CN116426417A - Lactobacillus curvatus LC02 and application thereof in preparing medicines for treating or preventing allergic diseases - Google Patents
Lactobacillus curvatus LC02 and application thereof in preparing medicines for treating or preventing allergic diseases Download PDFInfo
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- CN116426417A CN116426417A CN202310249439.7A CN202310249439A CN116426417A CN 116426417 A CN116426417 A CN 116426417A CN 202310249439 A CN202310249439 A CN 202310249439A CN 116426417 A CN116426417 A CN 116426417A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses Lactobacillus curvatus LC02 and application thereof in preparing medicines for treating or preventing allergic diseases, and belongs to the technical field of microorganisms. The preservation number of the Lactobacillus curvatus LC02 is CGMCC No.22986. Lactobacillus curvatus LC02 can remarkably inhibit compound 48/80 from stimulating zebra fish to secrete Tryptase in vivo, can inhibit in vivo mast cells or basophils from degranulation, and has the potential of being applied to in vivo treatment or prevention of anaphylactic reaction. The Lactobacillus curvatus LC02 disclosed by the invention has a great potential application prospect in the aspect of treating or preventing allergic reaction.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to Lactobacillus curvatus LC02 and application thereof in preparing medicines for treating or preventing allergic diseases.
Background
Allergy is a phenomenon that an allergen is stimulated to produce antibodies after entering the body, and when the allergen enters the body again, the allergen is combined with the antibodies in the body, so that the body is induced to generate allergy. Common allergic diseases are allergic rhinitis, which is manifested by sneezing, nasal discharge, and nasal itching. Also allergic asthma, which develops after exposure to allergens, and even dyspnea. Also common allergies are the occurrence of wind clusters, erythema, pimples, even erosion, exudation on the skin, which severely lead to anaphylactic shock.
In order to alleviate allergic reactions, besides suggesting patients to avoid allergens, antihistamines (antihistamines) or pararenal corticoids (also called steroids) are mostly used in clinical medicines for treating allergic diseases to alleviate allergic reactions of patients to allergens. Although the antihistaminic and the parathyroid cortisone can achieve remarkable curative effects on allergic diseases, the antihistaminic and the parathyroid cortisone can have adverse side effects on human bodies after long-term use or arbitrary use without following medical advice. The prevention and treatment effects of probiotics on allergic diseases such as atopic dermatitis, allergic rhinitis, food allergy, etc. have also been studied extensively. However, since the function of probiotics is strain-specific, only specific strains have the function of suppressing allergy.
Accordingly, providing Lactobacillus curvatus LC02 and its use in the manufacture of a medicament for the treatment or prevention of allergic diseases is a problem to be solved by the person skilled in the art.
Disclosure of Invention
In view of this, the present invention provides Lactobacillus curvatus LC02 and its use in the preparation of a medicament for the treatment or prevention of allergic diseases.
Mast cells are the main effector cells of the anaphylactoid reaction, the number of activated mast cells being related to the degree of allergy, the more activated mast cells, the more severe the degree of allergy. Tryptase (Tryptase) is a pre-synthesized neutral protease in mast cells, is the most abundant medium, is released outside cells through degranulation of mast cells, has high selectivity in storage and expression in mast cells, and can be used as a marker for activation and degranulation of mast cells. The Tryptase is normally secreted only in small amounts into the body fluid, and in large amounts into the body fluid when the hypersensitivity causes degranulation of mast cells. The predictive accuracy of the acute allergic positive and negative reactions of Tryptase was 92.6% and 54.3%, respectively, and the half-life of the Tryptase was 2 hours, so that the expression level of the Tryptase was frequently used as one of the detection markers of the allergic reaction.
Mast cells of zebra fish, which are structurally and functionally similar to mammals, are involved in immune and allergic reactions in the body. N-benzoyl-DL-arginine paranitroanilide hydrochloride (BAPNA) is a substrate specific to the Tryptase, and the expression level of the Tryptase in the zebra fish can be detected. The sensitization and antiallergic effects of a substance can be quantitatively analyzed by detecting the expression level of the Tryptase in the zebra fish body within 24 hours after the substance induces the zebra fish body.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
lactobacillus curvatus (Lactobacillus curvatus) LC02 with the preservation number of CGMCC No.22986 is preserved in China general microbiological culture center, CGMCC for short, and has the preservation date of 2021, 07 month and 30 days, and is classified and named Lactobacillus curvatus Lactobacillus curvatus.
Further, the Lactobacillus curvatus LC02 is applied to the preparation of medicines for treating or preventing allergic diseases.
Further, the application of the Lactobacillus curvatus LC02 in preparing medicines for inhibiting mast cells or basophils degranulation.
Further, the Lactobacillus curvatus LC02 is a bacterial suspension.
Compared with the prior art, the invention discloses the Lactobacillus curvatus LC02 and the application thereof in preparing medicines for treating or preventing allergic diseases, wherein the Lactobacillus curvatus LC02 is separated and screened from the faeces of long-life aged people in the city of abaca county, the Guangdong province, the Lactobacillus curvatus LC02 can obviously inhibit compound 48/80 from stimulating the zebra fish to secrete the Tryptase in vivo, can inhibit the degranulation of mast cells or basophils in vivo, has the potential of being applied to treating or preventing allergic reactions in vivo, and provides theoretical reference and guiding basis for developing probiotic preparations for pre-treating or preventing allergic reactions by using the Lactobacillus curvatus LC 02.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a drawing showing colony morphology of Lactobacillus curvatus LC02 according to the invention on MRS agar plates;
FIG. 2 is a graph showing the effect of Lactobacillus curvatus LC02 of the invention on the expression level of compound 48/80 stimulated zebra fish to secrete Tryptase;
FIG. 3 is a graph showing the inhibition rate of Compound 48/80 stimulated zebra fish to secrete Tryptase by Lactobacillus curvatus LC02 according to the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Sodium cromolyn and N-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA) were purchased from Beijing Warce chemical Co., ltd, and Compound 48/80 was purchased from Sigma Co., USA.
EXAMPLE 1 Lactobacillus curvatus LC02 isolation, identification and preservation
(1) Separating:
1) The feces (about 0.1 g) of the elder with long life are dissolved in a 1.5mL centrifuge tube filled with 1mL sterile physiological saline, and are fully blown and evenly mixed by a 1mL sterile gun head for standby.
2) Into each of 6 sterile 1.5mL centrifuge tubes, 900. Mu.L of sterile physiological saline was added.
3) Diluting the sample to 10 by adopting a gradient dilution method -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 。
4) From the package 10 -4 100 mu L of sample diluent is sucked into a centrifuge tube of the sample diluent and respectively inoculated on an MRS solid culture medium and a BS solid culture medium, and 100 mu L of bacterial liquid is spread and dried, and the coating method is mild, fast in action and needs to be operated near the flame of an alcohol lamp. After coating, the side of the dish was marked, including information on name, sample number, medium name, incubation time, dilution gradient, incubation conditions (anaerobic/aerobic), etc.
5) Repeating the above steps to obtain 10 -5 、10 -6 、10 -7 Dilution coating of dilution gradient.
6) After the coating, the dishes were cultured at 37℃under anaerobic conditions for 48 hours, and then were subjected to observation and recording.
7) Single colony on the plate is picked up by an inoculating loop and streaked into MRS solid culture medium, anaerobic culture is carried out for 48 hours at 37 ℃, and pure colony is obtained by separation.
8) Pure bacterial colony on the flat plate is inoculated in MRS liquid culture medium, anaerobic culture is carried out for 12-16 h at 37 ℃, 20% glycerol is added, and the flat plate is placed in a refrigerator at-80 ℃ for preservation.
(2) Molecular biological identification of strains: genomic DNA was extracted from the obtained strain, and a full-length fragment of 16S rDNA was amplified by PCR technique using the universal primers 27F and 1492R of 16S rDNA, followed by sequencing to identify the species of the strain.
The primer sequences of the universal primers 27F and 1492R are as follows:
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；SEQ ID NO.1；
1492R：5’-GGTTACCTTGTTACGACTT-3’；SEQ ID NO.2。
experimental results: the strain screened from the faeces of the elderly in the city of the abaca county of the Guangdong province is identified by morphological observation and 16S rDNA, wherein the strain LC02 is identified as Lactobacillus curvatus, and the 16S rDNA sequence is shown as SEQ ID NO. 3.
CTTAGACGGCTGGCTCCGAGGTTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTAAGAGATTAGCTAAACCTCGCGGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTAAATGCTGGCAACTAGTAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTTTGTCCCCGAAGGGAAAGCTCTATCTCTAGAGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACTACCTGATCAGTTACTATCAAATACGTTCTTCTCCAACAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATTACCCTCTCAGGTCGGCTATGCATCACGGTCTTGGTGAGCCTTTACCTCACCAACTAACTAATGCACCGCGGGTCCATCCTAAAGTGATAGCCGAAACCATCTTTCAACCTTGCACCATGCGGTGCTAGGTTTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTTAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCACTCAAATGTTATCAATCAGAAGCAAGCTTCTTCAATCTAACGAGAGTGCGTCGACTGCATGTATAG；SEQ ID NO.3。
The strain LC02 single colony is inoculated on MRS solid culture medium, grows well under the aerobic condition of 37 ℃, and has milky white, round shape, regular edge and smooth surface (figure 1). The strain LC02 is preserved in China general microbiological culture Collection center (CGMCC), and has a preservation date of 2021, 07 month and 30 days, and is classified and named as Lactobacillus curvatus Lactobacillus curvatus, and a preservation number of CGMCC No.22986.
EXAMPLE 2 preparation of Lactobacillus curvatus LC02 bacterial suspension (thallus)
Inoculating Lactobacillus curvatus LC02 after activation culture in MRS liquid culture medium, culturing at 37deg.C for 24 hr, and centrifuging at 4deg.C for 10min at 6000r/min to obtain thallus precipitate; after the bacterial cell precipitate is washed twice by PBS, the bacterial cell is resuspended by PBS, and the cell concentration is regulated to be 1 multiplied by 10 4 CFU/mL、1×10 5 CFU/mL、1×10 6 CFU/mL gave a bacterial suspension (cell).
Example 3 Effect of Lactobacillus curvatus LC02 on Compound 48/80 stimulation of Tryptase secretion by zebra fish
Healthy wild-type AB-line zebra fish that developed to 5dpf (days post fertilization) were selected and placed in 96-well cell culture plates, 10 strips/well. Experimental setting Normal group, model group, intervention group (positive control group, 1×10) 4 CFU/mL Lactobacillus curvatus LC02, 1X 10 5 CFU/mL Lactobacillus curvatus LC02, 1X 10 6 CFU/mL lactobacillus curvatus LC 02), solvent zeroing groups, each group was provided with 6 duplicate wells. PBS was added to the normal group, PBS was added to the model group, sodium cromolyn solution (100. Mu.g/mL) was added to the positive control group, and 1X 10 4 CFU/mL Lactobacillus curvatus LC02 intervention group addition 1X 10 4 CFU/mL Lactobacillus curvatus LC02, 1X 10 5 CFU/mL Lactobacillus curvatus LC02 intervention group addition 1X 10 5 CFU/mL Lactobacillus curvatus LC02, 1X 10 6 CFU/mL Lactobacillus curvatus LC02 intervention group addition 1X 10 6 CFU/mL bendLactobacillus reuteri LC02, adding PBS into solvent zeroing group (without zebra fish), placing 100 μl of the solution into biochemical incubator at 28deg.C for incubation, and replacing the new solution after 24 hr; after 48h incubation, 150. Mu.L PBS was added to the normal group, and the model group, positive control group, lactobacillus curvatus LC02 interference group (1X 10) 4 CFU/mL、1×10 5 CFU/mL、1×10 6 CFU/mL), solvent zeroing groups were added to compound 48/80 (8. Mu.g/mL), 150. Mu.L per well was incubated at 28℃for 2h, 150. Mu.L per well was placed in 2mL centrifuge tubes, 150. Mu.L BAPNA solution (20 mg/mL) was added to each centrifuge tube, incubated at 37℃for 48h, 200. Mu.L per centrifuge tube was placed in 96 well cell culture plates, and absorbance (OD) was measured at 405nm using a microplate reader. The expression level and inhibition rate of the Tryptase are expressed as follows:
SPSS 19.0 software was used to statistically process the data, the experimental data were all expressed as x+ -SD data, and analyzed by T-test, as compared to the normal group: #### p<0.001; using one-way analysis of variance, compared to model set: * P (P)<0.05，**P<0.01，****P<0.001。
The results of the expression level and the inhibition rate of the Tryptase are shown in fig. 2 and 3, and the results show that compared with the normal group (100.00+/-10.81%), the expression level of the Tryptase in the model group (342.14 +/-23.43%) is obviously increased, which indicates that the compound 48/80 induction allergy model is successfully established.
The expression level of the Tryptase in the positive control group (sodium cromolyn) is 134.12 +/-18.69%, the inhibition rate of the Tryptase is 85.91 +/-7.72%, and the difference is obvious compared with the model group (Tryptase expression level: 342.14 +/-23.43%) (P<0.001). Therefore, the cromolyn sodium has antiallergic effect, and is consistent with clinical results, which shows that the antiallergic test is effective. Lactobacillus curvatus LC02 concentration of 1X 10 4 CFU/mL、1×10 5 CFU/mL、1×10 6 At CFU/mL, the expression level of the Tryptase in the zebra fish is 256.76 +/-32.24%, 235.22 +/-39.24% and 152.83 +/-19.86%, and the inhibition rate of the Tryptase is 35.26+/-13.32%, 44.16+/-16.20% and 78.18+/-8.20%, respectively, and the difference is obvious compared with the model group (the expression level of the Tryptase is 342.14 +/-23.43%) (P<0.01 Shows good probiotic efficacy in inhibiting degranulation of mast cells or basophils. Thus, the above results indicate that Lactobacillus curvatus LC02 has an efficacy of preventing and/or alleviating allergic reactions.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. Lactobacillus curvatus LC02, characterized in that it has a preservation number of CGMCC No.22986.
2. Use of lactobacillus curvatus LC02 according to claim 1 for the manufacture of a medicament for the treatment or prophylaxis of allergic diseases.
3. Use of lactobacillus curvatus LC02 according to claim 2 for the manufacture of a medicament for the treatment or prophylaxis of allergic diseases, wherein said lactobacillus curvatus LC02 is a bacterial suspension.
4. Use of lactobacillus curvatus LC02 as claimed in claim 1 in the manufacture of a medicament for inhibiting degranulation of mast cells or basophils.
5. Use of lactobacillus curvatus LC02 according to claim 4 for the manufacture of a medicament for inhibiting degranulation of mast cells or basophils, wherein said lactobacillus curvatus LC02 is a bacterial suspension.
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