CN109481475A - Application of the bacteroides fragilis in the reinforcing agent of preparation enhancing gut barrier function - Google Patents

Application of the bacteroides fragilis in the reinforcing agent of preparation enhancing gut barrier function Download PDF

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CN109481475A
CN109481475A CN201710814290.7A CN201710814290A CN109481475A CN 109481475 A CN109481475 A CN 109481475A CN 201710814290 A CN201710814290 A CN 201710814290A CN 109481475 A CN109481475 A CN 109481475A
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bacteroides fragilis
intestinal
reinforcing agent
barrier
barrier function
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智发朝
王晔
刘洋洋
白杨
王从峰
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Guangzhou Zhiyi Biotechnology Co Ltd
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Guangzhou Zhiyi Biotechnology Co Ltd
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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Abstract

The present invention relates to application of the bacteroides fragilis in the reinforcing agent of preparation enhancing gut barrier function.Inventor reduces the function of the permeability of intestinal mucosa, can effectively enhance Intestinal mucosal barrier, protect human health by repairing enteric epithelium mucus barrier experimental results demonstrate: bacteroides fragilis has.

Description

Application of the bacteroides fragilis in the reinforcing agent of preparation enhancing gut barrier function
Technical field
The present invention relates to the applied technical fields of bacteroides fragilis, and in particular to bacteroides fragilis enhances intestinal mucosa in preparation Application in the reinforcing agent of barrier function.
Background technique
With going deep into for basic medical research, people gradually recognize that intestinal mucosa not only only has digestion and absorption function, and And there is important defensive barrier function.Enteron aisle is maximum contact surface between human body and external environment, is had in enteric cavity a large amount of Bacterium and a variety of toxin, but under normal circumstances, serious infection or poisoning disease do not occur for people.This is mainly intestinal mucosa Barrier effectively blocks the invasion of external microorganism and toxin, ensure that the health of body.However, in the case that it is a variety of stress Or intestinal mucosa, locally when being destroyed, this barrier function weakens, the invasive organism and toxin being present in enteric cavity There is bacterial translocation across Gut barrie r into lymphatic system and blood circulation, causes bacteremia or toxaemia, or even occur Inflammatory cascade reaction leads to multiple organ dysfunction.
Gut barrier is made of the mechanical barrier of intestinal mucosa, chemical barrier, immunization barrier and biological barrier.
1, the mechanical barrier of intestinal mucosa: there are structures several in this way on intestinal mucosa surface layer: not moving water layer, slime layer (including most The hydrophobic layer on upper layer), epithelial layer (close-coupled including epithelial cell).In addition, the endothelium and blood flow of intestines capillary are also right The barrier function of intestinal mucosa has a major impact.(1) do not move water layer: it is the outermost layer of mechanical barrier, and depth is about 100 ~800 μm, it is the rate-limiting step of many nutriments and drug absorption.After fat-soluble object has to pass through micronized, Cai Nengtong Cross this layer.(2) hydrophobic layer of mucous membrane surface: this is one layer of active phospholipid layer, is lining in the top layer of mucus.Certain detergents, NSAIDs can remove this phospholipid layer, to reduce the hydrophobicity of mucous membrane surface, the mucous membrane for increasing hydrophilic macromers seeps Permeability.(3) slime layer: this is one layer of viscose spline structure.It can prevent physical friction, chemical digestion and the resistance of epithelium villus Only pathogenic bacteria stick with intestinal mucosa, are colonized.In addition, there is the receptor for specializing in anaerobic bacteria combination in slime layer, obligate anaerobe is made to dwell Breath, plays its colonization resistance.Inhibit goblet cell to generate mucus using colchicine, glutinous permeability of the membrane can be dramatically increased.It is long Phase total parenteral nutrition can also destroy mucigel.(4) epithelial layer: it is a highly selective barrier, only allows nutritional substance It absorbs, limits the absorption of noxious material.This selection may be related with the size and its physicochemical property for penetrating substance.Enteric epithelium There are two approach, i.e. transepithelial approach and paracellular pathway (close-coupled) for permeability.The latter under normal circumstances, only allows 2 μm Small-molecule substance below passes through, and is that control macromolecular substances (such as endotoxin and opsonigenous substance) enters intracorporal key. Osmotic pressure increases in the studies have shown that enteric cavity of rat, can promote permeability by the cells of macromolecular substances, eat hypertonic saline and After glucose, Intestinal permeabiligy increases.It maintains epithelium and the complete primary structure of paracellular pathway is the silk for constituting cytoskeleton Shape structure: parapeptone silk, micro-pipe and intermediate filament.Colchicine, ethyl alcohol can destroy micro-pipe, and oxidant destroys flesh fibre egg It is white, cause Intestinal permeabiligy to increase.(5) endothelial barrier: capillary endothelium barrier plays an important role on maintaining intestinal barrier function. This may be related to supply of blood flow, and enough blood supplies are likely to provide nutrition, oxygen and energy (ATP), be possible to by Damaging agents exclude rapidly (such as oxygen radical).
2. chemical barrier: bile, lysozyme and acetic acid of enteron aisle probiotics secretion etc. have certain sterilization and bacteriolysis With constituting the chemical barrier of enteron aisle.
3. immunization barrier: enteron aisle is the maximum immune organ of human body.The immune system of intestinal mucosa include lymphonodi mesenterici, Thick liquid cell, B cell, helper T lymphocyte and M cell (mast cell).Signal is transmitted to by M cell after absorbing bacterial antigens T, B lymphoblast, the latter are colonized again by blood circulation in intestinal mucosa lower layer, and mature T, bone-marrow-derived lymphocyte are further divided into. The average secretion 3gIgA immunoglobulin daily of intestinal mucosa, it can inhibit in enteron aisle bacterial adsorption to Intestinal epithelial cells surface, It is prevented to be colonized in epithelial cell.In addition, IgA immunoglobulin package bacterium (can wrap up in enteron aisle 60%~80% G- it is thin Bacterium), weaken bacterium to the ability that Intestinal epithelial cells receptor is mobile and combines, and further stimulate intestinal secretion mucus, has Help drain the bacterium in enteron aisle and toxin.Macrophage positioned at intestinal mucosa lamina propria has phagocytosis alien bacteria and toxin Ability.Macrophage can generate TNF-α to antibacterium again after being stimulated by toxin.
4. biological barrier: parasitizing about 500 kinds of bacterium of human body, be mostly present in enteron aisle.Intestinal bacterium is about The 20%~30% of excrement weight in wet base is accounted for, wherein most (99.9%) is anaerobic bacteria.The ancestral home bacterium (probiotics) of these human bodies Stick with intestinal mucosa, in conjunction with, the chimeric biological barrier for forming a complete mycoderm composition enteron aisle.Under normal circumstances, probiotics By occupy-place protection, acidic materials and bacteriocin, the field planting for fighting for the modes antagonism pathogenic bacteria such as nutrition are generated, play a kind of biology Barrier action.
In addition, nontoxic, harmless, non-pathogenic oxygen consumption microorganism (such as Bacillus cereus, Bacillus subtillis) is temporarily It is colonized in enteron aisle, makes the reduction of local environment oxygen molecule concentration, oxidation-reduction potential decline causes to be suitble to normal bowel dominant bacteria The microenvironment of group-anaerobic bacteria growth, promotes the growth of anaerobic bacteria, finally restores normal microecological balance, also play biology Protective effect.Beneficial bacteria of intestinal tract also can produce a variety of antigenicity substances, promote body immunity, and stimulation immunocyte promotes to gulp down Phagocyte vigor promotes bone-marrow-derived lymphocyte to generate antibody.Beneficial bacteria of intestinal tract also manufactures the ability of multivitamin and protein, It generates glycuronidase, vulcanization enzymatic and enters hepato-enteric circulation into cholesterol.Beneficial bacteria of intestinal tract also participates in N- nitrite degradation, Play antitumaous effect.
Gut barrier destruction will lead to the consequences such as bacterial translocation, endotoxemia.Bacterial translocation refers to: parasitizing large intestine Pathogenic bacteria, such as Escherichia coli can translocate to small intestine (lateral transposition), can also enter mesenteric lymph across intestinal mucosa Knot and portal vein, subsequently into body circulation (longitudinal transposition).Either it is equal to enter circulation for small bowel bacterial overgrowth or bacterium It can cause serious systemic physiologic dysfunction.Endotoxemia refers to: the mass propagation of intestinal bacterium causes excessive poison Element enters portal vein and liver, once liver removes the ability decline of toxin, endotoxemia just occurs.And endotoxin not only can be straight Connect damage important organ, can also activating complement and clotting mechanism, there is Respiratory Distress Syndrome(RDS) and disseminated intravascular coagulation, very To generation multiple organ failure.
Therefore, drug or the method for enhancing function of intestinal mucosa barrier in patient are extremely important for people's health.With enteric epithelium The biological function research of cell tight junction is goed deep into, to the close-connected study on prevention also gradually weight by many scholars Depending on, but be lack of pertinence mostly.It has been most of that glutamine plays a significant role in maintaining enterocyte barrier function Scholar is received, but its mechanism is still not very clear.Therefore, the new means tool that can effectively enhance function of intestinal mucosa barrier in patient is studied It is significant.
Summary of the invention
Based on this, the present invention provides a kind of new opplications of bacteroides fragilis.
Specific technical solution is as follows:
Bacteroides fragilis (bacteroides fragilis) is in the reinforcing agent of preparation enhancing gut barrier function Using.
Bacteroides fragilis (bacteroides fragilis) be Gram-negative, without gemma, obligate anaerobic it is small Bacillus, belongs to Bacteroides, is normally lodged in human body intestinal canal, oral cavity, respiratory tract etc..According to whether there is or not carry the gene of bft1,2,3 point For toxic strain and avirulent strain.Not enterotoxigenic bacteroides fragilis (bacteroides fragilis) has many effects, tool There are the potentiality as new probiotics.
In wherein some embodiments, the bacteroides fragilis (bacteroides fragilis) is for deposit number Bacteroides fragilis (bacteroides fragilis) ZY-312 of CGMCC No.10685.
In wherein some embodiments, the reinforcing agent is drug or health food.
In wherein some embodiments, the dosage form of the drug is pill, tablet, granule, capsule, oral solution, aerosol Agent or tube feed preparation.The drug includes people's medication or animal-use drug, can be used for human or animal.
In wherein some embodiments, the health food is milk powder, ice cream, milk base fermented food or cereal fermentation food Product.The food can also be animal foodstuff, such as feed etc..
In wherein some embodiments, the milk base fermented food is cheese, curdled milk, yoghourt.
In wherein some embodiments, the food is baby food or pet food.
The present invention also provides a kind of reinforcing agents for enhancing gut barrier function.
Specific technical solution is as follows:
A kind of reinforcing agent enhancing gut barrier function, it is CGMCC No.10685 that the reinforcing agent, which contains deposit number, Bacteroides fragilis (bacteroides fragilis) ZY-312.
In wherein some embodiments, the reinforcing agent is drug or health food.
In wherein some embodiments, the dosage form of the drug is pill, tablet, granule, capsule, oral solution, aerosol Agent or tube feed preparation.
In wherein some embodiments, the health food is milk powder, ice cream, milk base fermented food or cereal fermentation food Product.
In wherein some embodiments, the bacteroides fragilis (bacteroides fragilis) can be with other adjustings The Drug combination of intestinal mucosal barrier, described other adjust the drugs of intestinal mucosal barriers including but not limited to Salazosulfamide Pyridine Salicylic Acid Formulations (such as Etiasa, mesalazine), corticosteroid (such as prednisone, dexamethasone) and/or immunosupress Agent (such as imuran).
Bacteroides fragilis (bacteroides fragilis) ZY-312 of the invention, is preserved on April 2nd, 2015 China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number are CGMCC No.10685, Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The present inventor accumulates by protracted experience and bacteroides fragilis has been excavated in a large amount of creative experiments researchs (bacteroides fragilis) new purposes has opened up a new application field for bacteroides fragilis.Inventor passes through Experimental results demonstrate: bacteroides fragilis, which has, repairs enteric epithelium mucus barrier, reduces the function of the permeability of intestinal mucosa, can have Effect enhancing Intestinal mucosal barrier, protects human health.Bacteroides fragilis provided by the invention does not have any secondary work to body With, while the Drug combination of intestinal mucosal barrier can also be adjusted with other, to have predictive of bacteroides fragilis fine Edible and prospect in medicine, provide a kind of health care that suitable human body is taken for clinic and prevent non-defective unit.
Detailed description of the invention
Fig. 1 is the colony characteristics figure of bacteroides fragilis (bacteroides fragilis) ZY-312 of embodiment 1；
Fig. 2 is after bacteroides fragilis (bacteroides fragilis) ZY-312 of embodiment 1 carries out Gram's staining Micro- sem observation figure；
Fig. 3 is-the 14 day the 0th day stools scored curve graph of embodiment 2；
Fig. 4 is the colon HE colored graph of each group experiment in embodiment 3；
Fig. 5 is the expression feelings of barrier GAP-associated protein GAP ZO-1, occludin, b-catenin and claudin-1 in embodiment 3 Condition figure.
Specific embodiment
The present invention is described in further details below by way of specific embodiment, these embodiments are only used to illustrate this hair It is bright, it does not limit the scope of the invention.
Bacteroides fragilis used in following embodiment is bacteroides fragilis (bacteroides fragilis) ZY-312, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation are preserved on April 2nd, 2015 Number is CGMCC No.10685, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The separation of 1 bacteroides fragilis of embodiment and the preparation of sample
(1) preparation of the Zengjing Granule and living bacterial liquid of bacteroides fragilis
By strain streak inoculation in blood plate, Anaerobic culturel 48h.Observe colony morphology characteristic, dyeing property, size, ball Rod-shaped and distribution situation etc..
Colony characteristics: after bacteroides fragilis ZY-312 cultivates 48h on blood plate, round dimpling, translucent, white is presented Color, surface be smooth, not haemolysis, colony diameter between 1-3 mm, referring to Fig. 1.
Form under microscope: bacteroides fragilis ZY-312 carries out gram stain microscopy, is gram-negative bacteria, allusion quotation is presented Rod-shaped, both ends blunt circle and the dense dye of type, not colored part is shaped like vacuole among thallus, referring to fig. 2.
Selection single bacterium colony, which is inoculated in the tryptone meat soup of improvement, carries out Zengjing Granule, gained bacterium solution centrifugation, Revolving speed 3000r/min is centrifuged 15min, removes supernatant, normal saline dilution is used after sediment brine, with Maxwell ratio Turbid pipe does Counting alive microbial, is diluted to 106CFU/mL, 108CFU/mL, 1010CFU/mL is respectively labeled as sample A, sample B and sample Product C, saves backup.
Curative effect of 2 bacteroides fragilis of embodiment to rat ulcer enteritis
One, experimental method
The present embodiment is established big by 2,4- dinitrobenzene sulfonic acid (dinitrobenzenesulfonate, DNBS) induction Mouse ulcerative enteritis (ulcerative colitis, UC) model.110 Wistar rats of the present embodiment, half male and half female, Xiang great The DNBS (DNBS is dissolved in 30% ethyl alcohol, ready-to-use) that perfusion 0.5mL concentration is 50mg/mL in mouse enteron aisle induces modeling. Every experiment is assigned with a unique number with rat.Before being grouped to rat, it should be marked on the label of mouse cage Project number, kind/strain, gender, cage number and animal number.Using BioBook software according to the original body mass of rat carry out with Machine grouping.
Day-2 starts, Rat Fast 48h.5% glucose salt solution (10mL/kg) is as experimental animal in the fasting phase Between energy extender.
After the day 0 of experiment, fasting rat free from worries (25mg/kg) anesthesia (extremely from rat anus to left flexure of colon Anus about 8cm) at inject 0.5mL, the DNBS of 50mg/mL carrys out induced rat enteritis.Group1 group rat injects 30% ethyl alcohol.Greatly Mouse keeps posture 15min upside down until it is revived, to prevent DNBS from flowing backwards.
Specific experiment grouping and dosage regimen are as follows:
1 experimental group of table and dosage regimen
Two, observation index and judgement
1, stools scored and collection
It in experimentation, scores daily rat excrement state, (0=is normal, and 1=is moist/viscous, and 2=is soft, 3= Liquid)；Every group of animal is divided into two batches, is denoted as day 0 on the day of modeling.
2, serum collection observes Colon and rectum
Every batch of animal collects peripheral blood before dissection, is placed at room temperature for after 30 minutes with 2000g, 4 DEG C are centrifuged 10 minutes. Separate about 1mL supernatant, cryo-conservation.Two batches animal solves after free from worries (50mg/kg) anesthesia in day 7 and day 14 days respectively It cuts open, cuts off abdominal cavity, take out Colon and rectum, longitudinally split, score the fecal character in Colon and rectum, rinse Colon and rectum well Afterwards, ulcer surface, record ulcer area, human colorectal length and weight are observed, and is integrally taken pictures.Rat colon damage score criteria It is shown in Table 2.
Whole section of colorectal carcinoma (from caecum to anus) of experimental animal is taken, vertical to be divided into two, wherein half colorectal carcinoma is set In liquid nitrogen, -80 DEG C of preservations；The other half is fixed with 10% formalin solution, with spare.
2 Macroscopic Evaluation rat colon Injury score of table
Three, result and analysis
The 0th day-the 14th day stools scored of table 3
The 7th day intestinal adhesion degree of table 4, intestinal obstruction degree, ulcer level, the scoring of intestinal wall thickened degree
The macroscopic evaluation of the 14th day intestines length of table 5, weight and ulcer area
Using the weight of mean value ± standard error (Mean ± S.E.M) statistics animal, human colorectal length, weight and burst Ulcer area.Colon and rectum correlated variables carries out multiple analysis with ANOVA combination Dunnett ' s.The weight of animals related data uses ANOVA carries out Multiple range test.As P < 0.05, then it is assumed that statistically variant.
This experiment induces it to generate ulcerative colitis, colitis performance Wistar rat with DNBS intracolonic administration Are as follows: colon weight increases, colon weight: weight ratio (i.e. CW/BW) increases, and colonic ulcer area increases, and daily excrement is commented Divide and increase, intestinal adhesion degree and intestinal wall thicken.In this experiment, positive drug control group (sulfasalazine) and the present invention mention Low (10 supplied6CFU/mL), (10 in8CFU/mL), height (1010CFU/mL) dosage bacteroides fragilis reduces Wistar rat Daily stools scored, colon weight and ulcer area, showing significantly reduces the effect that intestinal adhesions and intestinal wall thicken, and is detailed in Table 3, table 4, table 5 and Fig. 3.
Bacteroides fragilis low (106CFU/mL), (10 in8CFU/mL), height (1010CFU/mL) dosage group is shown very well Inhibition ulcerative enteritis drug effect.By to each group laboratory test results data analyze as it can be seen that low dosage (experimental group 1) i.e. The drug effect for showing inhibition ulcerative enteritis, by improving dosage (experimental group 2, experimental group 3), to the medicine for inhibiting ulcerative enteritis Effect is substantially close to Normal group or positive drug control group.Bacteroides fragilis low (106CFU/mL), (10 in8CFU/mL), high (1010CFU/mL) CW/BW (colon weight/weight * 100), CW/CL/BW (colon weight/colon can be effectively reduced in dosage group Length/weight * 1000), wherein the bacteroides fragilis of high dose significantly reduces CW/BW (colon weight/weight * 100), CW/CL/ BW (colon weight/colon lengths/weight * 1000), see Table 5 for details.
In the animal dissect of Day 7 and Day 14, bacteroides fragilis low (106CFU/mL), (10 in8CFU/mL), high (1010CFU/mL) three dosage groups of dosage can significantly reduce intestinal adhesion degree and intestinal wall thickened degree, score ulcer area Also there is certain inhibitory effect.In conclusion bacteroides fragilis provided by the invention has the DNBS rat colonitis induced Apparent therapeutic effect has the function of enhancing intestinal mucosal barrier.
Embodiment 3, intestinal permeability detection
Intestinal permeability is at a normal level the invasion that can effectively stop external microorganism and toxin, is conducive to body Interior ambient stable, be the guarantee of body health.The lesion that Day7, Day14 are collected in the present embodiment selection example 2 is most obvious Colonic segment detect bacteroides fragilis provided by the invention to the adjustment effect of intestinal permeability, detect following items:
1) histopathologic change: HE dyeing observation tissue change, inflammatory infiltration situation；Transmission electron microscope observing lesion ultra micro knot Structure variation closely connects between observation epithelial cell form, epithelium, epithelial permeability variation.The present embodiment is selected in embodiment 2 just The colon that Day7 is collected in normal control group, model control group, positive drug control group and experimental group 1, conventionally carries out HE dyeing observation, as a result such as Fig. 4.It is observed that the epithelium of intestinal mucosa layer integrality of model control group is by broken under microscope It is bad, with inflammatory cell infiltration, oedema under crypt distorted deformation and mucous membrane.And Normal group, positive drug control group and reality The rat colon tissue for testing group 1 has no apparent pathological change.
2) intestinal permeability: using everted intestinal sac, and the intestines capsule of preparation is placed in and is added to lactulose and mannitol In HBSS buffer (pH7.4), the intracapsular culture solution of intestines after cultivating is collected, is measured through high performance liquid chromatograph.
Specific steps:
(1) preparation of everted intestinal sac: respectively in Example 2 in model control group, positive drug control group and experimental group 2 The most apparent colonic segment of Day7 lesion, removes mesenterium, is rinsed well with physiological saline (or buffer), turn up make intestinal mucosa to Outside, ligation one end forms intestines cryptomere, and perfusion HBSS buffer (pH7.4) ligatures the other end afterwards, and marks.
(2) everted intestinal sac prepared by step (1) is placed in the culture solution for being added to lactulose and mannitol, is passed through 95% The mixed gas of oxygen and 5% carbon dioxide cultivates 2h.
(3) culture solution extracted respectively with syringe in intestines capsule carries out high performance liquid chromatograph measurement.
As a result: the ratio of lactulose and mannitol content is in model control group, positive drug control group and experimental group 2 0.079 ± 0.51,0.021 ± 0.003 and 0.026 ± 0.007, positive drug control group and experimental group 2 are found from testing result Being substantially reduced in the odds ratio model control group of middle lactulose and mannitol content, significant difference (P < 0.05) have statistics Learn meaning.As it can be seen that the permeability of rat colon section intestinal mucosal barrier obviously increases after DNBS is handled, and through willow nitrogen sulphur pyrrole It can obviously reduce the permeability of rat colon section intestinal mucosal barrier after pyridine and bacteroides fragilis treatment.
3) after intestinal tissue homogenate, TNF-α, IFN-γ, IL-13, IL-17 intestinal wall inflammation: are detected.
Specific step is as follows:
TNF-α, IFN-γ, IL-13, the measurement of IL-17 level cut intestinal segment by kit requirement respectively, remove fatty mesentery Tissue, 10% homogenate of physiological saline preparation on the rocks, -20 DEG C of storages are to be measured；
In addition to blank well, the standard items (100 hole μ L/) of sample or various concentration are added in corresponding aperture respectively, use sealing plate Gummed paper seals reacting hole, and 37 DEG C of insulating boxs are incubated for 90min；
Get rid of liquid in enzyme mark version, board-washing 4 times, every time 5 minutes；
In addition to blank well, it is added in biotin antibody working solution (100 hole μ L/), seals reacting hole, 37 DEG C of perseverances with sealing plate gummed paper Incubation 60min；
With 0.01M PBS board-washing 4 times, every time 5 minutes；
In addition to blank well, Avidin-peroxide complex (ABC) working solution (100 hole μ L/) is added and is sealed with sealing plate gummed paper Firmly reacting hole, 37 DEG C of insulating boxs are incubated for 30min；
With 0.01M PBS board-washing 4 times, every time 5 minutes；
It is added in color developing agent (100 hole μ L/), is protected from light 37 DEG C of insulating boxs and is incubated for 10~15min；
It is added in terminator (100 hole μ L/), measurement OD value at 450nm is engraved in after mixing；
After each sample OD value subtracts the OD value of blank well, standard curve is established, is calculated in each sample according to standard curve TNF-α, IFN-γ, IL-13, IL-17 content.
The results are shown in Table 6: TNF-α in the homogenate of Normal group mouse Colon, IFN-γ, IL-13, IL-17 content point It Wei not 9.34 ± 3.45ng/g, 7.94 ± 1.52ng/g, 8.05 ± 1.34ng/g, 15.04 ± 3.77ng/g；Model control group is small TNF-α in the homogenate of mouse colon, IFN-γ, IL-13, IL-17 content is respectively 35.42 ± 5.36ng/g, 21.84 ± 4.96ng/ g,18.09±3.12ng/g,38.97±5.67ng/g.Compared with rats in normal control group, the homogenate of model control group rat colon Middle TNF-a, IFN-γ content obviously increase (P < 0.01), prompt Intestinal Mucosa in immune imbalance, proinflammatory cytokine water Dawn, which shows, increases.Compared with model control group rat, by willow ammonia sulphur pyridine (positive drug control group) and provided by the invention Bacteroides fragilis (experimental group) treatment after, rat colon homogenate in TNF-a, IFN-γ content be substantially reduced (P < 0.01), with TNF-a, the content of IFN-γ are suitable in the homogenate of rats in normal control group colon, and difference is not significant (P > 0.05).
TNF-α in the homogenate of 6 colon of table, IFN-γ, IFN-13, IL-17 content
Cell factor Normal group Model control group Positive drug control group Experimental group 1
TNF-α(ng/g) 9.34±3.45 35.42±5.36* 10.06±2.39 9.88±3.20
IFN-γ(ng/g) 7.94±1.52 21.84±4.96* 8.65±2.03 9.74±3.41
IFN-13(ng/g) 8.05±1.34 18.09±3.12* 8.53±1.67 10.21±2.19
IL-17(ng/g) 15.04±3.77 38.97±5.67* 16.78±4.31 16.33±4.01
Note: * indicates that Normal group compares P < 0.05, significant difference.
4) intestinal mucosal barrier GAP-associated protein GAP mainly has ZO-1, occludin, b-catenin and claudin-1, the present embodiment Use the Normal group in Day14 in immunohistochemistry, Westernblot method detection embodiment 2, model control group, positive drug The difference of object control group and intestinal mucosal barrier the GAP-associated protein GAP ZO-1, occludin, b-catenin and claudin-1 of experimental group 1 It is different.As a result as shown in Figure 5.
Fig. 5 is as the result is shown: the intestinal mucosa screen of ZO-1, occludin, b-catenin and claudin-1 in model control group Hinder GAP-associated protein GAP expression be significantly lower than Normal group, and positive drug control group and experimental group 1 respectively through willow ammonia sulphur pyridine, After bacteroides fragilis treatment, the expression quantity of intestinal mucosal barrier GAP-associated protein GAP ZO-1, occludin, b-catenin and claudin-1 It obviously increases, wherein the expression quantity in experimental group 1 is suitable with Normal group.As it can be seen that bacteroides fragilis provided by the invention can The expression quantity of intestinal mucosal barrier GAP-associated protein GAP is significantly improved, the effect of intestinal mucosal barrier can be effectively enhanced.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Claims (9)

1. bacteroides fragilis (bacteroidesfragilis) answering in the reinforcing agent of preparation enhancing gut barrier function With.
2. application according to claim 1, which is characterized in that the bacteroides fragilis (bacteroides fragilis) For bacteroides fragilis (bacteroidesfragilis) ZY-312 that deposit number is CGMCC No.10685.
3. application according to claim 1 or 2, which is characterized in that the reinforcing agent is drug or health food.
4. application according to claim 3, which is characterized in that the dosage form of the drug is pill, tablet, granule, glue Capsule, oral solution, aerosol or tube feed preparation.
5. application according to claim 3, which is characterized in that the health food is milk powder, ice cream, milk base fermentation food Product or cereal fermented food.
6. a kind of reinforcing agent for enhancing gut barrier function, which is characterized in that the reinforcing agent contains deposit number and is Bacteroides fragilis (bacteroidesfragilis) ZY-312 of CGMCCNo.10685.
7. the reinforcing agent of enhancing gut barrier function according to claim 6, which is characterized in that the reinforcing agent is medicine Object or health food.
8. application according to claim 7, which is characterized in that the dosage form of the drug is pill, tablet, granule, glue Capsule, oral solution, aerosol or tube feed preparation.
9. application according to claim 7, which is characterized in that the health food is milk powder, ice cream, milk base fermentation food Product or cereal fermented food.
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Application publication date: 20190319