CN115300531B - Lactobacillus paracasei JY062 composition and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a lactobacillus paracasei JY062 composition, which comprises lactobacillus paracasei JY062 (Lactobacillus paracasei JY 062) and lactobacillus paracasei JY 062; the content of the lactobacillus paracasei JY062 is not less than 10 9 CFU/mL; the concentration of the exopolysaccharide is not lower than 30mg/mL. The invention also discloses a preparation method of the lactobacillus paracasei JY062 composition. The invention also discloses application of the lactobacillus paracasei JY062 composition in preparation of a medicine for improving intestinal mucosa barrier injury. The invention has the functions of obviously enhancing the intestinal immunocompetence and improving the intestinal mucosa barrier damage through the NF-kB signal path.
Description
Technical Field
The invention relates to a lactobacillus paracasei JY062 composition, a preparation method and an application thereof, and belongs to the technical field of microorganisms.
Background
The lactobacillus is generally regarded as safe food-grade bacteria, most of the lactobacillus is regarded as probiotics, lactobacillus paracasei is a probiotic which is researched more recently, belongs to lactobacillus, widely exists in traditional fermented dairy products and human gastrointestinal tracts, can effectively adhere to and colonize intestinal mucosa, regulates and controls intestinal flora, epithelial cells and immune factors in intestinal microenvironment steady state, performs immune regulation, and maintains intestinal steady state; the extracellular polysaccharide which is an important metabolite of the lactobacillus is a carbohydrate compound secreted by the lactobacillus in a tightly combined capsule or loosely attached mucus layer form, secreted outside a cell wall in a growth and metabolism process and often permeated in a culture medium, plays an important regulation role on an organism intestinal tract mainly by improving the adhesion of the intestinal mucosa, regulating the intestinal microecological balance and supplying energy to the intestinal tract in the interaction process of the extracellular polysaccharide and host intestinal tract cells, and realizes the probiotic function of the lactobacillus by the EPS function on one hand and the metabolism of the probiotics on the other hand.
The synbiotics are composed of probiotics and prebiotics, and play an important role in improving immune response, relieving intestinal inflammation, reducing oxidative stress, maintaining intestinal epithelial cell barrier function, adjusting short-chain fatty acid level, improving intestinal flora structure and the like by promoting the probiotics to colonize and proliferate in the intestinal tract of a host and simultaneously playing a role in promoting the physiological activity of the probiotics and the growth promotion of the prebiotics.
The NF-. Kappa.B/Rel protein family includes five members: c-Rel, relA (p 65), relB, NF-. Kappa.B 1 (p 50/p 105) and NF-. Kappa.B 2 (p 52/p 100), which exist as homo-or heterodimers, the NF-. Kappa.B 1 dimeric protein p65/p50 (most commonly) and the NF-. Kappa.B dimeric protein RelB/p52. When the organism is in a resting state, NF-kB is combined with an inhibitory protein I-kB (inhibit NF-kB) in cytoplasm to present an inactive state; when an organism is stimulated by external stimulus, the I-kappa B kinase (IKK) can phosphorylate the inhibitor I-kappa B of NF-kappa B, so that the I-kappa B is degraded, the NF-kappa B is released from an I-kappa B complex and separated from the I-kappa B, and free NF-kappa B enters a cell nucleus from cytoplasm and is combined with a specific binding site to regulate the transcription of a target gene, so that an NF-kappa B signal channel is activated. The NF-kB system can maintain the homeostasis of normal intestinal epithelium and plays a central role in mucosal immune response; but also can mediate pathogen specific response, and is a double-edged sword for the mucous membrane barrier.
The increase of intestinal permeability causes harmful bacteria to enter the inner layer of the mucous membrane, the secretion of mucin is greatly reduced, and a large amount of intestinal epithelial cells are apoptotic, thereby causing local environment disorder of the intestinal tract. Tight junctions, the most prominent way to connect between intestinal epithelial cells, are located at the boundaries of the top and outer membranes of the intestinal epithelial cells, and are a highly diverse structure composed of transmembrane and cytoplasmic proteins. Occluding proteins Occludin, occludin and Occludin (ZO-1, ZO-2 and ZO-3) are important protein molecules which constitute the barrier integrity of intestinal mucosa and determine the permeability of intestinal tracts, and serve as the most important 3 tight junction proteins. Mucus secreted in the intestinal tract is mainly composed of Mucin (Mucin) secreted from goblet cells and forms a mucus layer inside the intestinal lumen, wherein Mucin2 is a main substance constituting the mucus layer. A great deal of research shows that the traditional Chinese medicine composition can stimulate goblet cells to secrete mucin and increase the thickness of a mouse mucus layer to improve barrier function by preventing the increase of intestinal permeability; up-regulating the expression of the intestinal epithelial cell mucin MUC2, the tight junction protein Claudin-3, ZO-1 and E-cadherin to restore the integrity of the intestinal mucosal barrier.
Therefore, the lactobacillus paracasei JY062 composition, the preparation method and the application thereof have practical significance for realizing the function of accurately intervening lactobacillus and metabolic active substances thereof in regulating and controlling the health of host intestinal tracts, and provide theoretical basis for developing probiotics and potential prebiotics functional preparations.
Disclosure of Invention
The invention aims to provide a lactobacillus paracasei JY062 composition, which consists of lactic acid bacteria (namely lactobacillus paracasei JY 062) and extracellular polysaccharide.
Meanwhile, the invention provides a preparation method of the lactobacillus paracasei JY062 composition, and the composition obtained by the method has the functions of obviously enhancing intestinal immunocompetence and improving intestinal mucosa barrier injury through an NF-kB signal path.
Meanwhile, the invention provides application of the lactobacillus paracasei JY062 composition in preparation of a medicine for enhancing intestinal immunity activity.
Meanwhile, the invention provides application of the lactobacillus paracasei JY062 composition in preparation of a medicine for improving intestinal mucosa barrier injury.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
lactobacillus paracasei JY062 composition comprising lactobacillus paracaseiJY062（Lactobacillus paracasei JY 062) and lactobacillus paracasei JY 062; the content of lactobacillus paracasei JY062 is not less than 10 9 CFU/mL; the concentration of the exopolysaccharide is not lower than 30mg/mL.
A method of preparing a lactobacillus paracasei JY062 composition, the method comprising the steps of:
step one, inoculating lactobacillus paracasei JY062 into a sterilized MRS liquid culture medium according to the inoculation amount of 5 percent, wherein the initial concentration is 1.5 multiplied by 10 9 CFU/mL；
Step two, adding the optimized composite carbon source MRS liquid culture medium in an equivalent manner; culturing at 34 deg.C in a fermenter at 100rpm/min for 25h, and supplementing 20% w/v ammonia water during fermentation; after fermenting for 12 hours, starting to supplement 100mL/30min of MRS liquid culture medium until the end, and collecting bacterial liquid;
centrifuging the bacterial liquid, taking the supernatant I, and repeating the step twice;
step four, taking the supernatant I, and concentrating the volume to 1/3 of the original volume to obtain a culture medium;
step five, inactivating enzyme of the culture medium, cooling to room temperature, and collecting supernatant liquid II;
removing protein from the supernatant fluid II, and centrifuging to obtain a supernatant fluid III;
precipitating the supernatant III with ethanol, centrifuging and taking the precipitate;
step eight, resuspending the pellet in deionized water to obtain polysaccharide solution, placing the polysaccharide solution in an MD34 dialysis bag, wherein the molecular weight cut-off is as follows: 8KD-14KD, dialyzing in deionized water at 4 deg.C for 48h, and replacing deionized water every 8 h;
and step nine, pouring the dialyzed polysaccharide supernatant into a flat plate, placing the flat plate in a refrigerator at the temperature of-80 ℃ for pre-freezing overnight, then carrying out freeze drying for 24h, and carrying out freeze drying to obtain the extracellular polysaccharide.
In the first step, the MRS liquid culture medium comprises the following components: 10g/L peptone, 8g/L beef powder, 4g/L yeast powder, 20g/L glucose, 2g/L diammonium hydrogen citrate, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 0.2g/L magnesium sulfate, 0.04g/L manganese sulfate and 1g/L Tween-80; dissolving 52.24g of the components in 1000mL of distilled water, stirring by a glass rod, carrying out autoclaving at 121 ℃ for 15min, and obtaining the MRS liquid culture medium.
In the second step, the optimized composite carbon source MRS liquid culture medium comprises the following components: 10g/L peptone, 8g/L beef powder, 4g/L yeast powder, 2g/L diammonium hydrogen citrate, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 0.2g/L magnesium sulfate, 0.04g/L manganese sulfate, 1g/L Tween-80, 3.33g/L fructose, 3.33g/L mannose, 3.33g/L trehalose, 3.33g/L glucose, 3.33g/L galactose and 3.33g/L lactose; dissolving 52.24g of the components in 1000mL of distilled water, stirring with a glass rod, carrying out high-pressure sterilization at 121 ℃ for 15min, and obtaining the optimized composite carbon source MRS liquid culture medium.
The centrifugal process comprises the following steps: centrifuging at 10000r/min and 4 deg.C for 15min.
In the fifth step, the enzyme activity killing process comprises the following steps: heating in boiling water for 10min.
In the sixth step, the protein removing process comprises the following steps: 80% w/v of trichloroacetic acid was added to the supernatant II to a final concentration of 10% w/v, followed by stirring for 5min and standing at 4 ℃ for 10h,10000r/min, and centrifuging at 4 ℃ for 15min to remove proteins.
In the seventh step, the alcohol precipitation process comprises: adding 2-3 times volume of anhydrous ethanol into the supernatant III to make the final concentration 70%, standing overnight at 4 deg.C, centrifuging at 10000r/min at 4 deg.C for 15min.
Application of a lactobacillus paracasei JY062 composition in preparation of a medicament for improving intestinal mucosa barrier injury.
The invention has the following beneficial effects:
the invention provides a composition with intestinal immunoregulation function according to the result of synbiotics immunoregulation related research, and the subtype and distribution condition of colon macrophages are evaluated by an immunofluorescence staining method; detecting the content of Th17 and Treg cells in spleen and peripheral blood of the mouse by flow cytometry; ELISA detected 7 immune-related genes and 3 immunoglobulins. The invention overcomes the defects of the existing lactobacillus paracasei and the evaluation of the immunoregulation function of the extracellular polysaccharide generated by the lactobacillus paracasei, and can objectively evaluate the intestinal tract immunoregulation function of the lactobacillus paracasei JY062 and the produced extracellular polysaccharide on the host; discloses a deep mechanism for synergistically regulating intestinal immunity by virtue of lactobacillus paracasei JY062 and exopolysaccharide produced by the lactobacillus paracasei.
Meanwhile, the invention provides a composition for improving the barrier damage function of the intestinal mucosa through an NF-kB signal path, the damage condition of mouse colon tissues is reflected through HE staining, the activity of the NF-kB signal path of the colon is determined by combining RT-PCR with a Western Blot method, and the expression quantity of colon tight junction protein and mucin is determined by combining RT-PCR with an immunohistochemical method. The invention deeply excavates the function of preventing the damage of the intestinal mucosa, analyzes the potential mechanism of the intestinal mucosa immunity, provides a theoretical basis for developing synbiotics with the function of improving the barrier damage of the intestinal mucosa, and can make up the defects of the existing application level to a certain extent.
Drawings
FIG. 1 shows the content of genes related to colon immunity in mice; wherein, a: TNF-alpha; b: IFN-gamma; c: IL-1 β; d: IL-6; e: TGF-beta;
FIG. 2 is the mouse serum immunoglobulin content; wherein, a: igA; b: igG; c: an IgM;
FIG. 3 is a graph showing the change in the number of Treg cells in the spleen and peripheral blood of mice;
FIG. 4 is a graph showing the change in the number of Th17 cells in the spleen and peripheral blood of mice;
FIG. 5 shows the IL-17 content of the specific cytokine associated with colon Th17 cells in mice;
FIG. 6 shows the IL-10 content of the specific cell factor related to the mouse colon Treg cells;
FIG. 7 shows the subtype and distribution of mouse colon macrophages; wherein, a: type M1;
FIG. 8 shows the subtype and distribution of mouse colon macrophages; wherein, b: m2 type;
FIG. 9 is a histopathological observation of the colon of mice; wherein, a: HE staining; b: (ii) histological lesion scoring;
FIG. 10 shows the relative expression amounts of genes related to TLR4/MyD88/NF- κ B signaling pathway mRNA; wherein, a: NF- κ B p65; b: myD88; c: i kappa B; d: TLR4;
FIG. 11 shows the expression level of key proteins in TLR4/MyD88/NF- κ B signaling pathway; wherein, a: NF- κ B p65; b: p-NF-kB p65; c: myD88; d: i kappa B; e: p-I κ B; f: TLR4;
FIG. 12 shows the relative expression levels of claudin and mucin mRNA in colon tissues of mice; wherein, a: claudin-1; b: claudin-2; c: occludin; d: ZO-1; e: MUC2;
FIG. 13 is a graph of immunohistochemical staining of claudin and mucin in colon tissues of mice;
FIG. 14 is the mean optical density values for claudin and mucin in murine colon tissue; wherein, a: claudin-1; b: claudin-2; c: occludin; d: ZO-1; e: MUC2.
Detailed Description
The present invention will be further described with reference to the accompanying drawings.
In the invention, the Latin name of lactobacillus paracasei JY062 isLactobacillus paracasei JY062, deposited in the northeast agriculture university dairy product focus laboratory, was isolated from Tibet traditional fermented dairy products, the Lactobacillus paracasei JY062 of the invention prior art sources were: adhesion and tolerance evaluation of high-yield exopolysaccharide hypoglycemic lactobacillus paracasei JY062 (TD 062) [ J]The chinese dairy industry, 2022, 50 (4).
Zhangyu, jiangyuyujun, dandangfang, and the like, lactobacillus paracasei TD062 has the hypoglycemic effect on type II diabetic mice [ J ] Chinese food academy 2020, v.20 (05): 112-118.
The public is available from northeast university of agriculture.
Example 1
A Lactobacillus paracasei JY062 composition comprises Lactobacillus paracasei JY062 (B), (B)Lactobacillus paracasei JY 062) and lactobacillus paracasei JY 062; the content of the lactobacillus paracasei JY062 is 10 9 CFU/mL; the concentration of the exopolysaccharide is 30mg/mL.
A method of preparing a lactobacillus paracasei JY062 composition, the method comprising the steps of:
(1) Lactobacillus paracasei JY062 was inoculated into a sterilized MRS liquid medium at an initial concentration of 1.5X 109 CFU/mL by 5% inoculation amount.
(2) And (4) adding the optimized composite carbon source MRS liquid culture medium in an equivalent manner. Culturing at 34 deg.C in a fermenter at 100rpm/min for 25 hr, supplementing 20% (w/v) ammonia water in time during fermentation, and supplementing MRS liquid culture medium after 12 hr fermentation, and collecting bacterial liquid.
(3) Centrifuging the bacterial solution at 10000r/min and 4 deg.C for 15min, collecting supernatant, and repeating the steps twice.
(4) The supernatant was concentrated on a rotary evaporator to 1/3 of the original volume.
(5) Heating the culture medium in boiling water for 10min to inactivate enzyme, cooling to room temperature, and collecting supernatant.
(6) Adding 80% (w/v) trichloroacetic acid into the supernatant to a final concentration of 10% (w/v), stirring for 5min, standing at 4 deg.C for 10h,10000r/min, centrifuging at 4 deg.C for 15min to remove protein, and collecting the supernatant.
(7) Adding 2-3 times volume of anhydrous ethanol into the supernatant to make its final concentration 70%, standing overnight at 4 deg.C, centrifuging at 10000r/min at 4 deg.C for 15min, and collecting precipitate.
(8) The precipitate was resuspended in deionized water, the polysaccharide solution was placed in an MD34 dialysis bag (molecular weight cut-off: 8KD-14 KD), dialyzed in deionized water at 4 ℃ for 48h, and the deionized water was changed every 8 h.
(9) Pouring the dialyzed polysaccharide supernatant into a flat plate, placing the flat plate in a refrigerator at minus 80 ℃ for pre-freezing overnight, and then carrying out freeze drying for 24 hours. And (5) freeze-drying to obtain the exopolysaccharide.
The composition of the lactobacillus paracasei JY062 obtained in the embodiment is applied to the preparation of the medicine for enhancing the intestinal immune activity.
Enhancing immunity can regulate immunity related gene content, inhibit excessive secretion of proinflammatory factors (TNF-alpha, IFN-gamma, IL-1 beta, IL-6), and increase level of antiinflammatory factor (TGF-beta).
The immunity enhancing effect is obtained by reducing immunoglobulin (IgA, igG, igM) content to normal level.
The immunity enhancing function is to reduce the number of Th17 cells and the content of the related specific cytokine IL-17, and increase the number of Treg cells and the content of the related specific cytokine IL-10.
Enhancing immunity can regulate M1/M2 type macrophage balance, inhibit colon macrophage polarization to M1 type, and promote colon macrophage polarization to M2 type.
The test animals are male C57BL/6J 8-week-old and 25-30g in weight, and purchased from Liaoning Biotechnology GmbH.
Animal grouping: the experimental animals selected 60 male C57BL/6J with age of 8 weeks and weight of 25-30g, the basal feed was adaptively fed for 1 week and then adapted to the environment, and the experimental animals were randomly divided into 5 groups (n = 12) which were blank Control (NC), model (3 DSS) and single-strain group (10% 9 CFU/mL, JY 062), monosaccharide group (600 mg/kg, EPS), saccharomycete complex group (10) 9 CFU/mL +600mg/kg, JEC), numbered with picric acid. Mice in the blank control group were fed with normal diet and free water throughout the experiment (1-21 d), and the dry control group (JY 062, EPS, JEC) was gavaged regularly every day for the whole experiment period (8-21 d), with a gavage dose of 200 μ L. After one week of gavage, in the modeling period (15-21 d), the rest groups except the blank control group were induced by adding 3% DSS aqueous solution.
1. ELISA method for determining in vivo immune related genes:
colon tissue (killed after the test period was completed and the colon tissue was placed in a-80 ℃ freezer) was taken out of the-80 ℃ freezer (50-100 mg), pre-cooled sterile PBS was used as a homogenization medium, and the weight of the colon tissue: the volume of the homogenization medium was 1. Transferring 10% of the tissue homogenate completely into a brand-new centrifuge tube, centrifuging (4 ℃,12000rpm, 10min), collecting the supernatant, and repeating the centrifugation twice, referring to the description of an enzyme linked immunosorbent assay kit, and detecting the contents of TNF-alpha, IFN-gamma, IL-1 beta, IL-6 and TGF-beta in the colon tissue by adopting a competition method.
2. Determination of the immunoglobulin content in vivo by ELISA:
blood sampling is carried out by removing the eyeballs of the mice, and the eyeballs are clamped after being engorged with blood to collect the eye blood in a 1.5mL sterile centrifuge tube. Placing the blood around the eyes of the mouse on ice for 2h to fully separate out the serum, centrifuging (4 ℃,12000rpm, 10min), taking the upper serum, unfreezing on ice, placing the ELISA kit at room temperature for 30min in advance, specifically operating according to the kit specification, measuring absorbance at the wavelength of 492nm by using a multifunctional microplate reader, drawing a standard curve (taking the concentration of a standard product as the horizontal coordinate and A492nm as the vertical coordinate) as required, and calculating the contents of immunoglobulin IgA, igG and IgM in the serum.
3. The ELISA method is used for determining the content of specific cytokines related to the Th17 and Treg cells in vivo:
taking 50-100mg of colon tissue out of a refrigerator at-80 ℃, using precooled sterile PBS as a homogenizing medium, and mixing the components according to the weight of the colon tissue: the volume of the homogenization medium was 1. Completely transferring 10% of the tissue homogenate to a brand-new centrifuge tube, centrifuging (4 ℃,12000rpm, 10min) to collect supernatant, and repeating the centrifugation twice, wherein the centrifugation can be repeated according to the instruction of an enzyme linked immunosorbent assay kit, and the contents of IL-17 and IL-10 in the colon tissue are detected by adopting a competition method.
4. Detecting the content of Th17 and Treg cells in spleen and peripheral blood of the mouse by flow cytometry:
removing the eyeball of the mouse and taking blood, collecting the blood into an anticoagulation tube; adding 1 Xerythrocyte lysate, gently beating blood, mixing, centrifuging at 4 deg.C (600g, 10 min), and removing supernatant until erythrocyte lysis is complete; adding PBS, resuspending, centrifuging at 4 deg.C (600g, 10 min), washing for 1 time, adding appropriate amount of 1 XPBS (100 uL per tube); according to the weight ratio of 1.25: adding PE/Cyanine7 anti-mouse CD3, 0.5: adding FITC anti-mouse CD4 and 5 in a volume ratio of 100: adding APC/Cyanine7 anti-mouse CD25 surface antibody at the volume ratio of 100, and keeping out of the light for 30min at 4 ℃; after adding PBS for resuspension, the cells were centrifuged at 4 ℃ for 600g and 10min to break the nuclei. In this example, g of 600g represents the rotation speed, which can be converted to rpm, involved in the centrifugal operation.
Taking out eyeballs, killing the mice by a neck-removing method, dissecting, quickly separating out spleens by using forceps, putting the spleens into a 5mL EP tube, and shearing; adding collagenase IV, and digesting in a constant temperature shaking box (37 ℃,25min, 100rpm); adding FBS to stop digestion when the tissue fluid is turbid and is in a wiredrawing state; filtering, grinding, washing with PBS, centrifuging at 4 deg.C (600g, 10 min), discarding supernatant, adding 1 × erythrocyte lysate, gently blowing, mixing, and lysing for 2min; discarding the supernatant; adding PBS, resuspending, centrifuging at 4 deg.C (600g, 10 min), washing for 1 time, adding appropriate amount of 1 XPBS, 100uL per tube; according to the weight ratio of 1.25: adding PE/Cyanine7 anti-mouse CD3, 0.5: adding FITC anti-mouse CD4 and 5 in a volume ratio of 100: adding APC/Cyanine7 anti-mouse CD25 according to the volume ratio of 100, and keeping out of the sun for 30min at 4 ℃; after adding PBS for resuspension, the cells were centrifuged at 4 ℃ for 600g and 10min to break the nuclei.
Adding True-nucleic acid 1 × Fix Concentrate into each tube, vortexing, and incubating at room temperature in dark for 55min; adding True-nucleic acid 1 XPerm Buffer to each tube, centrifuging at room temperature (400g, 5 min), and discarding the supernatant, and repeating the step twice; add the True-Nuclear ™ 1 XPerm Buffer for resuspension, following 1.25: adding APC anti-mouse IL-17RB and 5: adding PE anti-mouse FOXP3 according to the volume ratio of 100, and incubating for 30min at room temperature in a dark place; adding True-Nuclear: (1 XPerm Buffer), centrifuging at room temperature (400g, 5 min), and discarding the supernatant; adding cell staining buffer solution, centrifuging at room temperature (400g, 5 min), and then removing supernatant; adding cell staining buffer solution for resuspension, and performing on-machine detection on the levels of Treg cells and Th17 cells by an Agilent flow cytometer.
5. The subtype and distribution of colon macrophages are evaluated by immunofluorescence staining:
the colon tissue after embedding the section is dewaxed by xylene to water, antigen is repaired, the immunohistochemical strokes form a tissue circle, 3 percent BSA serum is blocked, primary antibody is added for incubation overnight at 4 ℃ (CD 68, CD86 and CD 206), secondary antibody (HRP enzyme label) which is added with corresponding species of primary antibody is added for incubation at room temperature for 20min, TSA reagent is added, DAPI counterstain cell nucleus and mounting are carried out, and finally observation and photographing recording are carried out under a fluorescence microscope.
6. Results and analysis:
as can be seen from FIG. 1, compared with the NC group, the levels of proinflammatory cytokines (TNF-alpha, IFN-gamma, IL-6 and IL-1 beta) in colon tissues of mice in the DSS group are obviously increased, and the level of TGF-beta is obviously reduced (P < 0.0001); compared with DSS group, the levels of TNF-alpha, IL-1 beta, IFN-gamma and IL-6 in JEC complex group are reduced to different degrees (P <0.0001, P <0.001, P < 0.05), and the level of TGF-beta of anti-inflammatory cytokine is increased significantly (P < 0.0001). The JEC compound can effectively regulate the level of mouse cell factors, maintain the balance of proinflammatory factors and anti-inflammatory factors in colon tissues, relieve the inflammation of mice and enhance the intestinal resistance of the mice.
As can be seen from FIG. 2, compared with the NC group, the levels of IgG, igM and IgA in the serum of mice in the DSS group are significantly increased (P < 0.0001). Compared with the DSS group, the levels of IgG and IgM in the JEC complex group are reduced to different degrees (P <0.0001 and P < 0.001). The level of the immune globulin reflects the activity degree of B cell reaction, has important regulation and control effects on the excitation and maintenance of B cell immunity, and the JEC compound can relieve the abnormal expression of serum IgG, igM and IgA levels, thereby showing that the JEC compound has an immune regulation function and corrects the immune dysfunction of intestinal mucosa.
As can be seen from fig. 3 (which is a graph of CD4+ CD25+ Foxp3+ Treg cells in the spleen and peripheral blood of each group), the levels of CD4+ CD25+ Foxp3+ Treg cells in the DSS group were the lowest, 14.08% and 1.17%, respectively, in the spleen and peripheral blood, and thus it was also seen that there was a difference in the Treg cell content in different immune organs. The JEC complex increased the Treg cell levels in spleen and peripheral blood to different extents (P <0.001, P-were-woven 0.0001) by 38.23%, 5.87%, respectively, compared to the DSS group. The spleen Treg cell content after the JEC compound is dried is increased by nearly 3 times compared with the DSS group, and the peripheral blood is increased by nearly 5 times. The JEC complex can promote Treg cell differentiation.
As can be seen from FIG. 4 (the CD3+ CD4+ IL-17+ Th17 cell map in spleen and peripheral blood of each group), the levels of IL-17+ CD4+ Th17 cells in the DSS group in spleen and peripheral blood are the highest, respectively 18.83% and 24.34%, and are different from Treg cells, and the content difference of Th17 cells in different immune organs is not very large. JEC complexes down-regulated Th17 cell levels in spleen and peripheral blood (P < 0.01), 7.40%, 17.27% compared to DSS group, respectively. The spleen Th17 cell content after JEC compound drying is reduced by 2.5 times compared with the DSS group, while the peripheral blood is reduced by nearly 1.5 times, and the Th17 cell content in the peripheral blood is close to that in the NC group (14.58%). The balance between Th17 and Tregs is crucial to immune homeostasis, and Th17 and Treg can be transformed with each other in the differentiation process, and the results show that the JEC compound can inhibit the differentiation of Th17 cells and further maintain immune homeostasis.
As can be seen from FIG. 5, the ELISA detected the IL-10 content of the specific cytokine related to the mouse colon Treg cells, compared with the NC group, the level of IL-10 in the colon tissue of the mice in the DSS group was significantly reduced (P < 0.0001), compared with the DSS group, the IL-10 content in the JEC complex group was significantly increased (P < 0.0001), and the content was 242.863pg/mL, even the IL-10 content in the NC group (233.027 pg/mL). The specific anti-inflammatory cytokine IL-10 mainly secreted by Treg cells plays an important role in inhibiting inflammatory reaction and autoimmune diseases.
As can be seen from FIG. 6, the ELISA detected the IL-17 content of the Th17 cell-associated specific cytokine in the colon of mice, compared with the NC group, the level of IL-17 in the colon tissue of mice in the DSS group was significantly increased (P < 0.0001), and compared with the DSS group, the IL-17 content in the JEC complex group was significantly decreased (P < 0.0001), which was 29.0067pg/mL and was close to the IL-17 content in the NC group (35.18 pg/mL). The specific cytokine IL-17 secreted by Th17 cells is involved in mediating tissue inflammation and inducing autoimmune diseases.
As can be seen from FIGS. 7 to 8, the JEC complex inhibits the polarization of colonic macrophages to M1 type and promotes the polarization of colonic macrophages to M2 type. Co-localized mean optical density values of M1 macrophages represented by CD86 decreased from 659.281 (DSS group) to 421.199 (JEC complex group); co-localized mean optical density values of M2 macrophages represented by CD206 were upregulated from 350.794 (DSS group) to 648.394 (JEC complex group). Macrophages are important members of innate immunity, have strong plasticity as important immune cells of the body, play a significant role in maintaining the homeostasis of the body, and M1 macrophages have been shown to destroy the integrity of epithelial barriers. Taken together, JEC complexes can modulate the inflammatory response by modulating the subtype and distribution of colonic macrophages.
Example 2
The composition and the preparation method of this example are the same as example 1.
The Lactobacillus paracasei JY062 composition obtained by the embodiment is applied to preparation of a medicine for improving intestinal mucosa barrier injury.
The NF-kB signaling pathway regulated by the composition is a TLR4/MyD 88/NF-kB signaling pathway.
The related genes of the regulated signal channel are NF-kB p65, p-NF-kB p65, myD88, I kB, p-I kB and TLR4.
The functional package for improving the barrier damage of the intestinal mucosa can up-regulate the expression quantity of IkB protein and mRNA, and down-regulate the expression quantities of NF-kB p65, p-NF-kB p65, myD88, p-IkB and TLR4 protein and the expression quantities of NF-kB p65, myD88 and TLR4 mRNA.
The improvement of the barrier injury function of the intestinal mucosa comprises the improvement of the phenomena of epithelial cell shedding of the mucosal layer and intestinal permeability increase, the increase of the number of goblet cells, the improvement of histological injury score and the improvement of the injury of the colon mucosa tissue structure of the mouse.
Improving the barrier damage function of the intestinal mucosa comprises regulating the expression level and the average optical density value of Claudin-1, claudin-2, occludin, ZO-1 and mucin MUC2 mRNA.
Improving the barrier injury function of the intestinal mucosa comprises increasing expression level and average optical density value of Claudin-1, occludin, ZO-1 and MUC2mRNA, and reducing expression level and average optical density value of Claudin-2 mRNA.
Application of lactobacillus paracasei and exopolysaccharide thereof in preparing medicines for improving intestinal mucosa barrier injury function.
The dosage form of the medicine is any one of tablets, capsules, granules, powder and liquid preparations.
The test animals are male C57BL/6J 8-week-old and 25-30g in weight, and purchased from Liaoning Biotechnology GmbH.
Animal grouping: the experimental animals selected 60 male C57BL/6J with age of 8 weeks and weight of 25-30g, the basal feed was adaptively fed for 1 week and then adapted to the environment, and the experimental animals were randomly divided into 5 groups (n = 12) which were blank Control (NC), model (3 DSS) and single-strain group (10% 9 CFU/mL, JY 062), monosaccharide group (600 mg/kg, EPS), saccharomycete complex group (10) 9 CFU/mL +600mg/kg, JEC), numbered with picric acid. Mice in the blank control group were fed with normal diet and free water throughout the experiment (1-21 d), and the dry control group (JY 062, EPS, JEC) was gavaged regularly every day for the whole experiment period (8-21 d), with a gavage dose of 200 μ L. After one week of gavage, in the modeling period (15-21 d), the rest groups except the blank control group were induced by adding 3% DSS aqueous solution.
1. Histopathological observation of the colon of mice:
taking out eyeballs, killing mice by a cervical-removing method, disinfecting the abdomen by 75% alcohol, opening the abdominal cavity in a sterile environment, finding out the tail end of the cecum, separating the tail end from the anus of the mice, taking out complete colon tissues, measuring pathological changes, taking the colon tissues of the mice fixed by 4% paraformaldehyde, washing, cutting, trimming, dehydrating by gradient ethanol, embedding by paraffin, slicing tissue slices (slices with the thickness of about 4 mu m), fixing glass slides, placing the slices in a 60 ℃ baking machine for 1h for preliminary dewaxing, HE dyeing (the specific operation is shown in the following table 1), observing morphological changes of the colon tissues under an optical microscope and taking pictures, and grading pathological damage conditions of various groups of colon tissues in the pictures mainly from four aspects: inflammatory cell infiltration, crypt morphology and lesion extent, the specific scoring criteria are shown in table 2:
TABLE 1 HE staining procedure
TABLE 2 Colon histopathology scoring criteria
The final score is a superposition of the scores of the items.
2. Detecting the mRNA expression quantity of the tight junction protein and the TLR4/MyD 88/NF-kB signal channel by Real-time RT-PCR:
(1) Extraction of total RNA from mouse colon tissue:
100mg of colon tissue was removed from a-80 ℃ freezer, and pre-chilled sterile PBS was used as the homogenization medium, according to tissue weight: the volume of the homogenization medium was 1.
Taking the prepared mouse colon tissue homogenate, extracting the mouse colon tissue total RNA according to a method of a Simply P total RNA extraction reagent, collecting the RNA by using a 1.5mLRnase-free centrifugal tube after the extraction is finished, and performing subsequent experiments as soon as possible after the extraction because the RNA is easily interfered by factors such as environment and the like, if the RNA cannot be used immediately, placing the RNA on an ice box, sealing the RNA by using a sealing film, labeling the name, and placing the RNA in a refrigerator for temporary storage at-80 ℃; the experimental super clean bench is wiped and disinfected by chloroform in advance, the used instruments are treated by DEPC water, and the whole process is carried out on an ice box in a dark place during RNA extraction. The specific procedure was as above, using NanoDrop to determine OD 260 、OD 280 And OD 260 /OD 280 Value, purity and concentration of RNA determined.
(2) Reverse transcription cDNA Synthesis:
the total RNA with qualified extraction quality is reversely transcribed into cDNA by using a PrimeScriptTM RT Master Mix (Perfect Real Time) kit of TaKaRa company, a reverse transcription system is prepared according to the dosage specified by the kit in the kit, and the specific reaction system is shown in Table 3:
TABLE 3 reagent name and dosage table for kit
Reverse transcription was performed using a 7000 PCR amplification apparatus using a pre-warmed machine, and the cDNA obtained by reverse transcription was stored at-20 ℃ for subsequent testing, with the specific reverse transcription reaction conditions shown in Table 4:
TABLE 4 reverse transcription reaction conditions
(3) RT-PCR reaction
Selecting a plurality of genes such as Claudin-1, occludin, ZO-1, MUC2, claudin-2, NF-kB p65, myD88, I kB, TLR4 and the like as target genes for RT-PCR research, and taking beta-actin as an internal reference gene; the information of the required genes is searched on NCBI, the Primer 5.0 software is utilized to design each gene specific Primer, PCR primers are synthesized by Shanghai Biotechnology Limited company, and the specific genes and Primer sequences are shown in Table 5.
TABLE 5 mouse primer information
Adjusting the concentration of the cDNA to a proper range, carrying out Real-time RT-PCR reaction on the cDNA obtained by reverse transcription by using a SYBR Premix Ex TaqTM II (Perfect Real-time) kit of TaKaRa company, and setting 3 parallel tests for each group, wherein the specific reaction system and the reaction conditions are shown in tables 6 and 7:
TABLE 6 Real-time RT-PCR reaction System (operating on ice)
TABLE 7 Real-time RT-PCR reaction conditions
Performing RT-PCR reaction by using an ABI QuantStudio 3 real-time fluorescent quantitative PCR instrument, performing Meng curve analysis on an amplification product of each gene, judging whether the PCR product is specifically amplified, and determining the specificity and the purity of the PCR product; analyzing the Ct value of each gene, calculating the delta Ct value and the delta Ct value of each gene, and evaluating the relative expression quantity of the target gene by adopting a 2-delta Ct relative quantification method.
Δ Ct = Ct sample-Ct internal reference
Δ Δ Ct = Δ Ct experimental group- Δ Ct control group.
3. Western Blotting detection of the expression level of key proteins of TLR4/MyD 88/NF-kB signaling pathway:
and analyzing signal channel key proteins in the colon tissues of the mice by adopting a Western blotting method. Extracting total protein in colon tissue, utilizing BCA protein kit to quantitatively determine protein concentration, and performing western blotting operation after protein denaturation, wherein the specific steps are as follows. The optical density values of the target bands were analyzed using a GelDoc Go gel imaging system exposure development, IPWIN60 software processing system. By taking GAPDH as an internal reference, the expression changes of target proteins NF-kB p65, p-NF-kB p65, myD88, I kB, p-I kB and TLR4 are determined.
Target protein relative content = target band gray value/internal reference band gray value.
4. Immunohistochemistry method detects the expression of tight junction protein and mucin in colon tissue:
the colon tissue embedded and sliced by paraffin is incubated for 2 hours in an oven at 70 ℃, and then is subjected to xylene dewaxing, antigen retrieval, circle drawing around the tissue by a tissue pen, serum sealing, dropwise adding primary antibody prepared according to a certain proportion and incubating overnight at 4 ℃, dropwise adding secondary antibody of a corresponding species and incubating in a dark place for 50 minutes, DAB color development by a color developing agent, hematoxylin counterstaining cell nucleus, 1% hydrochloric acid alcohol differentiation, ammonia water rewet, absolute ethyl alcohol dehydration and xylene transparent neutral gum sealing. The target proteins are Claudin ZO-1, claudin-2, occludin and mucin MUC-2 in colon, and are scored by using Image J software.
5. Results and analysis
As can be seen from FIG. 9, the NC group mice had a substantially normal intestinal tissue structure, intact mucosal layer structure, regular arrangement, and no degeneration or shedding of epithelial cells; edema and inflammatory cell infiltration are not seen in the submucosa; the number of goblet cells did not decrease and black arrows indicate goblet cells. Histological lesions scored lowest. The DSS group has serious abnormal intestinal tissue structure, partial crypt structure of a mucous layer disappears, and a large number of inflammatory cells and fibrous tissues proliferate as shown by yellow arrows; expansion of the remaining crypts, as indicated by the green arrows; mucosal epithelial cell erosion sloughs off as indicated by red arrows; edema and inflammatory cell infiltration were seen in the submucosa as indicated by blue arrows. The number of goblet cells was greatly reduced. Histological lesions scored highest with significant differences from both NC and JEC groups (p < 0.0001). JEC can reduce the shedding phenomenon of epithelial cells on a mucous layer and ensure that the arrangement is more compact; inflammatory cell infiltration is reduced; goblet cell numbers increased and histological lesion scores increased.
As can be seen from FIG. 10, compared with the NC group, the mRNA expression levels of NF-kB p65, myD88 and TLR4 in colon tissues of mice after DSS induction are obviously increased, and the mRNA expression level of IkB is obviously reduced (p < 0.05), which indicates that the transcription levels of TLR4, myD88 and NF-kB p65 genes in the colon of the mice after modeling are increased, and the IkB is opposite to the above. Compared with the DSS group, the mRNA expression levels of TLR4, myD88 and NF-kappa B p65 in the JEC group are obviously reduced, and the mRNA expression level of I kappa B is obviously increased and is close to the level of mice in the NC group.
As can be seen from FIG. 11, compared with the NC group, the expression levels of NF- κ B p65, p-NF- κ B p65, myD88, p-I κ B and TLR4 proteins in colon tissues of mice after the DSS induction are significantly increased, and the expression level of I κ B proteins is significantly reduced. In contrast to the DSS group, the JEC group inhibited expression of NF-. Kappa.B p65, p-NF-. Kappa.B p65, myD88, p-Ikappa.B, TLR4 protein, and Ikappa.B in contrast. And the result is consistent with the result of RT-PCR, which shows that the JEC has the function of inhibiting a TLR4/MyD 88/NF-kB signal channel of a mouse.
As can be seen from FIG. 12, the expression level of Claudin-2 gene negatively correlated to the tight junction structure was significantly increased and ZO-1, claudin-1, occludin, MUC-2 were significantly decreased (p < 0.05) in the mice of the DSS group, as compared with the NC group. JEC can prevent the loss of TJ protein and mucin in intestinal tracts of DSS-induced colitis mice, protect the permeability of colon tissues of the mice, maintain the steady state of the intestinal tracts, and has more obvious protective effect on the TJ protein.
As can be seen from FIGS. 13 to 14, compared with the NC group, the average optical density of Claudin-2 negatively correlated to the tight junction structure in the mice of the DSS group is greatly increased, and ZO-1, claudin-1, occludin and MUC-2 are greatly reduced (p < 0.0001), which indicates that the intestinal mucosal barrier of the mice is seriously damaged; it is worth noting that the JEC group has the most obvious effect, and the intestinal mucosa barrier function can be further improved by adjusting the average optical density values of the Claudin ZO-1, claudin-1, occludin and MUC-2 up and down.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (7)
1. The lactobacillus paracasei JY062 composition is characterized by comprising lactobacillus paracasei JY062 (B: (B) (B))Lactobacillus paracasei JY 062) and lactobacillus paracasei JY 062; the content of the lactobacillus paracasei JY062 is not less than 10 9 CFU/mL; the concentration of the exopolysaccharide is not lower than 30mg/mL;
the preparation method of the exopolysaccharide of the lactobacillus paracasei JY062 comprises the following steps:
step one, inoculating lactobacillus paracasei JY062 into a sterilized MRS liquid culture medium according to the inoculation amount of 5 percent, wherein the initial concentration is 1.5 multiplied by 10 9 CFU/mL；
Step two, adding the optimized composite carbon source MRS liquid culture medium in an equivalent manner; culturing at 34 deg.C in a fermenter at 100rpm/min for 25h, and supplementing 20% w/v ammonia water during fermentation; after fermenting for 12 hours, starting to supplement 100mL/30min of MRS liquid culture medium until the end, and collecting bacterial liquid;
centrifuging the bacterial liquid, taking the supernatant I, and repeating the step twice;
step four, taking the supernatant I, and concentrating the volume to 1/3 of the original volume to obtain a culture medium;
step five, inactivating enzyme of the culture medium, cooling to room temperature, and collecting supernatant liquid II;
removing protein from the supernatant fluid II, and centrifuging to obtain a supernatant fluid III;
precipitating the supernatant III with ethanol, centrifuging and taking the precipitate;
step eight, resuspending the pellet in deionized water to obtain polysaccharide solution, placing the polysaccharide solution in an MD34 dialysis bag, wherein the molecular weight cut-off is as follows: 8KD-14KD, dialyzing in deionized water at 4 deg.C for 48h, and replacing deionized water every 8 h;
step nine, pouring the dialyzed polysaccharide supernatant into a flat plate, placing the flat plate in a refrigerator at the temperature of-80 ℃ for pre-freezing overnight, then carrying out freeze drying for 24h, and obtaining extracellular polysaccharide after freeze drying;
in the second step, the optimized composite carbon source MRS liquid culture medium comprises the following components: 10g/L peptone, 8g/L beef powder, 4g/L yeast powder, 2g/L diammonium hydrogen citrate, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 0.2g/L magnesium sulfate, 0.04g/L manganese sulfate, 1g/L Tween-80, 3.33g/L fructose, 3.33g/L mannose, 3.33g/L trehalose, 3.33g/L glucose, 3.33g/L galactose and 3.33g/L lactose; dissolving 52.24g of the components in 1000mL of distilled water, stirring with a glass rod, carrying out high-pressure sterilization at 121 ℃ for 15min, and obtaining the optimized composite carbon source MRS liquid culture medium.
2. The lactobacillus paracasei j062 composition according to claim 1, wherein in step one, the composition of MRS liquid medium comprises: 10g/L peptone, 8g/L beef powder, 4g/L yeast powder, 20g/L glucose, 2g/L diammonium hydrogen citrate, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 0.2g/L magnesium sulfate, 0.04g/L manganese sulfate and 1g/L Tween-80; dissolving 52.24g of the components in 1000mL of distilled water, stirring by a glass rod, carrying out autoclaving at 121 ℃ for 15min, and obtaining the MRS liquid culture medium.
3. The lactobacillus paracasei j062 composition according to claim 1, wherein the centrifugation process is: centrifuging at 10000r/min and 4 deg.C for 15min.
4. The lactobacillus paracasei JY062 composition according to claim 1, wherein in step five, the process of inactivating enzyme is as follows: heating in boiling water for 10min.
5. The lactobacillus paracasei j062 composition according to claim 1, wherein the process of protein removal in step six is: 80% w/v of trichloroacetic acid was added to the supernatant II to a final concentration of 10% w/v, followed by stirring for 5min and standing at 4 ℃ for 10h,10000r/min, and centrifuging at 4 ℃ for 15min to remove proteins.
6. The lactobacillus paracasei j062 composition according to claim 1, wherein in step seven, the alcohol precipitation process is: adding 2-3 times volume of anhydrous ethanol into the supernatant III to make the final concentration 70%, standing overnight at 4 deg.C, centrifuging at 10000r/min at 4 deg.C for 15min.
7. The use of a lactobacillus paracasei JY062 composition according to claim 1 in the preparation of a medicament for improving gut mucosal barrier injury.
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| CN110893195A (en) * | 2019-09-30 | 2020-03-20 | 内蒙古伊利实业集团股份有限公司 | Lactobacillus paracasei ET-22 with function of relieving intestinal inflammation |
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