CN113181221A - Application of selenium-enriched lactic acid bacteria in relieving barrier damage of chicken small intestine caused by aflatoxin - Google Patents
Application of selenium-enriched lactic acid bacteria in relieving barrier damage of chicken small intestine caused by aflatoxin Download PDFInfo
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- CN113181221A CN113181221A CN202110503113.3A CN202110503113A CN113181221A CN 113181221 A CN113181221 A CN 113181221A CN 202110503113 A CN202110503113 A CN 202110503113A CN 113181221 A CN113181221 A CN 113181221A
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Abstract
The invention discloses application of selenium-enriched lactic acid bacteria in relieving barrier damage of chicken small intestine caused by aflatoxin; the relieving of the barrier damage of the chicken small intestine caused by the aflatoxin refers to the following steps: the selenium-enriched lactic acid bacteria inhibit the aflatoxin to cause the protein level of chicken small intestine epithelial cell tight junction protein Occludin to be reduced. The experimental results show that: for chicken small intestine epithelial cells, 0.01-0.1 mu mol/L selenium-enriched lactic acid bacteria is used, and the decrease of the level of Ocplus protein of the chicken small intestine epithelial cells caused by aflatoxin can be inhibited when the concentration of the aflatoxin is 200 mu mol/L and 300 mu mol/L, so that the effect of weakening the barrier damage of the aflatoxin to the chicken small intestine intestinal tract is achieved, and technical support is provided for the application of the selenium-enriched lactic acid bacteria in the aspects of preventing and treating aflatoxin poisoning, particularly relieving the barrier damage of the intestinal tract and maintaining the intestinal tract function.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a cell engineering technology, and particularly relates to an application and a method of selenium-enriched lactic acid bacteria for inhibiting barrier damage of chicken small intestine caused by aflatoxin.
Background
The toxic effects of Aflatoxin (AF) mainly include: growth and development toxicity, hepatotoxicity, immunotoxicity and genetic toxicity, wherein AF causes damage to the liver and kidney of an organism and also generates corresponding toxic action on intestinal tracts; the gut is the initial barrier to mycotoxins and barrier protection of the intestinal mucosa is critical to gut health.
Selenium is used as a necessary trace element of an organism, has multiple biological functions of resisting oxidation, improving the immunity and disease resistance of the organism, regulating the metabolism of the organism, influencing the breeding function of animals and people, resisting tumor, delaying aging, antagonizing toxic elements and the like, and has certain improvement effects on intestinal flora imbalance and intestinal inflammation improvement. The lactobacillus can convert inorganic selenium into organic selenium through the biological action in vivo and can be prepared into selenium-enriched lactobacillus, which has antagonistic action on the toxicity of AF and can also inhibit the growth of aspergillus flavus.
The normal intestinal barrier plays an important role in maintaining homeostasis in the body and the external environment. Tight junctions are complexes of various proteins that are critical to the permeability, nature and function of the intestinal epithelial barrier and are of great importance to the transport, absorption and utilization of nutrients. Tight junction protein is important for maintaining the intestinal epithelial structure, can close the intercellular space and prevent harmful substances from entering the body, and is a mechanical barrier for regulating the permeability of epithelial cells; tight junction proteins not only play a role in regulating barriers, but also participate in regulating cell morphology, cell polarity, and the like. Occludin (Occupudin) is an important transmembrane tight junction protein and has close relation with epithelial mechanical barriers; if the Occludin protein level is reduced, the intestinal barrier is damaged, and the toxin enters the blood circulation through the intestinal cavity. Research finds that AF can reduce Occludin protein level, increase intestinal tract cell bypass permeability, damage the mechanical barrier of the intestinal tract and further influence the normal function of the intestinal tract.
The selenium-enriched lactic acid bacteria are used for inhibiting the decrease of the protein level of chicken small intestine epithelial cell tight junction protein Occludin caused by AF, and the effects of resisting the damage of AF to an intestinal barrier and maintaining the normal function of an intestinal tract are achieved. At present, no patent publication documents or reports on the aspect of inhibiting barrier injury of chicken small intestine intestinal tracts caused by AF by selenium-enriched lactic acid bacteria are found at home and abroad.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides the application and the method of the selenium-enriched lactic acid bacteria for relieving the barrier damage of the chicken small intestine caused by AF, and provides technical support for the application of the selenium-enriched lactic acid bacteria in preventing and treating chicken AF poisoning, particularly relieving the barrier damage of the intestinal tract and maintaining the intestinal function.
In order to achieve the purpose, the invention discloses the following technical contents:
application of selenium-rich lactobacillus in relieving barrier injury of chicken small intestine caused by AF; the relieving of the barrier damage of the chicken small intestine caused by AF refers to the following steps: the selenium-enriched lactic acid bacteria inhibit the decrease of the level of the chicken small intestine epithelial cell tight junction protein Occludin protein caused by AF.
The application of the invention is characterized in that: for the chicken small intestine epithelial cells, when the AF concentration is 200-300 mu mol/L, 0.01-0.1 mu mol/L selenium-enriched lactic acid bacteria (the final concentration in a culture solution is calculated by selenium) are used, the decrease of the level of Occludin in the chicken small intestine epithelial cells caused by AF can be inhibited, and therefore the effect of weakening the damage of AF to the intestinal barrier of the chicken small intestine is achieved. The experimental results show that: for the chicken small intestine epithelial cells, 0.01-0.1 mu mol/L selenium-enriched lactic acid bacteria (the final concentration in a culture solution is calculated by selenium) is used, and the concentration of AF is 200-300 mu mol/L, so that the decrease of the level of Occludin protein of the chicken small intestine epithelial cells caused by AF can be inhibited, the effect of AF on the barrier damage of the chicken small intestine is weakened,
the invention further discloses a method for relieving barrier injury of chicken small intestine caused by AF by selenium-enriched lactic acid bacteria, which comprises the following steps:
for the chicken small intestine epithelial cells, selenium-enriched lactic acid bacteria with selenium concentration of 0.01-0.1 mu mol/L (final concentration in culture solution) are added, after 6-12h of culture, AF is added into the chicken small intestine epithelial cells to enable the concentration to be 200-300 mu mol/L, after 12-24h of continuous culture, the culture is finished, the decrease of the level of Occludin in the chicken small intestine epithelial cells caused by AF can be inhibited, and therefore the effect of AF on the damage of the chicken small intestine intestinal barrier is weakened.
The invention also discloses a verification method for relieving the barrier damage of the chicken small intestine caused by AF by the selenium-enriched lactic acid bacteria, which comprises the following steps:
s1: inoculating chicken small intestine epithelial cells into a six-hole plate, and adjusting the cell concentration to 2 × 105Per well; the test is divided into a control group, an AF toxicity counteracting group and a selenium-enriched lactobacillus group; replacing the culture solution when the cells are converged to 60-70%; replacing the control group and AF challenge group with common culture solution, and replacing the selenium-rich lactobacillus group with culture solution containing selenium-rich lactobacillus with selenium concentration of 0.01-0.1 μmol/L; continuously culturing for 8-12h, taking out the six-hole plate, adding AF into the AF challenge group and the selenium-rich lactobacillus group to ensure that the concentration of AF is 200-;
s2: the change of Occludin protein level was determined by Western blotting:
extracting total protein of chicken small intestine epithelial cell, determining protein concentration by BCA method, adding 5 × buffer, boiling denatured protein at 100 deg.C; proteins were separated by SDS-PAGE, transferred to PVDF membrane, primary antibody was routinely incubated and Occludin protein levels were determined by ImageLab visualization.
The specific steps of step S2 are as follows:
(1) protein extraction
The cells were washed 3 times with pre-cooled PBS, 150 μ L of RIPA cell lysate containing 1mM PMSF was added to each well, after the cells were fully contacted, the cells were scraped off and transferred to a 1.5mL centrifuge tube, and the cells were continued to be lysed at 4 ℃ for 1h, and shaken 10s every 15min to fully lyse the cells. Centrifuging at 12000g at 4 deg.C for 10min, and collecting supernatant; storing at-20 deg.C;
(2) BCA method for determining protein concentration
Diluting the protein standard substance in the kit to 0.5 mg/kg;
preparing a BCA working solution: mixing the solution A and the solution B according to a ratio of 50: 1;
adding the standard substance to a 96-well plate according to 0, 1, 2, 4, 8, 12, 16 and 20 muL, and supplementing the standard substance to 20 muL by using physiological saline; adding 2 muL of samples to be detected into a 96-well plate, and supplementing the samples to 20 muL with physiological saline;
adding 200 mu LBCA working solution into each hole, and incubating for 30min at 37 ℃;
measuring the OD value at 540nm, and calculating the protein concentration of the sample;
adding 30 mu L of 5 XLoadingBuffer into a sample to be detected, boiling for 10min to denature protein, and storing at-20 ℃ for later use;
(3) SDS-PAGE electrophoresis
Correctly assembling an electrophoresis tank, and putting prefabricated glue; pouring an electrophoresis buffer solution, removing the comb, and sequentially adding the multicolor pre-dyed protein marker and a sample to be detected; performing constant-pressure electrophoresis at room temperature of 110V until the bottom of the gel is finished;
(4) rotary film
1) Soaking the PVDF membrane in methanol for 5min, and soaking the filter paper in a membrane transfer buffer solution;
2) taking out the gel, and cutting off the gel containing the target protein by contrast with a Marker;
3) paving the filter paper-PVDF membrane-gel-filter paper in sequence, and removing bubbles to ensure that the gel is fully contacted with the membrane;
4) rotating the membrane for 45min by 15V voltage;
(5) sealing of
After the membrane transfer is finished, taking out the PVDF membrane, and washing the PVDF membrane for 3 times with TBST on a shaking table at 70 r/min, wherein each time is 5 min; preparing 5% skim milk as a sealing liquid, fully contacting the PVDF membrane with the sealing liquid, and sealing overnight at 4 ℃;
(6) binding antibodies
In combination with an antibody:
the Occludin antibody was diluted with blocking solution according to the antibody instructions at a dilution of 1: 600; the dilution of rabbit-derived beta-actin antibody is 1: 1000. Placing the sealed PVDF membrane in the diluted primary antibody, incubating at room temperature for 90min, taking out the PVDF membrane, washing with TBST for 5min each time for 3 times;
binding of secondary antibody:
according to the antibody specification, the secondary antibody is diluted by using a blocking solution, and the dilution of the goat anti-rabbit IgG antibody is 1: 1000; incubating the PVDF membrane after primary incubation and the diluted secondary antibody, incubating at room temperature for 60min, taking out the PVDF membrane, and washing with TBST for 3 times, 5min each time;
(7) color development by ECL method
And (3) taking the equal amount of the solution A and the solution B in the ECL kit, fully mixing, contacting with a PVDF membrane, and reacting for 5min at room temperature in a dark place. Images were collected with a BIO-RAD gel imager, gray scale scanned with ImageLab software, and Occludin protein levels were determined.
The beneficial effects of the selenium-enriched lactic acid bacteria disclosed by the invention on relieving the barrier injury of chicken small intestine caused by AF are as follows:
the invention provides application of selenium-enriched lactic acid bacteria in relieving barrier damage of chicken small intestine caused by AF; the experimental results show that: for the chicken small intestine epithelial cells, 0.01-0.1 mu mol/L selenium-enriched lactic acid bacteria (the final concentration in a culture solution is calculated by selenium) is used, and the decrease of the level of Occludin protein of the chicken small intestine epithelial cells caused by AF can be inhibited at the AF concentration of 200 mu mol/L and 300 mu mol/L, so that the effect of weakening AF on the barrier damage of the chicken small intestine intestinal tract is achieved, and the technical support can be provided for the application of the selenium-enriched lactic acid bacteria in the aspects of preventing and treating chicken AF poisoning, particularly relieving the barrier damage of the intestinal tract and maintaining the intestinal tract function.
The selenium-enriched lactic acid bacteria capable of relieving AF-induced damage to intestinal barriers of chickens are used for protecting the epithelial cells of the chickens simultaneously subjected to AF challenge, and test results show that compared with an AF challenge group, the selenium-enriched lactic acid bacteria can up-regulate the reduction of Occludin expression of the epithelial cells of the chickens caused by AF, so that the increase of Lactate Dehydrogenase (LDH) activity in a culture solution caused by the damage of the epithelial cells of the chickens is inhibited, the damage of AF to the intestinal cells is relieved, and the integrity of the intestinal cells is maintained (see figure 1).
The invention mainly solves the problem of how to apply the selenium-enriched lactic acid bacteria to relieve the barrier damage of the chicken small intestine caused by AF and the application; the effect of selenium-enriched lactic acid bacteria serving as substances capable of inhibiting cell damage on weakening the barrier toxicity effect of AF on chicken small intestine is mainly investigated; the main difficulty is that the time and the optimal application concentration of the selenium-rich lactic acid bacteria with the optimal protection effect on the barrier damage of the chicken small intestine caused by AF are determined; therefore, the conditions of the influence of AF on the growth of chicken small intestine epithelial cells, toxin attacking dose, attacking time, application dose and application time of selenium-enriched lactic acid bacteria and the like are examined in sequence; the final scheme determined is: culturing chicken small intestine epithelial cells for 12h, and protecting intestinal barrier with culture solution containing selenium-rich lactobacillus (final concentration of 0.01-0.1 μmol/L in culture solution, calculated as selenium) at logarithmic phase of growth; continuously culturing for 6-12h, adding 200-300 mu mol/L AF for counteracting the toxicity after the selenium-enriched lactic acid bacteria fully play a role, and counteracting the toxicity for 12-24h, wherein under the condition, the selenium-enriched lactic acid bacteria have the best effect of inhibiting the barrier damage of the chicken small intestine caused by AF.
Drawings
FIG. 1 selenium-enriched lactic acid bacteria alleviate the effect of AF on LDH activity of chicken small intestine epithelial cells; note: the difference is significant (P < 0.05) with different lower case letters; the difference of different capital letters is obvious (P is less than 0.01); the same letter or no letter represents no significant difference (P > 0.05); the same applies below;
FIG. 2 is a determination result of barrier injury of small intestine of a chicken caused by AFB1(200 mug/mL) relieved by selenium-enriched lactic acid bacteria (0.01 mug/L); wherein the upper graph is a strip graph of the change of the protein level of chicken small intestine epithelial cells Occludin caused by the selenium-enriched lactic acid bacteria (0.01 mu mol/L) to AFB1(200 mu mol/L), and the lower graph is a statistical result graph of the change of the protein level of chicken small intestine epithelial cells Occludin caused by the selenium-enriched lactic acid bacteria (0.01 mu mol/L) to AFB1(200 mu mol/L).
FIG. 3 shows the measurement results of barrier injury of chicken small intestine caused by AFB1 (300. mu. mol/L) relieved by selenium-enriched lactic acid bacteria (0.1. mu. mol/L); wherein the upper graph is a strip graph of the change of the protein level of chicken small intestine epithelial cells Occludin caused by the selenium-enriched lactic acid bacteria (0.1 mu mol/L) to AFB1(300 mu mol/L), and the lower graph is a statistical result graph of the change of the protein level of chicken small intestine epithelial cells Occludin caused by the selenium-enriched lactic acid bacteria (0.1 mu mol/L) to AFB1(300 mu mol/L).
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention. The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
Preferably, the chicken small intestine epithelial cell culture solution of the present invention is commercially available from GIBCO, and AF is commercially available from Fermentek Ltd. The selenium-enriched lactic acid bacteria are prepared by a method of clinical veterinary medicine laboratory reference documents (Yaoan, Zhao Fang hong, Liu Lina, etc. research on the growth inhibition and toxin production effects of the selenium-enriched lactic acid bacteria with different selenium concentrations [ J ] Chinese feed 2016 (12):19-21+ 26.) of Tianjin agriculture institute, and raw materials are prepared: lactobacillus (lactobacillus rhamnosus, purchased from China general microbiological culture Collection center) is stored in a laboratory, and sodium selenite can be purchased from chemical reagents institute of Tianjin; the selenium content in the prepared selenium-enriched lactic acid bacteria is 9.69 mug/mL.
The application of the selenium-enriched lactic acid bacteria in relieving the barrier damage of chicken small intestine caused by AF.
Preferably, the application is the application of the selenium-enriched lactic acid bacteria in improving the decrease of the protein level of chicken small intestine epithelial cells Occludin caused by AF.
Preferably, for the chicken small intestine epithelial cells, when the AF concentration is 200-300 mu mol/L, selenium-enriched lactic acid bacteria with the final concentration of 0.01-0.1 mu mol/L of selenium in the culture solution are used, the decrease of the level of Occludin in the chicken small intestine epithelial cells caused by AF can be inhibited, and therefore the effect of weakening the damage of AF to the chicken small intestine intestinal barrier is achieved.
The method for relieving the barrier injury of chicken small intestine caused by AF by the selenium-enriched lactic acid bacteria comprises the following steps:
for the chicken small intestine epithelial cells, selenium-enriched lactic acid bacteria with selenium concentration of 0.01-0.1 mu mol/L (final concentration in culture solution) are added, after 8-12h of culture, AF is added into the chicken small intestine epithelial cells to enable the concentration to be 200-300 mu mol/L, after 12-24h of continuous culture, the culture is finished, the decrease of the level of Occludin in the chicken small intestine epithelial cells caused by AF can be inhibited, and therefore the effect of AF on the damage of the chicken small intestine intestinal barrier is weakened.
The verification method for relieving the barrier injury of the chicken small intestine caused by AF by the selenium-enriched lactic acid bacteria comprises the following steps:
s1: inoculating chicken small intestine epithelial cells into a six-hole plate, and adjusting the cell concentration to 2 × 105Per well; the test is divided into a control group, an AF toxicity counteracting group and a selenium-enriched lactobacillus group; and (3) replacing the culture solution when the cells are converged by 60-70 percent: replacing the control group and AF challenge group with common culture solution, and replacing the selenium-rich lactobacillus group with culture solution containing selenium-rich lactobacillus with selenium concentration of 0.01-0.1 μmol/L; continuously culturing for 8-12h, taking out the six-hole plate, adding AF into the AF challenge group and the selenium-rich lactobacillus group to ensure that the concentration of AF is 200-;
s2: the change of Occludin protein level was determined by Western blotting:
extracting total protein of chicken small intestine epithelial cell, determining protein concentration by BCA method, adding 5 × buffer, boiling denatured protein at 100 deg.C; proteins were separated by SDS-PAGE, transferred to PVDF membrane, primary antibody was routinely incubated and Occludin protein levels were determined by ImageLab visualization.
The specific steps of step S2 are as follows:
(1) protein extraction
The cells were washed 3 times with pre-cooled PBS, 150 μ L of RIPA cell lysate containing 1mM PMSF was added to each well, after the cells were fully contacted, the cells were scraped off and transferred to a 1.5mL centrifuge tube, and the cells were continued to be lysed at 4 ℃ for 1h, and shaken 10s every 15min to fully lyse the cells. Centrifuging at 12000g at 4 deg.C for 10min, and collecting supernatant; storing at-20 deg.C;
(2) BCA method for determining protein concentration
Diluting the protein standard substance in the kit to 0.5 mg/kg;
preparing a BCA working solution: mixing the solution A and the solution B according to a ratio of 50: 1;
adding the standard substance to a 96-well plate according to 0, 1, 2, 4, 8, 12, 16 and 20 muL, and supplementing the standard substance to 20 muL by using physiological saline; adding 2 muL of samples to be detected into a 96-well plate, and supplementing the samples to 20 muL with physiological saline;
adding 200 mu LBCA working solution into each hole, and incubating for 30min at 37 ℃;
measuring the OD value at 540nm, and calculating the protein concentration of the sample;
adding 30 mu L5 multiplied by LoadingBuffer into a sample to be detected, boiling for 10min to denature protein, and storing at-20 ℃ for later use;
(3) SDS-PAGE electrophoresis
Correctly assembling an electrophoresis tank, and putting prefabricated glue; pouring an electrophoresis buffer solution, removing the comb, and sequentially adding the multicolor pre-dyed protein marker and a sample to be detected; performing constant-pressure electrophoresis at room temperature of 110V until the bottom of the gel is finished;
(4) rotary film
1) Soaking the PVDF membrane in methanol for 5min, and soaking the filter paper in a membrane transfer buffer solution;
2) taking out the gel, and cutting off the gel containing the target protein by contrast with a Marker;
3) paving the filter paper-PVDF membrane-gel-filter paper in sequence, and removing bubbles to ensure that the gel is fully contacted with the membrane;
4) rotating the membrane for 45min by 15v voltage;
(5) sealing of
After the membrane transfer is finished, taking out the PVDF membrane, and washing the PVDF membrane for 3 times with TBST on a shaking table at 70 r/min, wherein each time is 5 min; preparing 5% skim milk as a sealing liquid, fully contacting the PVDF membrane with the sealing liquid, and sealing overnight at 4 ℃;
(6) binding antibodies
In combination with an antibody:
according to the antibody specification, the Occludin antibody was diluted with blocking solution at a dilution of 1: 600; the dilution of rabbit-derived beta-actin antibody is 1: 1000. Placing the sealed PVDF membrane in the diluted primary antibody, incubating at room temperature for 90min, taking out the PVDF membrane, washing with TBST for 5min each time for 3 times;
binding of secondary antibody:
according to the antibody specification, the secondary antibody is diluted by using a blocking solution, and the dilution of the goat anti-rabbit IgG antibody is 1: 1000; incubating the PVDF membrane after primary incubation and the diluted secondary antibody, incubating at room temperature for 60min, taking out the PVDF membrane, and washing with TBST for 3 times, 5min each time;
(7) color development by ECL method
And (3) taking the equal amount of the solution A and the solution B in the ECL kit, fully mixing, contacting with a PVDF membrane, and reacting for 5min at room temperature in a dark place. Images were collected with a BIO-RAD gel imager, gray scale scanned with ImageLab software, and Occludin protein levels were determined.
Example 1
The method for relieving barrier injury of chicken small intestine caused by AFB1 (aflatoxin B1) by selenium-enriched lactic acid bacteria comprises the following steps:
adding selenium-rich lactobacillus with selenium concentration of 0.01 μmol/L (final concentration in culture solution) into chicken small intestine epithelial cells, culturing for 8 hr, adding AFB1 to make its concentration 200 μmol/L, continuously culturing for 12 hr, and terminating the culture to inhibit AFB1The level of protein of chicken small intestine epithelial cell Occludin is reduced, and the effect of AFB1 on the damage of chicken small intestine intestinal barrier is weakened.
The method for verifying the inhibition of AFB 1-induced damage to the intestinal barrier of chicken small intestine by selenium-enriched lactic acid bacteria comprises the following steps:
1.1 Chicken intestinal epithelial cell culture and group treatment
Inoculating chicken small intestine epithelial cells into 6-well plate, and adjusting cell concentration to 2 × 105And (4) blowing and beating for multiple times slowly before sample adding and distributing. The test was divided into 3 groups, i.e., control group C, AFB1 challenge group (AFB 1 toxin 200. mu. mol/L) and selenium-enriched lactic acid bacteria group (0.01. mu. mol/LSe + AFB1 toxin 200. mu. mol/L), each of which was 6 replicates. After the cells grow to 60% -70%, the culture solution is discarded and washed for 2 times, and the culture solution is replaced: replacing common culture solution for the control group and the AFB1 challenge group; the selenium-enriched lactobacillus group is replaced by selenium-enriched lactobacillus culture solution containing 0.01 mu mol/L (calculated by selenium). And (4) continuously culturing for 8 h, taking out the cell culture plate, adding AFB1 into the selenium-enriched lactobacillus group and the AFB1 group to enable the concentration of the selenium-enriched lactobacillus group and the AFB1 group to be 200 mu mol/L, and continuously culturing for 12h to finish the culture.
1.2 protein extraction
The cells were washed 3 times with pre-cooled PBS, 150 μ L of RIPA cell lysate containing 1mM PMSF was added to each well, after the cells were fully contacted, the cells were scraped off and transferred to a 1.5mL centrifuge tube, and the cells were continued to be lysed at 4 ℃ for 1h, and shaken 10s every 15min to fully lyse the cells. Centrifuging at 12000g at 4 deg.C for 10min, and collecting supernatant; storing at-20 deg.C;
1.3 BCA assay for protein concentration
Diluting the protein standard substance in the kit to 0.5 mg/kg;
preparing a BCA working solution: mixing the solution A and the solution B according to a ratio of 50: 1;
adding the standard substance to a 96-well plate according to 0, 1, 2, 4, 8, 12, 16 and 20 muL, and supplementing the standard substance to 20 muL by using physiological saline; adding 2 muL of samples to be detected into a 96-well plate, and supplementing the samples to 20 muL with physiological saline;
adding 200 mu LBCA working solution into each hole, and incubating for 30min at 37 ℃;
measuring the OD value at 540nm, and calculating the protein concentration of the sample;
adding 30 mu L of 5 XLoadingBuffer into a sample to be detected, boiling for 10min to denature protein, and storing at-20 ℃ for later use;
1.4 SDS-PAGE electrophoresis
Correctly assembling an electrophoresis tank, and putting prefabricated glue; pouring an electrophoresis buffer solution, removing the comb, and sequentially adding the multicolor pre-dyed protein marker and a sample to be detected; performing constant-pressure electrophoresis at room temperature of 110V until the bottom of the gel is finished;
1.5 transfer film
(1) Soaking the PVDF membrane in methanol for 5min, and soaking the filter paper in a membrane transfer buffer solution;
(2) taking out the gel, and cutting off the gel containing the target protein by contrast with a Marker;
(3) paving the filter paper-PVDF membrane-gel-filter paper in sequence, and removing bubbles to ensure that the gel is fully contacted with the membrane;
(4) rotating the membrane for 45min by 15v voltage;
1.6 sealing
After the membrane transfer is finished, taking out the PVDF membrane, and washing the PVDF membrane for 3 times with TBST on a shaking table at 70 r/min, wherein each time is 5 min; preparing 5% skim milk as a sealing liquid, fully contacting the PVDF membrane with the sealing liquid, and sealing overnight at 4 ℃;
1.7 binding antibodies
In combination with an antibody:
the Occludin antibody was diluted with blocking solution according to the antibody instructions at a dilution of 1: 600; the dilution of rabbit-derived beta-actin antibody is 1: 1000. Placing the sealed PVDF membrane in the diluted primary antibody, incubating at room temperature for 90min, taking out the PVDF membrane, washing with TBST for 5min each time for 3 times;
binding of secondary antibody:
according to the antibody specification, the secondary antibody is diluted by using a blocking solution, and the dilution of the goat anti-rabbit IgG antibody is 1: 1000; incubating the PVDF membrane after primary incubation and the diluted secondary antibody, incubating at room temperature for 60min, taking out the PVDF membrane, and washing with TBST for 3 times, 5min each time;
1.8 color development by ECL method
And (3) taking the equal amount of the solution A and the solution B in the ECL kit, fully mixing, contacting with a PVDF membrane, and reacting for 5min at room temperature in a dark place. Images were collected with a BIO-RAD gel imager, gray scale scanned with ImageLab software, and Occludin protein levels were determined.
The results of the determination that selenium-enriched lactic acid bacteria (0.01. mu. mol/L) can relieve the barrier injury of the small intestine of the chicken caused by AFB1 (200. mu. mol/L) are shown in FIG. 2.
The result shows that 200 mu mol/L of AFB1 can obviously reduce the expression level of Occludin protein in chicken small intestine epithelial cells (PLess than 0.05), and 0.01 mu mol/L (calculated by selenium) of selenium-enriched lactic acid bacteria can obviously improve the level reduction of the egg level of chicken small intestine epithelial cells Occludin caused by AFB1 (the content of the protein is reduced by the selenium-enriched lactic acid bacteria)P＜0.05）。
Example 2
The method for relieving the barrier injury of the chicken small intestine caused by AFB1 by the selenium-enriched lactic acid bacteria comprises the following steps:
adding selenium-rich lactobacillus with selenium concentration of 0.1 μmol/L (final concentration in culture solution) into chicken small intestine epithelial cells, culturing for 12 hr, adding AFB1 to make its concentration 300 μmol/L, and culturing for 24 hr to inhibit AFB1The level of protein of chicken small intestine epithelial cell Occludin is reduced, and the effect of AFB1 on the damage of chicken small intestine intestinal barrier is weakened.
The method for verifying the inhibition of AFB 1-induced damage to the intestinal barrier of chicken small intestine by selenium-enriched lactic acid bacteria comprises the following steps:
2.1 Chicken Small intestinal epithelial cell culture and group treatment
Inoculating chicken small intestine epithelial cells into 6-well plate, and adjusting cell concentration to 2 × 105And (4) blowing and beating for multiple times slowly before sample adding and distributing. The test was divided into 3 groups, i.e., control group C, AFB1 challenge group (AFB 1 toxin 300. mu. mol/L) and selenium-enriched lactic acid bacteria group (0.1. mu. mol/LSe + AFB1 toxin 300. mu. mol/L), each of which was 6 replicates. CellsAfter 60% -70% of the growth, abandoning the culture solution and cleaning for 2 times, replacing the culture solution: replacing common culture solution for the control group and the AFB1 challenge group; the selenium-enriched lactobacillus group is replaced by selenium-enriched lactobacillus culture solution containing 0.1 mu mol/L (calculated by selenium). And (4) continuously culturing for 12h, taking out the cell culture plate, adding AFB1 into the selenium-enriched lactobacillus group and the AFB1 group to enable the concentration of the selenium-enriched lactobacillus group and the AFB1 group to be 300 mu mol/L, and continuously culturing for 24h to finish the culture.
2.2 protein extraction
The cells were washed 3 times with pre-cooled PBS, 150 μ L of RIPA cell lysate containing 1mM PMSF was added to each well, after the cells were fully contacted, the cells were scraped off and transferred to a 1.5mL centrifuge tube, and the cells were continued to be lysed at 4 ℃ for 1h, and shaken 10s every 15min to fully lyse the cells. Centrifuging at 12000g at 4 deg.C for 10min, and collecting supernatant; storing at-20 deg.C;
2.3 BCA assay for protein concentration
Diluting the protein standard substance in the kit to 0.5 mg/kg;
preparing a BCA working solution: mixing the solution A and the solution B according to a ratio of 50: 1;
adding the standard substance to a 96-well plate according to 0, 1, 2, 4, 8, 12, 16 and 20 muL, and supplementing the standard substance to 20 muL by using physiological saline; adding 2 muL of samples to be detected into a 96-well plate, and supplementing the samples to 20 muL with physiological saline;
adding 200 mu LBCA working solution into each hole, and incubating for 30min at 37 ℃;
measuring the OD value at 540nm, and calculating the protein concentration of the sample;
adding 30 mu L of 5 XLoadingBuffer into a sample to be detected, boiling for 10min to denature protein, and storing at-20 ℃ for later use;
2.4 SDS-PAGE electrophoresis
Correctly assembling an electrophoresis tank, and putting prefabricated glue; pouring an electrophoresis buffer solution, removing the comb, and sequentially adding the multicolor pre-dyed protein marker and a sample to be detected; performing constant-pressure electrophoresis at room temperature of 110V until the bottom of the gel is finished;
2.5 transfer film
(1) Soaking the PVDF membrane in methanol for 5min, and soaking the filter paper in a membrane transfer buffer solution;
(2) taking out the gel, and cutting off the gel containing the target protein by contrast with a Marker;
(3) paving the filter paper-PVDF membrane-gel-filter paper in sequence, and removing bubbles to ensure that the gel is fully contacted with the membrane;
(4) rotating the membrane for 45min by 15v voltage;
2.6 sealing
After the membrane transfer is finished, taking out the PVDF membrane, and washing the PVDF membrane for 3 times with TBST on a shaking table at 70 r/min, wherein each time is 5 min; preparing 5% skim milk as a sealing liquid, fully contacting the PVDF membrane with the sealing liquid, and sealing overnight at 4 ℃;
2.7 binding antibodies
In combination with an antibody:
according to the antibody specification, the Occludin antibody was diluted with blocking solution at a dilution of 1: 600; the dilution of rabbit-derived beta-actin antibody is 1: 1000. Placing the sealed PVDF membrane in the diluted primary antibody, incubating at room temperature for 90min, taking out the PVDF membrane, washing with TBST for 5min each time for 3 times;
binding of secondary antibody:
according to the antibody specification, the secondary antibody is diluted by using a blocking solution, and the dilution of the goat anti-rabbit IgG antibody is 1: 1000; incubating the PVDF membrane after primary incubation and the diluted secondary antibody, incubating at room temperature for 60min, taking out the PVDF membrane, and washing with TBST for 3 times, 5min each time;
2.8 color development by ECL method
And (3) taking the equal amount of the solution A and the solution B in the ECL kit, fully mixing, contacting with a PVDF membrane, and reacting for 5min at room temperature in a dark place. Images were collected with a BIO-RAD gel imager, gray scale scanned with ImageLab software, and Occludin protein levels were determined.
The results of the determination that selenium-enriched lactic acid bacteria (0.1. mu. mol/L) can relieve the barrier injury of the small intestine of the chicken caused by AFB1 (300. mu. mol/L) are shown in FIG. 3.
The result shows that 300 mu mol/L AFB1 can remarkably reduce the expression level of Ocplus protein in chicken small intestine epithelial cells (PLess than 0.01), and 0.1 mu mol/L (calculated by selenium) of selenium-enriched lactic acid bacteria can remarkably improve the level reduction of the egg level of chicken small intestine epithelial cells Occludin caused by AFB1 (the content of the Occludin protein is reduced by adopting the selenium-enriched lactic acid bacteria (the content of the selenium-enriched lactic acid bacteria is reduced by adopting the formula of (I))P＜0.01）。
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Claims (2)
1. The application of the selenium-enriched lactic acid bacteria in relieving the barrier damage of chicken small intestine caused by aflatoxin; the relieving of the barrier damage of the chicken small intestine caused by the aflatoxin refers to the following steps: the selenium-enriched lactic acid bacteria inhibit the aflatoxin to cause the protein level of chicken small intestine epithelial cell tight junction protein Occludin to be reduced.
2. The use of claim 1, wherein: for the chicken small intestine epithelial cell, when the concentration of the aflatoxin is 200-300 mu mol/L, the decrease of the level of the Occludin protein of the chicken small intestine epithelial cell caused by the aflatoxin can be inhibited by using the selenium-enriched lactic acid bacteria with the final concentration (calculated by selenium) of the selenium in the culture solution of 0.01-0.1 mu mol/L.
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Application publication date: 20210730 |