CN108866145A - Saccharomyces cerevisiae and DON interaction reduce detecting step and its application of IPEC-J2 Apoptosis - Google Patents

Abstract

The invention proposes the detection steps and application of Saccharomyces cerevisiae and DON for reducing the apoptosis of IPEC-J2 cells, and the problem of using Saccharomyces cerevisiae to repair pig intestinal epithelial cell damage. The application of the interaction between Saccharomyces cerevisiae and DON (vomitoxin) to reduce the apoptosis of IPEC-J2 cells has a slowing effect on DON-induced inflammation and necrosis of porcine intestinal epithelial cells. In this application, Saccharomyces cerevisiae (MOI=1) was co-cultured with DON, which promoted cell proliferation to a certain extent, alleviated the damage of DON to pig intestinal epithelial cells, and protected the integrity of intestinal cell membranes; when the concentration of DON was 1.2 At the concentration of μg/mL, the addition of Saccharomyces cerevisiae could significantly increase the proliferation rate of porcine intestinal epithelial cells and the expression of IL-10 anti-inflammatory factors, and significantly reduce the apoptosis rate and necrosis rate of cells, indicating that Saccharomyces cerevisiae can inhibit DON-induced Cytopathy has a certain relieving effect.

Description

The detection steps of the interaction between Saccharomyces cerevisiae and DON to reduce the apoptosis of IPEC-J2 cells and its application

technical field

The invention relates to the field of molecular biology, in particular to a detection step for reducing IPEC-J2 cell apoptosis through the interaction between Saccharomyces cerevisiae and DON and its application.

Background technique

Vomotoxin, also known as deoxynivalenol (DON), is one of the most common mycotoxins in grains and foods worldwide. A series of poisoning symptoms have been caused by humans and animals ingesting food containing DON.

Porcine intestinal epithelial cells (IPEC-J2) are the first physical barrier against external stimuli, and DON is the first recognized toxin that has pro-inflammatory and immunomodulatory effects on intestinal epithelial cells, so this test aims to screen out The DON-degrading bacterial strain was constructed to construct the DON-IPEC-J2 cell model, and the degrading effect of the screened microorganisms on DON and the alleviation of the toxin's effect on cell proliferation were studied.

DON is the first recognized toxin with pro-inflammatory and immunomodulatory effects on intestinal epithelial cells. The toxic effect of DON on cells is mainly to change cell morphology, DNA damage, inhibit protein synthesis, promote apoptosis and so on. The main mechanism of action of DON on intestinal epithelial cells is to up-regulate tight junction proteins, thereby showing up-regulation of RNA expression of related genes as a compensatory mechanism. Intestinal tests have proved that DON passes through the intestinal surface and is rapidly absorbed by intestinal epithelial cells, destroys the tight junction of intestinal epithelial cells, binds RNA peptidyl transferase, and inhibits protein synthesis. In addition, DON can promote apoptosis.

Yeast and other probiotics are widely used as feed additives, mainly to regulate the intestinal microbial flora of animals, protect the intestinal health, regulate the immune function of the body, play a direct role in the intervention of pathogenic bacteria to prevent infection of the body, and protect against pathogenic toxins. degradation etc. Probiotics can also enhance the intestinal epithelial barrier function by promoting the formation of mucus layer, secreting antibacterial factors, and enhancing the formation of tight junctions.

The invention studies the harm effect of Saccharomyces cerevisiae on alleviating the proliferation of intestinal epithelial cells by yeast at the cell level, and provides a basis for production practice.

Contents of the invention

The invention proposes the detection steps and application of the interaction between Saccharomyces cerevisiae and DON to reduce the apoptosis of IPEC-J2 cells, and solves the problem of using Saccharomyces cerevisiae to repair pig intestinal epithelial cell damage.

Technical scheme of the present invention is realized like this:

The detection steps for the interaction between Saccharomyces cerevisiae and DON to reduce the apoptosis of IPEC-J2 cells are as follows:

(1) Take the IPEC-J2 cells in the logarithmic phase, digest and count them, and inoculate them on a 6-well plate, 2 mL/well, the number of cells in each well is 5×10 ⁵ , discard the original medium after the cells are cultured to the wall , washed once with PBS;

(2) There are four groups in the detection test, which are respectively the blank control group, the Saccharomyces cerevisiae preparation group, the DON preparation group and the Saccharomyces cerevisiae preparation+DON preparation group, and each group has three repetitions;

(3) adding the four groups of preparations in step (2) to the 6-well plate equipped with IPEC-J2 cells in step (1);

(4) Place the 6-well plate treated in step (3) at 30-35° C., co-culture for 8 hours, discard the supernatant, wash twice with PBS, centrifuge at 2000 rpm for 5 minutes, and collect the cells;

(5) Add 500 μL of Binding Buffer to the cells collected in step (4) to suspend cells, then add 5 μL of Annexin V-FITC and mix well, then add 5 μL of Propidium Iodide and mix well, react at room temperature in the dark for 15 minutes, and then detect cell viability.

The yeast is Saccharomyces cerevisiae, and the preservation number is CGMCC No: 2.3866.

The concentration of DON in the step (2) is 0.075-0.15 μg/mL, and the cell number of Saccharomyces cerevisiae is the same as that of IPEC-J2 cells.

The processing of saccharomyces cerevisiae preparation in described step (2) is:

a. activating the stored Saccharomyces cerevisiae in YPD liquid medium for 24h, inoculating the activated bacterium liquid in fresh YPD liquid medium with an inoculum size of 2-5%, continuing to cultivate for 16-24h, and counting on plates, Store in refrigerator at 4°C;

b. Take the Saccharomyces cerevisiae culture medium preserved in step a, centrifuge at 8000rpm/min for 5min, discard the supernatant, pass through a 0.22μm filter membrane to sterilize, obtain bacterial cells, and store at 4°C for later use;

c. The bacterial cells after centrifugation are resuspended with DMEM/F-12 medium, and centrifuged at 8000 rpm/min for 5 minutes. After washing twice, the bacterial cells are suspended in an equal volume with DMEM/F-12 medium, namely The Saccharomyces cerevisiae preparation was obtained and stored in a 4°C refrigerator for later use.

The application of the interaction between Saccharomyces cerevisiae and DON to reduce the apoptosis of IPEC-J2 cells has a relieving effect on pig intestinal epithelial cells with inflammation induced by DON.

Each 2 mL mixture of Saccharomyces cerevisiae and DON contains 5×10 ⁵ Saccharomyces cerevisiae cells, 5×10 ⁵ porcine intestinal epithelial cells and 1.2 μg/mL DON.

Add 5-10mL of Saccharomyces cerevisiae and DON mixture per Kg of feed.

The beneficial effects of the present invention are:

(1) This application co-cultivates Saccharomyces cerevisiae (MOI=1) and DON, which promotes cell proliferation to a certain extent, can alleviate the damage of DON to pig intestinal epithelial cells, and protect the integrity of intestinal cell membranes; and DON Compared with the control group, the release of LDH was significantly reduced, and it had a good effect on the degradation of DON; when the concentration of DON was 1.2 μg/mL, the addition of S. The expression of the factor can significantly reduce the apoptosis rate and necrosis rate of cells, and at the same time reduce the expression of tight junction protein (TJP) and occludin (Occuldin) genes to a certain extent, indicating that Saccharomyces cerevisiae has a strong effect on DON-induced cell pathology. There is a certain palliative effect.

(2) In the present invention, the early apoptotic cells and late apoptotic cells of the DON group increased by 2.67 times and 4.53 times respectively (P<0.05) compared with the control group; the yeast group and the control group increased the number of early and late apoptotic cells, The number of viable cells decreased, but the difference was not significant (P>0.05).

(3) The proliferation inhibition of porcine intestinal epithelial cells (IPEC-J2) by deoxynivalenol (DON) showed a dose-time dependent effect, and the proliferation inhibition rate of PEC-J2 cells increased gradually with the increase of DON concentration and the prolongation of action time (P <0.05), and increased the release of LDH (lactate dehydrogenase) (P<0.05), indicating that DON destroys the cell membrane of IPEC-J2, increases cell permeability, and has a certain damage effect on pig intestinal epithelial cells.

(4) When Saccharomyces cerevisiae was co-cultured with DON and porcine intestinal epithelial cells, Saccharomyces cerevisiae could alleviate the inhibitory effect of DON on intestinal epithelial cell proliferation, reduce the release of lactate dehydrogenase (LDH) (P<0.05), and increase The degradation rate of DON significantly increased the expression of interleukin IL-8 (P<0.05); especially in the high-dose DON group (1.2 μg/mL), the addition of Saccharomyces cerevisiae significantly increased the expression of IL-6, IL -8. The expression of IL-10 gene (P<0.05), but there was no significant effect on the expression of tight junction protein (TJP) and occuldin (Occuldin) gene.

(5) Compared with the DON group, the number of early and late apoptotic cells in the yeast and DON co-culture group decreased by 44.78% and 46.37% (P<0.05), and the number of viable cells increased by 2.35% (P<0.05). The number decreased by 38.05% (P>0.05).

Description of drawings

Figure 1 shows the effect of different concentrations of DON on the viability and proliferation rate of IPEC-J2 cells for 24 hours.

Fig. 2 is the mRNA expression level of cytokine IL-6 by the co-culture of Saccharomyces cerevisiae and different concentrations of DON.

Fig. 3 is the expression level of cytokine IL-8 mRNA by the co-culture of Saccharomyces cerevisiae and different concentrations of DON.

Fig. 4 shows the mRNA expression level of cytokine IL-10 by the co-culture of Saccharomyces cerevisiae and different concentrations of DON.

Figure 5 shows the mRNA expression of cytokine TJP by co-culture of Saccharomyces cerevisiae and different concentrations of DON.

Fig. 6 shows the mRNA expression level of cytokine Occludin by co-culture of Saccharomyces cerevisiae and different concentrations of DON.

Figure 7 is a fluorescent double-stained two-dimensional dot diagram of the effect of co-culture of Saccharomyces cerevisiae and DON on apoptosis, wherein 7-1 is the control group, 7-2 is the DON group, 7-3 is the Saccharomyces cerevisiae group, 7-4 For Saccharomyces cerevisiae+DON group, the four quadrants represent the area ratio of cells; the abscissa is the number of IPEC-J2 cells stained with Annexin V (phospholipid-binding protein) and FITC (fluorescein isothiocyanate) fluorescently labeled (number of fluorescent channels ); the ordinate is the number of IPEC-J2 cells stained with PI (propidium iodide) and fluorescently labeled with PerCP (chlorophyll protein) (the number of fluorescent channels); where Q1 represents necrotic cells, Q2 represents late apoptotic cells, and Q3 represents viable cells. cells, Q4 represents early apoptotic cells.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

1 Materials and methods

1.1 Test material

Pig intestinal epithelial cells (IPEC-J2): donated by the Henan Provincial Key Research Laboratory of Animal-derived Food Safety, Henan Agricultural University. Saccharomyces cerevisiae is the strain preserved in this laboratory.

1.1.1 Reagents

DMEM/F-12 culture medium (Hyclone, USA); fetal bovine serum (Fetal Bovine Serum, FBS) (Hangzhou Sijiqing Bioengineering Materials Co., Ltd.); penicillin and streptomycin mixed solution, MTT, PBS buffer, 0.25% pancreatic Enzyme protease-EDTA digestion solution, 0.25% trypsin protease (Beijing Suo Lai Bao Technology Co., Ltd.); DMSO (dimethyl sulfoxide) (Sigma, the United States); Trizol, RNase-free water, reverse transcription kit, Real-time fluorescence quantification kit (Takara, Japan).

1.1.2 Main instruments and equipment (see Table 1)

Table 1 main equipment

1.1.3 Preparation of reagents

(1) Cell culture medium: 10% fetal bovine serum, 1% penicillin mixed solution and 90% DMEM/F-12 culture medium are fully mixed evenly (fetal bovine serum should be stored at -20°C, and should be stored at 4°C before use) After thawing in the refrigerator, inactivate in a water bath at 56°C for 30 minutes), and store at 4°C for later use.

(2) Cell cryopreservation medium: 40% fetal bovine serum, 10% DMSO and 50% DMEM/F-12 culture medium are mixed evenly, ready to use immediately.

(3) The preparation of YPD medium is the same as the previous chapter.

(4) Preparation of DON storage solution: Dissolve the pure DON in ethanol completely, prepare a 5 mg/mL stock solution, and store at -20°C in the dark. Dilute to 100 μg/mL with DMEM/F-12 culture medium before use, and store at 4°C for later use.

(5) Preparation of DON working solution: 100 μg/mL DON stock solution was prepared with DMEM/F-12 medium to 0, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 μg/mL The working solution was stored at 4°C in the dark for later use.

1.2 Cell culture

1.2.1 Cell recovery

Take out the IPEC-J2 cell line preserved in liquid nitrogen, quickly dissolve it in a 37°C water bath, transfer it to a cell culture medium containing 5 times the volume, centrifuge at 1000r/min for 5min, discard the supernatant, and use 1mL The complete medium containing 10% serum and 1% double antibody was evenly pipetted into a 25cm ² culture bottle, and 5mL of fresh medium was added, cultured at 37°C and 5% CO ₂ , and the time for changing the medium was based on the growth of the cells If the medium turns yellow or there are too many dead cells, replace it with fresh medium immediately. When the cells grow to 80%-90% of the flask wall, they are digested and passaged with 0.25% trypsin-EDTA digestion solution.

1.2.2 Cell culture and passage

Discard the old culture medium in the culture bottle, wash with 1mL PBS buffer solution 2-3 times respectively, add 1mL 0.25% trypsin protease-EDTA digestion solution, digest in the cell culture incubator for 3-5min, and observe the cells under an inverted microscope When the cells become round, the gaps become larger, or the cells are seen falling from the bottle wall like fine sand against a place with light, immediately add 1mL medium to stop the digestion; use a pipette to repeatedly blow the cells on the bottle wall, Form a cell suspension, centrifuge the suspension at 1000r/min for 5min, discard the supernatant, add medium to suspend the cells, passage according to 1:2 or 1:3, make up the medium to 5mL, 37℃, 5 Cultured under the condition of %CO ₂ , the medium was changed every other day, and the morphology of the cells was observed.

1.2.3 Cell cryopreservation

Discard the old culture medium in the culture bottle, wash with 1mL PBS buffer solution 2-3 times, add 1mL 0.25% trypsin protease-EDTA digestion solution, digest in the cell culture incubator for 3-5min, then add 1mL complete medium To terminate the digestion, pipette the cells on the bottle wall repeatedly to form a cell suspension, centrifuge the suspension at 1000r/min for 5min, discard the supernatant, add the cryopreservation solution, count the cell concentration on a hemocytometer Adjust to 1-3×10 ⁶ cells/mL, transfer to cryopreservation tubes, label, pre-cool at 4°C for 20 minutes, store at -20°C for 1 hour, then transfer to -80°C to freeze overnight, the next day Transfer to liquid nitrogen for long-term storage.

1.3 Activation and cultivation of strains

Activate the preserved Saccharomyces cerevisiae in YPD liquid medium for 24 hours respectively, re-insert the activated bacterial solution into fresh medium according to the inoculum size of 5% and 2% respectively, and continue to culture for 16 hours and 24 hours respectively, and count on plates, 4°C Store in the refrigerator for later use.

1.4 Establishment of IPEC-J2-DON cell proliferation model

1.4.1 Effect of DON on the proliferation of IPEC-J2 cells

Cells in the logarithmic growth phase were taken, routinely digested and counted, inoculated into 96-well plates, 100 μL/well, the number of cells per well was ¹ ×104, and the original medium was discarded after the cells were cultured for 24 hours, washed once with PBS, respectively. Add different concentrations of DON (0, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 μg/mL) in serum-free, double-antibody-free DMEM/F-12 medium, and make 6 wells for each concentration , repeated 3 times, the control group contained the same volume of ethanol as the DON group, and the group containing only medium and no cells was used as the zero-adjustment group, and the cell morphology was observed under the microscope when cultured to 12h, 24h, and 48h respectively, and then each well was Add 10 μL of MTT (concentration: 5 mg/mL) and incubate together in a 37°C, 5% _CO2 incubator for 4 hours, carefully suck off the culture medium, add 150 μL of DMSO and shake on a shaker for 10 minutes to fully dissolve the formazan. Measure the absorbance value (OD value) at a wavelength of 450nm with a microplate reader, express the cell viability with the OD value, and calculate the cell survival rate according to the OD value, the formula is as follows:

Cell survival rate=(OD value of test group-OD value of zeroing group)/(OD value of control group-OD value of zeroing group)×100%

1.4.2 Effects of different DON concentrations at different times on the proliferation of IPEC-J2 cells

Cells in the logarithmic growth phase were taken, routinely digested and counted, inoculated into 96-well plates, 100 μL/well, the number of cells per well was ¹ ×104, and the original medium was discarded after the cells were cultured for 24 hours, washed once with PBS, respectively. Serum-free and double-antibody-free DMEM/F-12 medium with different concentrations of DON (0, 0.075, 0.15, 0.3, 0.6, 1.2 μg/mL) was added, and six replicate wells were made for each concentration, repeated three times. Containing the same volume of ethanol as that of the DON group, using only culture medium and no cells as the zeroing well, cultured to 4h, 8h, 16h, and 32h respectively, observed the morphology and structure of the cells under a microscope, and then added 10μL MTT (5mg /mL) to detect cell viability.

1.5 Effect of Saccharomyces cerevisiae on the proliferation of IPEC-J2 cells

1.5.1 Treatment of Saccharomyces cerevisiae

Take the cultured yeast, centrifuge at 8000rpm/min for 5min, absorb the supernatant, pass through a 0.22μm filter membrane to sterilize, and store at 4°C for later use; the centrifuged bacterial cells are resuspended in DMEM/F-12 medium The cells were centrifuged at 8000rpm/min for 5min, washed twice in this way, and then suspended in an equal volume with DMEM/F-12 medium, and stored in a 4°C refrigerator for later use.

1.5.2 Effect of Saccharomyces cerevisiae on the viability of IPEC-J2 cells

Cells in the logarithmic growth phase were taken, routinely digested and counted, and inoculated into a 96-well plate, 100 μL/well, the number of cells per well was ¹ ×104, and the original medium was discarded after the cells were adhered to the wall for 24 hours, washed once with PBS, and tested Grouped as follows:

(1) Control group: DMEM/F-12 medium group;

(2) Yeast fermentation liquid (S-FL group): 5 μL of yeast fermentation liquid was added to each well, so that the MOI ratios of yeast and cells were 0.1, 1, and 10, respectively, that is, the yeast in each well was 1×10 ³ , 1 ×10 ⁴ , 1×10 ⁵ cells, the remaining 95 μL was supplemented with DMEM/F-12 medium;

(3) Yeast supernatant (S-CFS) group: Add 5 μL of the supernatant of the yeast culture solution with the same number of viable bacteria as in the previous step to each well, and supplement the remaining 95 μL with DMEM/F-12 medium;

(4) Yeast somatic cell (SC) group: Add 100 μL of yeast diluted with DMEM/F-12 to different numbers of viable cells in each well, so that the number of yeast cells in each well is 1×10 ³ and 1×10 ⁴ , 1×10 ⁵ , that is, the MOI ratios are 0.1, 1, and 10, respectively.

According to the above grouping, each group made 6 duplicate wells, repeated 3 times, cultured for 1h, 2h, 4h, 8h respectively, washed 2-3 times with PBS buffer to wash off the added bacteria, and then added 100μL to each well DMEM/F-12 medium with a final concentration of 0.5mg/mL MTT, incubate at 37°C in a 5% CO ₂ incubator for 4 hours. After 4 hours, carefully suck off the culture solution, add 150 μL of DMSO and shake on a shaker for 10 minutes to make the Formazan was fully dissolved, and its OD value was measured at a wavelength of 450 nm with a microplate reader.

1.5.3 Effect of Saccharomyces cerevisiae supernatant on the viability of IPEC-J2 cells

Centrifuge the fermentation broth of Saccharomyces cerevisiae cultured for 24 hours at 8000rpm/min for 5min, collect the supernatant, dilute it with DMEM/F-12 medium, and dilute to 2, 4, 8, 16, 32, 64, 128 times of the original solution respectively . Cells in the logarithmic growth phase were taken, routinely digested and counted, and inoculated into 96-well plates, 100 μL/well, the number of cells per well was ¹ ×104, the original medium was discarded after the cells were cultured for 24 hours, and washed once with PBS, every Add 100 μL of the above supernatant dilution to the wells, make 6 replicate wells for each treatment, repeat 3 times, and culture until 1h, 2h, 4h, 8h respectively, and measure the cell viability by MTT.

1.6 Effect of co-cultivation of Saccharomyces cerevisiae and DON on the proliferation of IPEC-J2 cells

1.6.1 Effect of co-cultivation of Saccharomyces cerevisiae and DON on the viability of IPEC-J2 cells

Take the cells in the logarithmic growth phase, routinely digest and count them and inoculate them into 96-well plates, 100 μL/well, the number of cells per well is 1×104, discard the original medium after the cells adhere to the wall for 24 hours, wash once with PBS, and divide into groups Same as 1.7. Each group had 6 replicate wells, repeated 3 times, cultured for 4h and 8h respectively, washed 2-3 times with PBS buffer until the added bacteria were washed away, and then added 100 μL of DMEM with a final concentration of 0.5 mg/mL MTT to each well /F-12 medium, 37°C, 5% CO ₂ incubator and incubate together for 4 hours. After 4 hours, carefully absorb the culture solution, add 150 μL of DMSO and shake on the shaker for 10 minutes to fully dissolve formazan. Measure its OD value at a wavelength of 450nm.

1.6.2 Effects of co-cultivation of Saccharomyces cerevisiae and DON on the release of lactate dehydrogenase and the residual amount of DON in IPEC-J2 cells

Take the cells in the logarithmic growth phase, routinely digest and count them, inoculate them into 6-well plates, 2 mL/well, the number of cells per well is ² ×105, discard the original medium after the cells are cultured to the wall, wash once with PBS, and divide into groups Same as 1.8.1, with 3 repetitions in each group. After co-cultivation for 8 hours, pipette the culture solution of each well into a 2mL centrifuge tube, centrifuge at 3000rpm/min for 5min, re-absorb 120μL of the supernatant and place it in a new centrifuge tube. Hydrogenase (LDH) kit steps to measure the release of LDH, take 500 μL to measure the content of DON, and calculate the degradation rate of DON.

1.7 Effect of co-cultivation of Saccharomyces cerevisiae and DON on the expression of IPEC-J2 cytokine genes

Take the cells in the logarithmic growth phase, routinely digest and count them, inoculate them into 6-well plates, 2 mL/well, and the number of cells per well is 2×10 ⁵ , discard the original medium after the cells are cultured to the wall, wash once with PBS, and divide into groups Same as 1.8.1, 3 replicates in each group, after co-cultivation for 8 hours, aspirate the supernatant, wash 2-3 times with PBS, and collect the cells.

1.7.1 Cell RNA extraction

(1) Add 1mL trizol to each well, pipette 3-5 times to fully lyse the cells, and place at room temperature for 5 minutes;

(2) Add 200 μL of chloroform to the lysate, shake vigorously by hand for 15 seconds, place at room temperature for 3 minutes, and centrifuge at 12,000×g for 15 minutes at 4°C;

(3) Carefully transfer the supernatant (~300 μL) to a new 1.5 mL centrifuge tube, add 600 μL of isopropanol, vortex to mix, let stand at room temperature for 10 min, and centrifuge at 12000×g for 10 min at 4°C to precipitate RNA;

(4) Discard the supernatant, add 1 mL of 75% ethanol, vortex and mix well, and centrifuge at 7500×g for 5 min at 4°C;

(5) Discard the supernatant, turn the centrifuge tube upside down on clean absorbent paper to absorb the remaining liquid, and air dry for 10-15 minutes.

(6) Add 20 μL of RNase-free water to the RNA pellet, vortex to resuspend the RNA pellet, place on ice for 10-30 minutes to fully dissolve the RNA, and store at -80°C for later use;

(7) Take 1 μL RNA stock solution to detect its concentration.

1.7.2 Synthesis of cDNA

The extracted RNA was reverse-transcribed into cDNA, and the reverse-transcription operation was carried out according to Bao Biological Engineering (Dalian) Co., Ltd. The reverse transcription kit was carried out according to the instructions of the RT reagent Kit, and the reverse-transcribed cDNA was stored at -20°C.

1.7.3 Real-time fluorescent quantitative PCR detection

(1) Primer design

Quantitative primers (Table 2) were designed with Primer Premier 5.0 software according to the porcine gene mRNA sequences already available on Genbank, and the primers were synthesized by Shanghai Bioengineering Technology Co., Ltd.

(2) Reaction system

According to the instructions of Baobio PrimeScriptTM RT reagent Kit Perfect Real Time kit, the reaction system is shown in Table 3.

(3) Reaction program: The reaction system was carried out on a Bio-Rad iQ5 fluorescent quantitative PCR instrument, and the reaction program is shown in Table 4.

Table 2 Fluorescent quantitative primers

Table 3 RT-PCR reaction system (μL)

Table 4 RT-PCR reaction program

1.8 Effect of co-culture of Saccharomyces cerevisiae and DON on apoptosis of IPEC-J2 cells

Cells in the logarithmic growth phase were taken, routinely digested and counted, and inoculated into a 6-well plate, 2 mL/well, the number of cells per well was 5×10 ⁵ , and the original medium was discarded after the cells were cultured until adherent, and washed once with PBS. The experiment was divided into control group, Saccharomyces cerevisiae group (with cell ratio of 1:1), DON added group (1.2 μg/mL), Saccharomyces cerevisiae+DON group, and each group had 3 repetitions. After co-cultivation for 8 hours, remove the supernatant, wash 2-3 times with PBS, digest with EDTA-free trypsin for 3 minutes, centrifuge at 1000 rpm for 5 minutes, wash twice with PBS, and centrifuge at 2000 rpm for 5 minutes to collect cells. Add 500 μL of Binding Buffer to suspend the cells, add 5 μL of AnnexinV-FITC and mix well, add 5 μL of Propidium Iodide and mix well, react at room temperature in the dark for 15 minutes, and then test on the machine.

1.9 Data Analysis

The test results were expressed as mean ± standard error, and SPSS 20.0 was used for analysis of variance and multiplicity test, and Turkey method was used for significant comparison, and P<0.05 indicated a significant difference. The data of the qPCR test results were analyzed and calculated by the comparative ct (2- ^△△Ct method) method of Bio-Rad CFX96 software.

2 result analysis

2.1 Effects of different DON concentrations on the viability of IPEC-J2 cells

The effect of different DON concentrations on IPEC-J2 cells after 24h on cell viability is shown in Figure 1. It can be seen from Figure 1 that the effect of DON on IPEC-J2 cells was dose-dependent, and the cell viability decreased significantly with the increase of DON concentration (P<0.05). When the DON concentration was greater than 0.075μg/mL, the cell proliferation rate decreased significantly (P<0.05). When the concentration of DON is 0.075μg/mL-19.2μg/mL, the inhibition rate of cell proliferation is 9.49% (P>0.05) and 16.19%, 22.74%, 36.18%, 38.96%, 40.46%, 43.00%, 46.70%, 50.94% (P<0.05).

2.2 Effects of different DON concentrations and different time on the viability of IPEC-J2 cells

It can be seen from Table 5 that at 2 hours, all DON concentrations were not significantly different from those in the control group (P>0.05). At 4 hours, the DON concentration was 0.075μg/mL and 0.15μg/mL and the control group had no significant difference (P>0.05); the DON concentration was 0.3μg/mL, 0.6μg/mL, 1.2μg/mL and the control group was significantly different Significantly (P<0.05), there was no significant difference among the concentrations of DON (P>0.05). At 8 hours, the DON concentration of 0.075μg/mL was not significantly different from the control group (P>0.05), and the other four concentrations were significantly different from the control group (P<0.05). At 16h and 32h, the five DON concentrations all significantly reduced cell viability (P<0.05), and the DON concentrations of 0.15μg/mL, 0.3μg/mL, 0.6μg/mL and 1.2μg/mL were significantly different at 32h (P<0.05). <0.05). Cell growth was dose-time dependent, the same toxin concentration, cell proliferation rate decreased with time; at the same time, cell proliferation rate decreased with time. Compared with the control group, the cell proliferation rates were 89.31% (P>0.05), 87.83% (P>0.05) and 86.53%, 85.00%, 85.78% (P<0.05) when treated with different concentrations of DON for 4 hours; Proliferation rates were 95.25% (P>0.05) and 82.27%, 89.01%, 82.12%, 78.11% (P<0.05); 16h cell proliferation rates were 88.42% (P<0.05), 91.14% (P>0.05) And 77.74%, 69.62%, 69.64% (P<0.05); 32h cell proliferation rate were 94.45%, 88.83%, 80.42%, 72.10%, 64.56% (P<0.05).

Table 5 Effects of different DON concentrations at different times on IPEC-J2 cell viability (OD value) (n=6)

Note: The uppercase English letters indicate the differences in the viability of IPEC-J2 cells treated with different DON concentrations for the same time; the lowercase letters indicate the differences in the viability of IPEC-J2 cells treated with the same DON concentration for different times. The same below.

2.3 Effect of Saccharomyces cerevisiae on the proliferation of IPEC-J2 cells

2.3.1 Effects of different components of Saccharomyces cerevisiae on the proliferation of IPEC-J2 cells

It can be seen from Table 6 that when the MOI is 0.1 and 1, the yeast supernatant and fermentation broth have no significant effect on the proliferation of cells at various time periods (P>0.05); at 8 hours, the yeast cells with MOI of 1 and 10 respectively There was no significant difference between the cell addition groups (P>0.05), but the cell viability increased by 25.71% (P>0.05) and 37.14% (P<0.05) respectively compared with the control group.

2.3.2 Effect of cultured supernatant of Saccharomyces cerevisiae on the proliferation of IPEC-J2 cells

As can be seen from Table 7, the Saccharomyces cerevisiae supernatant dilution was not significantly different from the control group at 1h, 2h, and 4h (P>0.05), and at 8h, when the supernatant was diluted 2 times, it significantly inhibited cell proliferation (P<0.05 ), the cell proliferation inhibition rate was 30.77% (P<0.05).

Table 6 Effects of different components of Saccharomyces cerevisiae on IPEC-J2 cell viability (OD value) (n=6)

The influence (n=6) of the supernatant liquid that table 7 Saccharomyces cerevisiae cultured on IPEC-J2 cell viability (OD value)

2.3.3 The effect of Saccharomyces cerevisiae interfering with DON on the proliferation of IPEC-J2 cells

It can be seen from Table 8 that at 4 hours, compared with the control group, the cell viability of the Saccharomyces cerevisiae addition group increased by 7.69% (P>0.05). increased by 21.05% (P<0.05); at 8 hours, the cell viability of the yeast addition group increased by 44.83% (P<0.05) compared with the control group, when the DON concentration was 0.075 μg/mL, 0.15 μg/mL, 0.3 μg/mL, At 0.6 μg/mL and 1.2 μg/mL, the cell viability increased by 29.63%, 11.11%, 20.83%, 4%, and 4.17% in the yeast-supplemented group compared with the non-yeast-supplemented group, but the differences were not significant (P>0.05 ).

The influence of table 8 Saccharomyces cerevisiae and DON co-culture on IPEC-J2 cell viability (OD value) (n=6)

2.3.4 Effects of co-culture of Saccharomyces cerevisiae and DON on the release of LDH and the residual amount of DON

It can be seen from Table 9 that when the concentration of DON is greater than 0.075 μg/mL, the release of LDH is significantly increased (P<0.05). %, 182.14%, 167.86% (P<0.05). Compared with the control group, Saccharomyces cerevisiae could reduce the release of LDH, but the difference was not significant (P>0.05); compared with the DON group, the release of LDH in the yeast and DON groups decreased by 26.67% (P>0.05), 66.67%, and 69.86% respectively , 72.15%, 68.00% (P<0.05). There was no significant difference in the concentration of DON between the yeast and DON addition group and the corresponding DON addition group (P>0.05), but they all reduced the concentration of DON to a certain extent, and the degradation rates were 57.14%, 46.15%, 42.31%, 26.67%, 13.51% (P>0.05).

Table 9 Effects of Saccharomyces cerevisiae and different concentrations of DON co-cultured on the release of LDH and the residual amount of DON in cells (n=3)

2.3.5 Effects of co-culture of Saccharomyces cerevisiae and DON on the expression of related genes in IPEC-J2 cells

See Figure 2 for the expression levels of cytokine mRNAs for Saccharomyces cerevisiae co-cultured with different concentrations of DON. It can be seen from Figure 2 that when the DON concentration is 1.2 μg/mL, compared with the control group, the gene expression of IL-6 is up-regulated by 67% (P<0.05), and other concentrations of DON have no significant effect on the expression of IL-6 (P>0.05); Yeast and 0.3, 0.6, 1.2 μg/mL DON co-supplemented group respectively increased the expression of IL-6 by 50.89%, 123.88%, 110.78% compared with the corresponding DON supplemented group (P<0.05) . It can be seen from Figure 3 that when the concentration of DON was 0.075 μg/mL and 0.3 μg/mL, the expression of IL-8 was down-regulated by 46% (P<0.05) and 36% (P<0.05); Compared with the DON group, the expression of IL-8 was significantly up-regulated, and the up-regulation rates were 83.33%, 73.44%, 59.22%, 153.33%, and 221.55% (P<0.05). It can be seen from Figure 4 that DON at different concentrations had no significant effect on the expression of IL-10 compared with the control group (P>0.05); The expression of IL-10 was up-regulated by 36.81 (P<0.05). It can be seen from Figure 5 that with the increase of DON concentration, the expression of TJP showed an increasing trend. Compared with the control group, the up-regulation rates of TJP expression were 18% (P>0.05) and 55%, 91%, and 80%. , 116% (P<0.05); yeast and DON co-culture group down-regulated the expression of TJP compared with DON group, when the DON concentration was 0.3μg/mL, the down-regulation rate of TJP was 36.13% (P<0.05). It can be seen from Figure 6 that the expression of Occludin was up-regulated in the DON addition group. Compared with the control group, the up-regulation rates of Occludin were 48% (P>0.05), 44% (P>0.05), and 175% (P<0.05) , 100% (P<0.05), 150% (P<0.05); the yeast and DON co-culture group all down-regulated the expression of Occludin compared with the DON group, and the down-regulation rates were 35.81% (P<0.05), 27.78% (P>0.05), 50.18% (P<0.05), 19%, 18.4% (P>0.05).

2.3.6 The effect of co-culture of Saccharomyces cerevisiae and DON on the apoptosis of IPEC-J2 cells

It can be seen from Figure 7 and Table 10 that the proportion of late apoptotic cells in the DON addition group was significantly increased compared with the control group (P<0.05), which was 4.53 times that of the control group, and the early apoptotic cells were 2.23 times that of the control group (P<0.05), the number of dead cells was 2.83 times that of the control group (P<0.05); compared with the DON group, the early and late apoptotic cells in the yeast and DON co-culture group decreased by 44.78% and 46.37% respectively (P< 0.05), the number of living cells increased by 2.35% (P<0.05), and the number of necrotic cells decreased by 38.05% (P>0.05); compared with the yeast group and the control group, there was no significant difference in each parameter (P>0.05).

Table 10 Saccharomyces cerevisiae and the influence of DON co-culture on IPEC-J2 proliferation and apoptosis (%)

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (6)

1. Saccharomyces cerevisiae and DON interaction reduce the detection step of IPEC-J2 cell apoptosis, it is characterized in that, the step is: (1) Take the IPEC-J2 cells in the logarithmic phase, digest and count them, and inoculate them on a 6-well plate, 2 mL/well, the number of cells in each well is 5×10 ⁵ , and discard the original culture after the cells are cultured to the wall base, washed once with PBS; (2) There are four groups in the detection test, which are blank control group, Saccharomyces cerevisiae preparation group, DON preparation group and Saccharomyces cerevisiae preparation+DON preparation group, with three repetitions in each group; (3) Add the four groups of preparations in step (2) to the 6-well plate containing IPEC-J2 cells in step (1); (4) Place the 6-well plate treated in step (3) at 30-35°C, co-culture for 8 h, discard the supernatant, wash twice with PBS, centrifuge at 2000 rpm for 5 min, and collect the cells; (5) Add 500 μL of Binding Buffer to the cells collected in step (4) to suspend the cells, then add 5 μL of Annexin V-FITC and mix well, then add 5 μL of Propidium Iodide and mix well, react at room temperature for 15 minutes in the dark Cell Viability Assay. 2. The detection step for the interaction between Saccharomyces cerevisiae and DON to reduce the apoptosis of IPEC-J2 cells according to claim 1, characterized in that: the concentration of DON in the step (2) is 0.075-0.15 μg/mL, Saccharomyces cerevisiae The number of cells was the same as that of IPEC-J2 cells. 3. The detection step for the interaction between Saccharomyces cerevisiae and DON to reduce the apoptosis of IPEC-J2 cells according to claim 1, characterized in that, the treatment of Saccharomyces cerevisiae preparation in the step (2) is: a. Activate the preserved Saccharomyces cerevisiae in YPD liquid medium for 24 h, inoculate the activated bacterial liquid into fresh YPD liquid medium at an inoculation amount of 2-5%, continue to cultivate for 16-24 h, and smear the plate Count and store in a refrigerator at 4°C; b. Take the Saccharomyces cerevisiae culture medium preserved in step a, centrifuge at 8000 rpm/min for 5 min, discard the supernatant, pass through a 0.22 μm filter membrane to sterilize, obtain bacterial cells, and store at 4°C for later use; c. The bacterial cells after centrifugation were resuspended with DMEM/F-12 medium, centrifuged at 8000 rpm/min for 5 minutes, and after washing twice, the bacterial cells were suspended in an equal volume with DMEM/F-12 medium. The Saccharomyces cerevisiae preparation was obtained and stored in a 4°C refrigerator for later use. 4. Saccharomyces cerevisiae as described in any one of claim 1-3 interacts and reduces the application of IPEC-J2 cell apoptosis, it is characterized in that: use Saccharomyces cerevisiae to relieve the porcine intestinal epithelial cells with inflammation induced by DON . 5. Saccharomyces cerevisiae as claimed in claim 4 interacts with DON to reduce the application of IPEC-J2 cell apoptosis, it is characterized in that: every 2 mL of Saccharomyces cerevisiae and DON mixture contains ⁵ * 10 Saccharomyces cerevisiae cells, 5 * 10 ⁵ porcine intestinal epithelial cells and 1.2μg/mL DON. 6. The application of Saccharomyces cerevisiae and DON interaction to reduce IPEC-J2 cell apoptosis as claimed in claim 5, characterized in that: 1-10 mL of Saccharomyces cerevisiae and DON mixture is added in every Kg feed.