CN111568912B - Glycyrrhizic acid and probiotics are combined to be used as feed additive for relieving vomitoxin harm - Google Patents

Abstract

The invention belongs to the technical field of feed additives, and particularly relates to a feed additive for relieving vomitoxin, which is prepared by mixing glycyrrhizic acid and probiotics. The invention clarifies a repairing mechanism of glycyrrhizic acid on the vomitoxin-induced cell inflammation and apoptosis by researching a molecular mechanism of vomitoxin-induced intestinal epithelial cell inflammation and apoptosis, provides an application of glycyrrhizic acid in preparing a medicine or feed additive for relieving the harm of vomitoxin on the basis, provides a feed additive prepared by mixing glycyrrhizic acid and probiotics, and lays a foundation for relieving the toxicity of vomitoxin to animals and improving the health condition of livestock and poultry.

Description

Glycyrrhizic acid and probiotics are combined to be used as feed additive for relieving vomitoxin harm

Technical Field

The invention belongs to the technical field of feed additives, and particularly relates to a feed additive for relieving vomitoxin, which is prepared by mixing glycyrrhizic acid and probiotics.

Background

Deoxynivalenol (DON), also known as vomitoxin, is a type B trichothecene toxin produced by gibberella zeae and mainly exists in cereals such as wheat, corn, oat, barley and the like. DON has toxic effects on humans and many animals, with pigs being one of the most sensitive animals to DON, resulting in vomiting, diarrhea, slow growth, immune system disturbances and economic losses. The intestinal tract of animals is the first barrier for the body to resist the invasion of external harmful substances, and the intestinal mucosa is an important barrier for absorbing and transporting various nutrient substances such as glucose, amino acid, trace elements and the like. DON can cause increased intestinal permeability, damaged and necrotic mucosa, apoptosis, decreased antioxidant capacity and mitochondrial dysfunction, leading to damage to porcine intestinal tract. Tight junction proteins are connective polyprotein complexes that primarily maintain the intestinal epithelial barrier, causing body injury and inflammation, and even affecting transport and absorption of nutrients, when they are disrupted by environmental conditions (mycotoxins, environmental changes, feed changes, etc.).

The licorice is a perennial herb and has good detoxification function, the licorice extract is widely applied to animal husbandry to promote animal growth, improve meat quality and prevent and treat various livestock and poultry diseases, and in addition, the licorice extract is reported in documents to be clinically used for treating liver injury. Glycyrrhizic Acid (GA) is an extract of liquorice, has various pharmacological activities including anti-inflammatory, immunity regulating, antioxidant, antiviral, anticancer, blood fat reducing and the like, and can be used for treating or relieving enterotoxicity and inflammation caused by DON, so that an effective and economical veterinary medicine or feed additive is provided.

The probiotics are live bacteria preparations or metabolites thereof which play a beneficial role by improving the micro-ecological balance of the gastrointestinal tract of a host and achieve the purpose of improving the health level and the health state of the host, and beneficial bacteria or fungi in an animal body mainly comprise: lactobacillus, bifidobacterium, actinomycetes, yeast, enterobacter and the like. The probiotics have the effects of regulating the animal gastrointestinal microflora, degrading mycotoxin, relieving immunosuppression, inhibiting the growth and reproduction of harmful bacteria, promoting animal production and the like. The invention provides a feed additive for effectively relieving harm of vomitoxin to intestinal cells, which is prepared by matching glycyrrhizic acid with probiotics, and the feed additive is not reported in the prior art, so that the invention is a main technical problem to be solved.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide glycyrrhizic acid and probiotics which are used as feed additives for relieving DON; the invention provides a feed additive prepared by mixing glycyrrhizic acid and probiotics on the basis of clarifying the repair mechanism of DON-induced cell inflammation and apoptosis by researching the molecular mechanism of DON-induced intestinal epithelial cell inflammation and apoptosis, thereby laying a foundation for relieving the toxicity of DON to animals and improving the health condition of livestock and poultry.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

application of glycyrrhizic acid in preparing medicament or feed additive for relieving DON harm is provided.

Preferably, glycyrrhizic acid relieves cell inflammation or cell premature senility caused by DON by regulating the relative expression of genes IL-6, IL-8, TNF-alpha, COX-2, NF-Kb, Bax, Caspase3 or Bcl-2.

Preferably, glycyrrhizic acid alleviates cellular oxidative stress caused by DON by adjusting the levels of lactate dehydrogenase, malondialdehyde, catalase and superoxide dismutase.

Further preferably, the cell is a porcine intestinal cell.

Preferably, the addition amount of the glycyrrhizic acid is 50-800 mug/mL.

Based on a general inventive concept, the invention also comprises a feed additive for alleviating the harm of DON, wherein the feed additive consists of glycyrrhizic acid, saccharomycetes and enterococcus.

Preferably, the addition amount of the feed additive is 200-400 mg/kg of glycyrrhizic acid feed and 0.5 multiplied by 10 of yeast ⁹ ～5× 10 ⁹ CFU/kg fodder, enterococcus 0.5 × 10 ⁹ ～5×10 ⁹ CFU/kg feed.

More preferably, the feed additive is added with glycyrrhizic acid 400mg/kg feed and yeast 1 × 10 ⁹ CFU/kg fodder, enterococcus 1X 10 ⁹ CFU/kg feed.

Preferably, the yeast is candida utilis; the enterococcus is enterococcus faecalis.

Based on a general inventive concept, the invention also comprises the use of said feed additive for alleviating the decline of piglet growth performance caused by DON.

The research result of the invention shows that the DON with the concentration of 0.5 mu g/mL can stimulate for 6 hours to cause the oxidative stress, inflammation or apoptosis of cells; compared with a DON treatment group, the cell activity and the activities of superoxide dismutase (SOD) and Catalase (CAT) can be obviously improved by adding 200-400 mu g/mL GA while adding DON, and the release of Lactate Dehydrogenase (LDH), the content of Malondialdehyde (MDA) and the apoptosis rate are reduced. GA can also obviously reduce the relative expression quantity of inflammatory genes such as IL-6, IL-8, TNF-alpha, COX-2, NF-Kb, Bax, Caspase3 and the like, obviously up-regulate the relative expression quantity of an anti-inflammatory gene Bcl-2, inhibit the generation of inflammatory factors and chemotactic factors by activating a Tumor Necrosis Factor (TNF) signaling pathway, a Toll-like receptor (Toll-like) signaling pathway and a nuclear transcription factor (NF-kB) signaling pathway, relieve DON-induced oxidative stress, inflammatory reaction, cytotoxicity and apoptosis, and protect the integrity of intestinal tracts and the health of animals.

The research result of the invention also shows that the addition of 400g glycyrrhizic acid/ton fodder and 1 x 10 candida utilis in the fodder of weaned pig is ¹² CFU/ton of feed and enterococcus faecalis is 1 × 10 ¹² CFU/ton of feed can effectively relieve the harm of DON to the growth of piglets. The invention lays a foundation for the glycyrrhizic acid and the probiotics to be used as the feed additive for relieving the DON toxicity and improving the health condition of the livestock and poultry.

Drawings

FIG. 1: the effects of GA and DON on the viability of IPEC-J2 cells; effect of different GA concentrations on IPEC-J2 cell viability at different action times; b, the influence of different concentrations of GA and 0.5 mu g/mL DON on the cell viability after co-culture for 6 hours;

FIG. 2: influence of GA on lactate dehydrogenase release amount and antioxidant parameters in IPEC-J2 cells after DON treatment; a, Lactate Dehydrogenase (LDH) release; b, Malondialdehyde (MDA) content; c, superoxide dismutase (SOD) viability value; d, Catalase (CAT) activity value;

FIG. 3: effect of GA on DON-induced apoptosis of IPEC-J2 cells; detecting Annexin V/FITC/PI stained apoptotic cells by using flow cytometry, wherein Q1, Q2, Q3 and Q4 represent late apoptotic cell rate, necrotic cell rate, viable cell rate and early apoptotic cell rate respectively; b, quantitative analysis of total apoptosis rate of cells; c, Q1, Q2, Q3 and Q4 under different conditions;

FIG. 4: influence of GA on IPEC-J2 cell-related gene expression after DON treatment; a, influence of GA on apoptosis-related genes (Bax, Bcl-2 and Caspase 3); b, the effect of GA on genes associated with cellular inflammation (IL-6, IL-8, TNF-alpha, COX-2 and NF-kappa B);

FIG. 5: the analysis result of the differentially expressed genes of the sample; a, PCA principal component analysis; b, differential expression gene number between the two treatment groups; c, carrying out clustering heat map analysis on the differentially expressed genes among the samples; d, Venn analysis.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

1. Test materials

Glycyrrhizic Acid (GA) was supplied by henna-nei biologies ltd. DON was purchased from Sigma-Aldrich (USA); phosphate Buffered Saline (PBS), 0.25% pancreatin-ethylenediaminetetraacetic acid (EDTA), penicillin/streptomycin (10,000 units/10,000. mu.g/ml), dimethyl sulfoxide (DMSO), and thiazolyl tetrazolium bromide (MTT) were purchased from Beijing Soilebao BioLimited; high glucose medium (HGDMEM) and Fetal Bovine Serum (FBS) were purchased from Biological Industries, Inc. (Israel); the Annexin V-FITC/PI kit is purchased from Higasat Biotech, Inc., Shanghai; superoxide dismutase (SOD), Catalase (CAT), intracellular Glutathione (GSH) and Malondialdehyde (MDA) kits are purchased from Beijing Solaibao biological Limited company; trizol reagent was purchased from Invitrogen corporation (USA); the reverse transcription kit and the TB GREEN kit were purchased from TaKaRa (Chinese Dalian).

The test pig is weaned ternary hybrid piglet (Duroc, large white and long white). The yeast is candida utilis (Canida utilis, purchased from China general microbiological culture Collection center (CGMCC) with the bacterial number of 2.615); the Enterococcus is Enterococcus faecalis (Enterococcus faecalis, purchased from China general microbiological culture Collection center (CGMCC), with a bacterial number of 1.2135).

2. Test method

1) Cell culture and sample preparation

The IPEC-J2 cell line was provided by the institute of animal science and technology, university of agriculture in Jiangxi. IPEC-J2 cell culture medium is HGDMEM medium containing 10% FBS and 1% penicillin-streptomycin, and is inoculated to 25cm ² In a culture flask, and placing at 37 deg.C and 5% CO ₂ The incubator of (2). When the cell fusion rate reaches 80% -90%, the cells are respectively inoculated in a 96-well plate and a 6-well plate for 24h, and then different treatments are carried out. The original DON was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1mg/mL stock solution, which was then sterilized by filtration through a 0.22 μm filter. GA and DON are diluted into different concentrations by culture solution without serum and antibiotics, and are prepared for use.

2) Data statistics and analysis

All data in the experiment are presented as mean ± standard deviation, and all plots were generated using GraphPad Prism 7. All data were statistically analyzed by one-way analysis of variance (ANOVA) using the SPSS 20.0 general linear model and multiple comparisons were performed using the Duncan test. P <0.05 is significantly different, and P >0.05 is not significantly different.

Example 1 Effect of GA on cell viability after DON treatment

The porcine small intestinal epithelial cells (IPEC-J2) are a cell model commonly used for researching intestinal functions, and in order to research the influence of GA and DON on the intestinal cells, the porcine small intestinal epithelial cells (IPEC-J2) are used as the cell model for research. First, the inventors used the MTT method to determine the effect of different GA concentrations and treatment times on cell proliferation:

IPEC-J2 cells were harvested at logarithmic growth phase, digested with pancreatin, and cultured at 1X 10 ⁴ cells/well density into 96-well plate, after 24 hours incubation, abandoning supernatant, washing twice with PBS, then adding GA with different concentrations (0 ug/mL, 50 ug/mL, 100 ug/mL, 200 ug/mL, 400 ug/mL and 800 ug/mL) for 3h, 6h, 12g and 24 h; then 10. mu.L of MTT solution (5mg/mL) was added to each well and incubated for 4 h. The supernatant was carefully discarded and 150. mu.L of DMSO was added to each well to dissolve. After shaking at room temperature for 10min, the absorbance value (OD) was measured at 490nm and 630 nm using a microplate reader. Each treatment was 6 replicates. The activity of IPEC-J2 cells under different conditions is shown in A in FIG. 1; control in the figure is blank group, and different superscripts indicate the degree of difference significance compared with blank group, wherein difference significance is used ^* (P<0.05) and ^** (P<0.01).

As can be seen from A in FIG. 1, the GA-promoting effects are dose-dependent and time-dependent on the cell proliferation; cell viability was significantly enhanced after 6h of GA treatment at different concentrations (P < 0.01). Therefore, 6h was used as the optimum processing time for GA.

Previous studies of the inventors show that the stimulation of IPEC-J2 cells with 0.5. mu.g/mL DON for 6h can significantly reduce the cell viability, in order to examine the alleviating effect of GA on the toxicity of DON, a control group with DON concentration of 0.5. mu.g/mL is added with different concentrations of GA while DON is added, and the proliferation of the cells after 6h treatment is shown as B in FIG. 1; the different superscripts in the figure represent the degree of significance of the difference compared to the control group, which is used as the significance of the difference compared to the control group ^* P<0.05 and ^** P<0.01 represents; comparison with DON group, significance of difference ^# P<0.05 and ^## P<0.01 indicates that ns represents no significant difference (P)>0.05)。

As can be seen from B in FIG. 1, under the conditions that the GA addition concentrations are respectively 50 μ g/mL, 100 μ g/mL, 200 μ g/mL and 400 μ g/mL, the cell viability decrease caused by DON is relieved, and the cell viability is improved compared with that of the control group, and particularly when the GA concentration reaches 400g/mL, the cell proliferation activity basically returns to the blank control level, which indicates that the cell viability decrease caused by DON can be relieved by the GA, and the cell proliferation activity is recovered.

Example 2 influence of GA on cellular oxidative stress response after DON treatment

IPEC-J2 cells were harvested in the logarithmic growth phase, digested with pancreatin, and cultured at 5X 10 ⁵ cells/well density cells were cultured in 6-well plates until the cell fusion rate reached 80%, and then cells were treated according to control, DON, GA + DON groups: wherein DON group was treated with 0.5. mu.g/mL DON for 6h, GA group was treated with 400. mu.g/mL GA for 6h, GA + DON group was co-treated with 400. mu.g/mL GA and 0.5. mu.g/mL DON for 6h, and untreated cell group was used as a control group, and 3 replicates were set for each group. And collecting cells after treating for corresponding time, and then measuring the SOD enzyme activity, CAT enzyme activity and MDA content of each treatment group according to the operation steps of the corresponding kit. The effect of GA on DON-induced LDH release rate and antioxidant parameters of IPEC-J2 cells is shown in FIG. 2.

As can be seen from the graph, DON significantly increased the release amount of Lactate Dehydrogenase (LDH) and the level of Malondialdehyde (MDA) (P <0.01), decreased the activities of Catalase (CAT) and superoxide dismutase (SOD) (P <0.01) compared to the control group; compared with a DON treatment group only by adding GA while treating DON, LDH release and MDA content in cells are obviously reduced (P is less than 0.01), and SOD content and CAT activity in the cells are obviously improved; the fact shows that the addition of GA can relieve the oxidative stress reaction caused by DON and restore the oxidation resistance of cells.

Example 3 influence of GA on DON-induced apoptosis

IPEC-J2 cells were harvested in the logarithmic growth phase, digested with pancreatin, and cultured at 5X 10 ⁵ cells/well density cells were cultured in 6-well plates until the cell fusion rate reached 80%; then treating the cells according to a control group, a DON group, a GA group and a GA + DON group respectively, wherein the DON group is treated with 0.5 mu g/mL DON for 6h, the GA group is treated with 400 mu g/mL GA for 6h, the GA + DON group is treated with 400 mu g/mL GA and 0.5 mu g/mL DON for 6h, and the untreated cell group is taken as the control group, and each group is repeated for 3 times; the harvested cells were then digested with trypsin without EDTA for 5min, placed in a 5mL centrifuge tube and centrifuged at 800rpm for 5 min. Cells were washed twice with PBS to eliminate dead cells and resuspended in 100. mu.L of 1Xbind buffer, then stain with 5. mu.L Annexin V-FITC and 5. mu.L PI at room temperature in the dark. Finally, 400 μ L of 1 × binding buffer was added and the level of apoptosis was analyzed by flow cytometry within 1 h; the effect of GA on DON-induced apoptosis and inflammatory responses of IPEC-J2 is shown in FIG. 3.

From the aspect of apoptosis, compared with a control group, the late apoptosis rate (Q1) and the early apoptosis rate (Q4) of IPEC-J2 cells after DON treatment are obviously increased, and the increase of the late apoptosis rate and the early apoptosis rate caused by DON can be obviously reduced by adding GA, namely, the increase of the late apoptosis rate and the early apoptosis rate caused by DON can be obviously relieved by adding GA. Correspondingly, DON can cause the rate of IPEC-J2 living cells to be obviously reduced, and the rate of necrotic cells is basically unchanged; and the addition of GA obviously improves the condition of low survival rate of the living cells caused by DON, and the necrotic cell rate is even lower than that of a blank control.

Example 4 Effect of GA on the expression levels of the relevant genes in IPEC-J2 cells after DON treatment

In order to further research the cause of GA relieving DON to cause apoptosis, the inventors carried out measurement on the expression levels of genes Bax, caspase-3 and Bcl-2 related to apoptosis in IPEC-J2 cells; in addition to the genes, the expression level changes of some inflammatory genes can also cause cell premature senescence and apoptosis, such as IL-6, IL-8, TNF-alpha, COX-2, NF-kappa B and the like, so the inventors have measured the expression level of the genes.

IPEC-J2 cells were first cultured at 5X 10 ⁵ cells/well were seeded in 6-well plates at a cell fusion rate of 80% in the above four treatment groups (control, DON, GA + DON), total RNA was extracted using Trizol, and the RNA pellet was dissolved in 50. mu.L RNase-free water and stored at-80 ℃. RNA concentration was measured by a NanoDrop ND-1000 spectrophotometer, OD260/OD280 and OD260/OD230 were calculated, and RNA integrity was verified by agarose gel. Using the TB GREEN kit, about 1. mu.g of total RNA in each sample was kept at 42 ℃ for 2min to remove gDNA, and cDNA was produced by reverse transcription. Using CFX Connect ^TM The real-time PCR detection system performs real-time PCR. PCR conditions were 95 ℃ pre-denaturation for 300sThe last 95 ℃ 20s, 60 ℃ 30s and 72 ℃ 30s were performed for 38 cycles. GAPDH was used as an internal reference gene, 2 ^-ΔΔCT The method analyzes the relative expression of each gene, and all the primer pairs used for determining the genes and amplifying the genes are listed in Table 1.

TABLE 1 genes to be tested and amplification primer sequences

The results of the measurement of the expression levels of the respective genes are shown in FIG. 4; as can be seen from A in FIG. 4, DON significantly up-regulates the relative mRNA abundance of Bax and caspase-3 genes (P <0.01), down-regulates the mRNA abundance of Bcl-2 (P < 0.05); compared with a DON group, the DON is added with GA, so that the mRNA abundance (P <0.01) of Bax and caspase-3 can be obviously reduced, and the mRNA abundance (P <0.05) of Bcl-2 is increased, which indicates that GA can improve the cell cycle shortening or apoptosis condition of cells caused by DON by regulating the expression quantity of Bax, caspase-3 and Bcl-2;

the B result in FIG. 4 shows that after the DON treatment is carried out for 6h, the relative mRNA abundance of inflammatory genes such as IL-6, IL-8, TNF-alpha, COX-2 and NF-kB is remarkably up-regulated (P is less than 0.01) in IPEC-J2 cells; however, the amount of the above gene expression (P <0.01) can be significantly reduced by the addition of GA, i.e., GA can improve the DON-induced inflammation of cells by regulating the expression of IL-6, IL-8, TNF-alpha, COX-2 and NF-kappa B.

Example 5 RNA-seq data analysis

In order to explore the molecular mechanism of GA for reducing DON-induced IPEC-J2 cell apoptosis and inflammation, RNA-seq analysis was carried out on four groups of treated cells of Control (CON), DON, GA and GAD (GA + DON), and Differentially Expressed Genes (DEGs) were screened, with 3 replicates in each group; the specific process is as follows:

IPEC-J2 cells at 5X 10 ⁵ cells/well density were seeded in 6-well plates until cell fusion rate reached 80%After the above four treatment groups (control, DON, GA + DON), total RNA was extracted using Trizol, and the RNA pellet was dissolved in 50. mu.L of RNase-free water and stored at-80 ℃. The quality and purity of the RNA were checked using an Agilent 2100 bioanalyzer and the integrity of the RNA was checked using agarose gel electrophoresis. The purified mRNA was enriched with oligo (dT) beads, Mg ²⁺ The ion disruption buffer disrupted it to 300 bp. The fragment mRNA was then reverse transcribed into cDNA using 6bp random primers. Then, second strand cDNA was synthesized using the first strand cDNA as a template. The synthesized product was purified using a PCR purification kit, followed by end repair, poly (a) addition, and Illumina sequencing adapter ligation. Sequencing libraries were selected for size and their size was measured using an Agilent 2100 bioanalyzer. Finally, sequencing was performed using the Illumina HiSeq 4000 platform (Illumina, San Diego, CA, USA) using a paired (PE 150bp) sequencing strategy.

Quality assessment of the original Reads was performed by removing the sequences with adaptors at the 3' end using Cutadapt, removing Reads with an average mass fraction below Q20. The filtered Reads were then aligned to the reference genome using HISAT2 software and the distribution of the Reads aligned to the genome was statistically analyzed. The Read Count value of each gene was statistically aligned using HTSeq as the original expression level of the gene. In order to make the expression level of genes comparable between different genes and different samples, the expression level was normalized by FPKM (fragments Per Kilo bases Per Million fragments), which is the number of fragments Per kilobase in length Per Million fragments. In addition, PCA principal component analysis was performed on each sample based on the expression level using the DESeq software package in R language. Differential expression analysis was performed on DESeq2(Bioconductor version 1.6.2) with P <0.05 as the differential selection threshold between groups and log2(fold change) >1 absolute as Differentially Expressed Genes (DEGs).

After filtering low quality reads, the Illumina Hiseq sequencing platform produced approximately 4200 million clean reads (93%), and over 96% of the clean reads aligned to the reference genome. Then analyzing the gene expression changes of the 12 samples, and performing PCA principal component analysis on each sample according to the gene expression quantity, wherein the specific result is shown in figure 5;

as can be seen from a in fig. 5, the PC1 axis accounts for 86% of the total variation and the PC2 axis accounts for 10% of the total variation. As can be seen from B in fig. 5, 1576 difference genes (668 up-and 908 down-regulated), 289 genes (211 up-and 78 down-regulated) and 1398 genes (734 up-and 664 down-regulated) were detected in the DON group, the GA group and the GA + DON group, respectively, compared to the control group; in the comparison of DON to the GA + DON group, 154 genes were detected, 103 up-regulated and 51 down-regulated. The clusters of deg were then analyzed in a heat map and as can be seen from C in fig. 5, there was more similarity between samples of the same treatment, 3 biological replicates in each treatment were hierarchically clustered using deg, clustered together at close distances from each other, indicating good reproducibility of each treatment. Analyzing the co-expression DEGs of the DON group, the GA group, the GAD group and the control group respectively and the GAD group and the DON group through a Venn diagram, and as can be seen from D in FIG. 5, 79 DEGs are co-expressed in the control group and the DON and GAD group, wherein 37 differential genes are down-regulated in the control group and the DON group and up-regulated in the DON and GAD group; there were 26 differential genes whose expression was up-regulated in the control and DON groups and down-regulated in the DON and GAD groups.

Example 6 GO and KEGG pathway enrichment analysis

To better understand the biological processes and pathways involved in DEGs, the inventors functionally annotated DEGs using the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The functional analysis of GO mainly comprises three parts: biological Processes (BP), Molecular Functions (MF) and Cellular Components (CC); and carrying out GO enrichment analysis by using topGO, calculating a gene list and the number of genes of each term by using the differential genes annotated by GO term during analysis, then calculating a P value (the standard of significant enrichment is P <0.05) by a super-geometric distribution method, and finding the GO term in which the differential genes are significantly enriched compared with the whole genome background, thereby determining the main biological functions performed by the differential genes.

By performing GO annotation on Control (CON) and DON groups, control and GA groups, control and GAD groups, and DON and GAD groups, it was found that, from the Biological Process (BP) level, deg was mainly focused on functions related to phylogeny and cell differentiation, except for some deg annotations on response to stimuli and immune system. At the Molecular Function (MF) level, the rest of the three groups of dees are almost all associated with sequence-specific DNA binding, except for the DON and GAD groups. From the Cellular Component (CC) level, the CON and DON groups, and CON and GAD groups, the role of DEGs is concentrated in intracellular organelles; while the DEGs of the CON and GA groups were almost annotated to the extracellular region or cell membrane and to some extent had similar GO annotations to the DON and GAD groups.

In the above four different comparisons, the inventors performed KEGG pathway enrichment analysis on DEGs, resulting in pathway enrichment that varied significantly under different experimental conditions. KEGG is a database related to pathways for understanding high-level functions and utilities of biological systems, with P <0.05 being the significance of the current analysis. All the DEGs were annotated for KEGG pathway based on cellular processes, environmental information processing, disease, metabolism and biological systems; as a result, it was found that the DEGs are involved in various signal transduction pathways in the CON and DON groups, including tumor necrosis factor, Notch, MAPK, Ras, and NF-kappa B signal transduction pathways, as well as Th1 and Th2 cell differentiation, B cell receptor, and Toll-like receptor signal transduction pathways, and further include three metabolic pathways of steroid biosynthesis, unsaturated fatty acid biosynthesis, and terpene skeleton biosynthesis. For the CON and GA groups, the pathways include Cell Adhesion Molecules (CAMs), leukocyte migration across endothelial cells, complement and coagulation cascades, PI3K-Akt signaling pathway, etc. Since the CON and DON groups, and the CON and GAD groups shared more than 900 DEGs, the pathways enriched between them were similar and related to signal transduction and the immune system. Interestingly, the DON and GAD groups found some of the same pathways, such as TNF, cAMP, leukocyte migration across endothelial cells, etc., as compared to the CON and DON groups, but the Jak-STAT signaling pathway was unique to the DON and GAD groups. In addition, it is also enriched in several immune systems, including chemokine signaling pathways, the Fc epsilon-RI signaling pathway, the complement and coagulation cascade, and Fc γ -R mediated phagocytosis.

The inventors functionally annotated and enriched the 79 deg's co-expressed in example 5 by GO and KEGG pathway enrichment in the control and DON groups, and DON and GAD groups. The GO annotation results show that most of the annotated BP bodies are focused on the migration of immune cells, such as myeloid leukocytes, neutrophils, monocytes, granulocytes, etc.; and some of the DEGs are annotated as external stimuli and inflammatory responses. In MF, DEGs are annotated to Notch binding, receptor binding, chemokines, chemokine receptors, IL8(CXCL8) receptor binding, and the like. After being enriched by KEGG, DEGs including CXCL8, CCL5, IL15 and the like are enriched in a chemokine, tumor necrosis factor signal pathway and a cytokine-receptor interaction pathway.

Example 7 Effect of glycyrrhizic acid in combination with probiotic on alleviating DON toxicity

The results of examples 1-6 show that GA can inhibit the production of inflammatory factors and chemokines, remarkably slow down oxidative stress and reduce necrotic cell apoptosis, and has a certain immunoregulation effect; therefore, when cells are affected by DON, the glycyrrhizic acid can be added to relieve DON-induced intestinal inflammatory reaction and improve DON-induced pathological activity. In order to obtain an economical and effective feed additive, the inventor combines GA with probiotics, and examines the effect of the obtained combined preparation on relieving the DON toxicity.

The probiotics are selected from candida utilis and enterococcus faecalis, wherein the enterococcus faecalis is selected from an MRS culture medium: tryptone 15g, yeast extract powder 10g, glucose 20g, Tween 801 mL, K ₂ HPO ₄ 2g, 5g of sodium acetate, 2g of ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 1000mL of water, wherein the pH value is 6.2-6.6; sterilizing the culture medium at 121 deg.C and 0.15MPa for 20 min; and inoculating enterococcus faecalis into an MRS liquid culture medium, and standing and culturing at the constant temperature of 37 ℃ for 24 hours.

The Candida utilis adopts YPD culture medium: 20g of peptone, 10g of yeast extract powder, 20g of glucose and 1000mL of water, wherein the pH value is 7.0-7.2; sterilizing the culture medium at 121 deg.C and 0.15MPa for 20 min; the Candida utilis is inoculated into YPD culture medium and put into a shaking table at 30 ℃ and 180r/min for shaking culture for 24 h.

The concentration of the used candida utilis and enterococcus faecalis is 1 multiplied by 10 ⁶ Colony Formed Unit (CFU)/mL; the results of the determination of the activity of the porcine intestinal epithelial cells under different treatment conditions are shown in table 2;

TABLE 2 Effect of different treatment conditions on the viability of porcine intestinal epithelial cells

Note: in the table, the difference of lower case letters after the same column data indicates that the difference is significant (P <0.05), and the difference is not significant (P >0.05) if the same column data indicates that the difference is not significant.

As can be seen from the data in Table 2, DON decreased the viability value of the cells from 100 in the control group to 78.14. Adding glycyrrhizic acid while adding DON, and increasing the cell viability value to 88.03; the cell viability value of the candida utilis is only 62.94 when the DON is added, and the cell viability value can be improved to 90.68 when the enterococcus faecalis is added when the DON is added. The results show that glycyrrhizic acid or enterococcus faecalis has a remarkable effect of relieving cell viability reduction caused by DON, and the Candida utilis alone can worsen the cell viability reduction tendency caused by DON.

As previous researches show that the candida utilis has the effect of degrading DON in an animal body, and glycyrrhizic acid, candida utilis, enterococcus faecalis and DON are used simultaneously, the cell viability value can be recovered to 89.13, the relieving effect is obvious, and therefore, the combination of glycyrrhizic acid, candida utilis and enterococcus faecalis selected in the following animal feeding experiments.

Example 8 Effect of glycyrrhizic acid in combination with probiotic bacteria on the alleviation of DON-induced decline in piglet growth performance

The weaned tripartite piglets were divided randomly into 4 groups of 4 replicates each, with 10 piglets per replicate. The treatment conditions for the 4 groups were respectively: control group (without GA and DON), negative control group (DON group, DON content is 1000 mg/ton of diet), glycyrrhizic acid and probiotic compatible group (glycyrrhizic acid content is 400 g/ton of diet + Candida utilis 1 × 10) ¹² CFU/ton diet + enterococcus faecalis 1X 10 ¹² CFU/ton diet), DON + glycerineOxalic acid and probiotics compatibility group (DON 1000 mg/ton diet + glycyrrhizic acid 400 g/ton diet + Candida utilis 1X 10) ¹² CFU/ton diet + enterococcus faecalis 1X 10 ¹² CFU/ton diet). The piglet diet formula was referenced to the recommendation of NRC (2012). The pilot period is 30 days, the pig is fed and drunk freely, and the feeding management is carried out according to the requirements of a pig farm. The growth of piglets under different treatment conditions is shown in table 3.

TABLE 3 comparison of growth of piglets under different treatment conditions

Note: the difference of lower case letters after the data of the same row in the table indicates that the difference is significant (P <0.05), and the same indicates that the difference is not significant (P > 0.05).

The test results in table 3 show that the daily gain and daily feed intake of piglets can be improved to a certain extent by feeding the feed additive with glycyrrhizic acid compatible with candida utilis and enterococcus faecalis alone; and the DON is used together with the glycyrrhizic acid, the candida utilis and the enterococcus faecalis, so that the negative influence of the DON on the growth performance of the piglets can be relieved, the daily gain of the piglets is increased by 12.56 percent (P is less than 0.05) compared with that of the piglets treated by the DON only, and the death rate of the piglets caused by the DON can be reduced.

In summary, GA can inhibit the production of inflammatory factors and chemotactic factors, remarkably slow down oxidative stress and reduce necrotic apoptosis, and has a certain immunoregulation function; therefore, when cells are affected by DON, the glycyrrhizic acid can be added to relieve DON-induced intestinal inflammatory reaction and improve pathological activity caused by DON. The glycyrrhizic acid and the probiotics can relieve the harm of DON on cell activity and piglet growth performance; therefore, the glycyrrhizic acid and the probiotics are combined to be expected to become a new feed additive, so that the toxicity of DON can be reduced, and the intestinal immunity and the production efficiency of animals can be improved.

Claims (4)

1. The application of a feed additive for relieving vomitoxin harm in preparing a medicine for relieving piglet growth performance reduction caused by vomitoxin comprises glycyrrhizic acid, saccharomycetes and enterococcus.

2. The use of claim 1, wherein: the additive amount of the feed additive is 200-400 mg/kg of glycyrrhizic acid feed and 0.5 multiplied by 10 of microzyme ⁹ ~5×10 ⁹ CFU/kg fodder, enterococcus 0.5 × 10 ⁹ ~5×10 ⁹ CFU/kg feed.

3. Use according to claim 2, characterized in that: the feed additive is prepared from glycyrrhizic acid 400mg/kg feed, yeast 1 × 10 ⁹ CFU/kg fodder, enterococcus 1X 10 ⁹ CFU/kg feed.

4. The use of claim 1, wherein: the yeast is candida utilis; the enterococcus is enterococcus faecalis.

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