Sample protein control The protein concentrations of the experimental groups (CON, D0.2, V50, D0.2V50) are shown in Table 2. The results of SDS-PAGE electrophoresis are shown in Figure 6 As shown: Table 2 Protein concentration determination results ;

[0047] The protein bands are clear, the protein bands are distributed normally, the protein is not degraded, the electrophoretic behavior of the groups is not significantly different, and there is a certain degree of high-abundance protein. The quality control is qualified.

[0048] ②Mass 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 the theoretical spectrum library was constructed using a deep learning algorithm. The anti-library was added to calculate the false positive rate (FDR) caused by random matching. In this protein mass spectrometry data analysis, the number of identified peptides was 84431, the number of identified unique peptides was 74984, the total number of identified proteins was 8870, and the number of quantified proteins was 8862, see Figure 7 .

[0049] After the database search is completed, the quality control evaluation results are visible Figure 8Most 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; most proteins are covered below 30%; the molecular weight of the identified proteins is widely and evenly distributed at different stages. It meets the quality control standards of proteomics.

[0050] ③ Sample repeatability test Three statistical analysis methods, namely Pearson correlation (PCC), principal component analysis (PCA) and relative standard deviation (RSD), were used to evaluate the repeatability, e.g. Fig. 9 As shown, in part A, it can be seen that the correlation between repeated samples is strong, in part B, it can be seen that the degree of aggregation of repeated samples is high, and in part C, it can be seen that the overall RSD value is small and the quantitative repeatability is good.

[0051] Embodiment 4 Analysis of differentially expressed proteins in BHEC cells damaged by DON induced by vitamin E Liquid chromatography-mass spectrometry was used to perform proteomic analysis on each group of samples. In order to determine the significance of the difference during the proteomic analysis, the relative quantitative values ​​of the proteins in each treatment group were subjected to T-test. P <0.05 indicated significant differences.

[0052] 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.

[0053] Through the above difference analysis, P When the difference is less than 0.05, the difference in expression is greater than 1.5 as the threshold for significant upregulation, and less than 1 / 1.5 as the threshold for significant downregulation. In data analysis, FC and P values ​​are used to draw volcano plots to show the significant differences in sample data. Fig.10 As shown in the figure, Part A is a volcano plot, the horizontal axis represents the fold change (log2 transformation); the vertical axis represents the p value (log10 transformation); red, up-regulated; blue, down-regulated; Part B is a statistical chart of up-regulated or down-regulated proteins in different groups; Part C is a Venn diagram of up-regulated or down-regulated proteins in different groups.

[0054] It can be seen that the number of down-regulated or up-regulated proteins in the 0.2D, 50V, and 0.2D50V groups partially overlapped, and each group had unique proteins. A total of 786 differentially expressed proteins were screened in the 0.2D / CON comparison group, of which 476 were up-regulated and 310 were down-regulated; a total of 289 differentially expressed proteins were screened in the 50V / CON comparison group, of which 182 were up-regulated and 107 were down-regulated; a total of 1004 differentially expressed proteins were screened in the 0.2D50V / CON comparison group, of which 553 were up-regulated and 451 were down-regulated; a total of 160 differentially expressed proteins were screened in the 0.2D50V / 0.2D comparison group, of which 57 were up-regulated and 103 were down-regulated.

[0055] Embodiment 5 Functional classification of differentially expressed proteins in BHEC cells damaged by DON induced by vitamin E In order to understand the possible biological functions of differentially expressed proteins in the process of vitamin E alleviating DON-induced BHEC cell damage, the functional analysis of differentially expressed proteins among different treatment groups was performed using Gene Ontology (GO), subcellular structure localization and COG functional classification.

[0056] (1) GO secondary classification of differentially expressed proteins The differentially expressed proteins were classified by GO and the GO functional annotation map was drawn. Figure 11~Figure 14 As shown, the differentially expressed proteins in different comparison groups were significantly enriched in biological process (BP), cellular component (CC), and molecular function (MF), among which the number of differentially expressed proteins in 0.2D50V / 0.2D was significantly reduced.

[0057] In terms of biological processes, the biological processes involved in the differentially expressed proteins in different comparison groups mainly include biological regulation processes, metabolic processes, cellular processes, stress responses and other processes; in terms of cellular components, the differentially expressed proteins are mainly distributed in intracellular anatomical structures, organelles, cytoplasm, etc.; in terms of molecular functions, the differentially expressed proteins play roles in binding ability, catalytic activity, enzyme regulatory activity, etc.

[0058] (2) Subcellular localization of differentially expressed proteins Mature proteins must be in specific subcellular organelles to exert stable biological functions. Subcellular organelles are important components of cells and are a type of micro-organ with certain morphology and function. 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 function. Protein subcellular localization is very important for studying protein function. Subcellular localization can locate a protein or expression product in a specific location in the cell, such as in the nucleus, various organelles, and on the plasma membrane, thereby providing a research direction for understanding the mechanism of gene action.

[0059] like Fig.15 The following table shows the annotations of the subcellular structures of the differentially expressed proteins in each group. It can be seen that the differentially expressed proteins are mainly located in the nucleus, cytoplasm, extracellular matrix, mitochondria, cell membrane, etc.

[0060] (3) COG / KOG functional annotation of differentially expressed proteins Through database comparison, the differentially expressed proteins were statistically classified according to COG / KOG functions. The COG functions were mainly divided into cell processes and signal transduction, storage and processing of information, metabolism, and unknown functions.

[0061] The results of differential protein classification in each comparison group are as follows Fig.16 As shown, the differentially expressed proteins were mainly concentrated in signal transduction mechanisms, post-translational modifications, protein turnover, chaperone proteins, transcription, RNA processing and modification, energy production and conversion, and amino acid transport and metabolism.

[0062] Embodiment 6 GO enrichment analysis of differentially expressed proteins in BHEC cells damaged by vitamin E induced by DON Based on the functional annotations of the identified differential proteins, GO enrichment analysis was performed on the differential proteins. Fisher's exact test was used to obtain the p value, and a bubble chart was used to display the top 20 significantly enriched classification results. The vertical axis of the bubble chart is the GO functional description information, and the horizontal axis is the functional enrichment degree after Log2 transformation. The larger the value, the higher the enrichment degree; the color of the point represents the enrichment significance P value, and the bluer the color, the stronger the enrichment significance; the size of the point represents the number of differential proteins in the GO function, and the larger the point, the more differential proteins there are.

[0063] like Fig.17 As shown in A, the 0.2D / CON group mainly affected the retinoid metabolism process, the regulation of protein activation cascade reaction, the diterpenoid metabolism process, the regulation of acute inflammatory response, coagulation, fibrin clot formation, the acute phase response, the regulation of sterol transport, the regulation of cholesterol transport, the remodeling of protein-lipid complexes, and the negative regulation of endopeptidase activity.

[0064] like Fig.17 As shown in B, the 50V / CON group mainly affected the regulation of acute inflammatory response, regulation of protein activation cascade, regulation of protein processing, regulation of protein maturation, coagulation, fibrin clot formation, regulation of triglyceride biosynthesis process, positive regulation of protein maturation, positive regulation of granulocyte chemotaxis, positive regulation of steroid metabolism process, and positive regulation of inflammatory response.

[0065] like Fig.17As shown in C, the 0.2D50V / CON group mainly affected the regulation of acute inflammatory response, regulation of protein activation cascade, coagulation, fibrin clot formation, regulation of protein processing, regulation of protein maturation, acute phase response, negative regulation of peptidase activity, regulation of proteolysis, negative regulation of endopeptidase activity, coagulation, and intrinsic pathway.

[0066] like Fig.17 As shown in D, the 0.2D50V / DON group mainly affected the regulation of acute inflammatory response, coagulation, intrinsic pathway, coagulation, fibrin clot formation, regulation of protein activation cascade, regulation of protein processing, regulation of protein maturation, negative regulation of inflammatory response, acute phase response, positive regulation of acute inflammatory response, and negative regulation of interleukin-6 production.

[0067] Embodiment 7 KEGG enrichment analysis of differentially expressed proteins in BHEC cells damaged by vitamin E induced by DON In order to further explore the mechanism of vitamin E in alleviating DON-induced liver cell damage in dairy cows, KEGG pathway enrichment analysis of differentially expressed proteins was performed using bioinformatics. Most pathways changed after BHEC cells were stimulated.

[0068] like Fig.18 As shown, signaling pathways such as cholesterol metabolism, drug metabolism-cytochrome P450, metabolism of exogenous substances by cytochrome P450, fat digestion and absorption, retinol metabolism, PI3K-Akt signaling pathway, PPAR signaling pathway and glutathione metabolism were significantly upregulated, while signaling pathways such as protein digestion and absorption, cell adhesion molecules, ubiquitin-mediated proteolysis, ECM receptor interaction, oocyte meiosis and endocytosis were significantly downregulated.

[0069] In 0.2D50V / CON, it was found that the addition of vitamin E upregulated the ECM receptor interaction pathway and downregulated the cholesterol metabolism pathway. In 0.2D50V / CON, the LDLR gene was significantly upregulated, and the APOA1, APOA2, and APOA4 genes were significantly downregulated and enriched in the cholesterol metabolism pathway.

[0070] The results of 0.2D50V / CON KEGG enrichment are shown in Table 3. The effects of vitamin E supplementation on cholesterol metabolism by enriching differential proteins to alleviate DON-induced liver cell damage in dairy cows are shown in Table 4: Table 3 0.2D50V / CON KEGG enrichment results ;

[0071] Table 4 Effect of vitamin E supplementation on cholesterol metabolism of differentially enriched proteins in dairy cow liver cell damage induced by DON ;

[0072] Embodiment 8 Targeted proteome quantification (PRM) In order to verify the reliability of the KEGG enrichment pathway results of proteomics differential proteins, targeted proteomics quantitative technology was used to verify the differential proteins on the screened pathways. PRM commissioned Jingjie Bioassay to first perform protein extraction and trypsin hydrolysis, then perform liquid chromatography-mass spectrometry tandem analysis, and finally perform database search and data processing.

[0073] The four target proteins shown in Table 4 were selected for PRM verification. The relative quantitative expression results of APOA1, APOA2 and APOA4 genes were summarized as follows: Fig.19 The results showed that the three candidate proteins showed similar trends in PRM verification and 4D label-free quantitative proteomics analysis, which also proved the rationality and reliability of proteomics data. The CDS sequence of APOA1 gene is shown in SEQ ID NO.19; the CDS sequence of APOA2 gene is shown in SEQ ID NO.20; and the CDS sequence of APOA4 gene is shown in SEQ ID NO.21.

[0074] In summary, vitamin E can effectively protect the cell membrane integrity of dairy cow liver cells and alleviate the decline in cell viability, oxidative stress, inflammatory response and cell apoptosis caused by vomitoxin. Vitamin E promotes cholesterol metabolism pathway by regulating APOA gene, thus protecting dairy cow liver cells.

[0075] 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 role of APOA gene in vitamin E in alleviating the damage of bovine liver cells to vomitoxin, characterized in that: The APOA gene refers to the APOA1 gene, the APOA2 gene and the APOA4 gene, The CDS sequence of the APOA1 gene is shown in SEQ ID NO.19; The CDS sequence of the APOA2 gene is shown in SEQ ID NO.20; The CDS sequence of the APOA4 gene is shown in SEQ ID NO.21; The effect is that vitamin E regulates the enrichment of APOA gene into the cholesterol metabolism pathway.

Images