A preparation method of probiotics and its use in the treatment of allergic rhinitis

Abstract

The present invention belongs to the field of biomedical technology, and particularly relates to a preparation method of probiotics and their use in the treatment of allergic rhinitis. The products of the present invention have a good therapeutic effect on allergic rhinitis, with good safety and remarkable curative effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of probiotics and their use in the treatment of allergic rhinitis. Background Art

[0002] Allergic rhinitis (AR), also known as allergic rhinitis, is a common non-infectious inflammatory disease of the nasal mucosa. After allergic individuals come into contact with different allergens, it triggers a type I allergic reaction mainly mediated by immunoglobulin E (IgE), and a variety of immunocompetent cells, cytokines, etc. are involved in the development of the disease. In AR, the rapid reaction caused by IgE-mediated mast cell degranulation and mediator release leads to symptoms mainly including nasal congestion, nasal itching, sneezing, runny nose, and sometimes accompanied by partial loss of nasal mucosal sensation. Eye symptoms may also occur simultaneously, mainly including itchy eyes, red eyes, tearing, a burning sensation, etc. In the late stage, an inflammatory reaction with eosinophil infiltration may occur.

[0003] Currently, the treatment of AR mainly includes drug therapy (① glucocorticoids, ② antihistamines, ③ anti-leukotriene drugs, ④ cromones, and ⑤ combination drug therapy), specific immunotherapy, surgery, and other treatments (such as laser irradiation, radiofrequency, and chemical cauterization of the nasal turbinate mucosa). The use of drugs such as nasal glucocorticoids and antihistamines can better control the symptoms of AR, but cannot change the natural course of the disease. The commonly used antigen-specific immunotherapy has a long treatment cycle and low safety, and there is a risk of exacerbating the AR condition and even threatening the patient's life. Although these treatments are effective in controlling the symptoms of most patients, at least 20% of patients still cannot control their symptoms well, and many patients need to take medications for life. Therefore, controlling the continuous increase in the incidence of allergic rhinitis and finding new treatment strategies remain a huge challenge for us.

[0004] In recent years, many new drugs and immunotherapies have been used in the clinical treatment of AR. The survey results at home and abroad show that probiotics can play a good role in the prevention and control of respiratory allergic diseases. For example, probiotics play a role in the prevention and treatment of hypersensitivity through multi-pathway and multi-level molecular biological mechanisms. The preventive and therapeutic effects of probiotics on hypersensitivity are mainly manifested in: 1. regulating the balance of immune cell subsets (the ratio of Th1 / Th2 cells); 2. promoting cell differentiation to develop in a non-inflammatory or regulatory direction (such as the induction of regulatory T cells); 3. inhibiting mast cell activation. These effects together contribute to the alleviation and prevention of hypersensitivity. For example, studies have shown that the supplementation of Bifidobacterium longum BB536 can regulate the Th1 / Th2 balance in patients with seasonal allergic rhinitis and reduce the severity of symptoms; the supplementation of Lactobacillus rhamnosus GG (LGG) can significantly increase the number of Treg cells in the nasal mucosa and reduce the expression of IL-4 and IL-13 in the nasal mucosa, thereby alleviating the symptoms of allergic rhinitis. In addition, probiotics have the effect of inhibiting the activation of mast cells and basophils, which play a key role in allergic reactions. Studies have shown that after stimulating mast cells derived from human peripheral blood with LGG, LGG can reduce the expression of FcεRI receptor and histamine H4 receptor (H4R) on the surface of mast cells to weaken the sensitivity of mast cells.

[0005] Histamine receptor-4 (HR4) is a newly discovered histamine receptor, which is a transmembrane G-protein-coupled receptor (GPCRs). Its function is to inhibit adenylate cyclase activity, activate phospholipase C and induce calcium ion flow. HR4 is preferentially distributed on immune organs and hematopoietic cells and is involved in all immune cells in the pathological immune response of AR. The expression of HR4 can be detected in mast cells, CD4 + T, CD8 + T cells, eosinophils, NK cells, dendritic cells, and monocytes, and they have been proven to have the function of mediating immune regulation. HR4 antagonists show an inhibitory effect on cytokines and chemokines such as IL-6, KC, MIP-α, and IP-10 produced during the activation process of TLR ligand-induced dendritic cells. HR4 antagonists and negative agonists have shown good anti-inflammatory prospects in in vitro experiments.

[0006] However, at present, there are not enough studies on the use of probiotics, especially in combination with drugs, for the treatment of allergies, and the available options are not rich enough, which is worthy of further research. Summary of the Invention

[0007] In view of the situation of the existing technology, the object of the present invention is to provide an active polypeptide that binds to HR4, its composition with probiotics, and its application in the treatment of allergic diseases, especially allergic rhinitis. The polypeptide of the present invention has a good therapeutic effect on allergic rhinitis, good safety, and remarkable curative effect.

[0008] The present invention first provides an active polypeptide that binds to HR4, which is characterized in that the amino acid sequence of the polypeptide is ADCNERVPTRCKFIRCGC, as shown in SEQ ID NO:1.

[0009] In some embodiments, the polypeptide with the ability to bind to HR4 is an HR4 antagonist polypeptide.

[0010] Another aspect of the present invention also provides the use of the polypeptide in the preparation of a drug for treating allergic diseases.

[0011] Another aspect of the present invention provides a pharmaceutical composition, which comprises a probiotic as an active ingredient and a polypeptide with the ability to bind to HR4, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1.

[0012] In some embodiments, the probiotic is Lactobacillus rhamnosus GG strain HN001.

[0013] The polypeptide of the present invention or the pharmaceutical composition containing the polypeptide is administered in the form of a freeze-dried powder.

[0014] In some embodiments, the pharmaceutical composition provided by the present invention further comprises vitamins, immunosuppressants, analgesics, steroids, non-steroidal anti-inflammatory agents (NSAIDs) or cytokine antagonists and combinations thereof.

[0015] Another aspect of the present invention also provides the use of the polypeptide or the pharmaceutical composition in the preparation of a drug for treating allergic diseases.

[0016] In some embodiments, the allergic diseases are food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria and allergic contact dermatitis.

[0017] The polypeptide of the present invention has an excellent therapeutic effect on allergic diseases and has certain clinical application value. In addition, the polypeptide 3G2 provided by the present invention is easy to synthesize, has a low cost, and is easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The shown result is that the polypeptide 3G2 inhibits the activation of the HR4 signaling pathway induced by histamine.

[0019] Figures 2A - 2C The results shown are the levels of serum IL4 ( Figure 2A ), IL6 ( Figure 2B ), and IFN-γ ( Figure 2C ) in a guinea pig allergy model after treatment with polypeptide 3G2 and / or probiotics. Detailed implementation manners

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0021] Except as otherwise indicated in the operating examples or otherwise noted, all numbers expressing amounts of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about". When used in conjunction with percentages, the term "about" can mean ±1%.

[0022] The term "probiotic" refers to "live microorganisms that confer a health benefit on the host when ingested in appropriate amounts", and generally probiotics are mainly part of the gut microbiota such as lactic acid bacteria and bifidobacteria. There are a wide variety of probiotics. In the early days, the term "probiotic" was limited to lactic acid bacteria. With the identification of new bacteria and the increase in research on probiotic species, probiotics also include some yeasts. Among them, lactic acid bacteria include 17 genera and 273 species: Enterococcus, Lactobacillus, Bacillus, Clostridium, Lactococcus lactis, Leuconostoc, Pediococcus, Carnobacterium, Vagococcus, Tetragenococcus, Bifidobacterium, Atopobium, Weissella, Abiotrophia, Granulicatella, Oenococcus, Paralactobacillus. In addition, it also includes some Saccharomyces such as Saccharomyces boulardii.

[0023] The term "atopic disease" refers to a group of diseases with a genetic predisposition to allergic reactions. Examples of atopic diseases can be asthma, atopic dermatitis, allergic rhinitis, urticaria, allergic conjunctivitis, anaphylactic reactions, angioedema, food allergies, etc. The common allergens in allergic rhinitis mainly include: 1. Drug or chemical allergens, such as penicillin, sulfonamides, organic iodides and other molecules and their degradation products. After entering the body and binding to a certain protein, they acquire immunogenicity and become allergens; 2. Inhaled allergens, such as pollen grains, dust mites, fungal hyphae, animal furs, etc.; 3. Food allergens such as milk, eggs, fish, shrimp and other food proteins.

[0024] The pathogenesis of atopic diseases is divided into the induction stage and the effector stage. Induction stage: The allergen is phagocytosed by antigen-presenting cells (APCs), and the antigen information is presented to T cells by major histocompatibility complex (MHC) class II molecules. T cells are activated and secrete inflammatory factors such as IL-4. IL-4 and others activate B cells and drive the production of IgE. IgE binds to the receptor FcεRI on the surface of mast cells in tissues and basophils in the blood. Effector stage: When the allergen contacts again, the multivalent antigen binds to the specific IgE on the cell surface, causing the cross-linking of the IgE-FcεRI complex, resulting in mast cell degranulation and the release of a large number of inflammatory factors. The activated mast cells can release more than 40 kinds of mediators, which are divided into 3 categories: pre-synthesized mediators, such as histamine, proteolytic enzymes, etc.; newly synthesized arachidonic acid metabolites, such as leukotrienes, prostaglandins, etc.; newly synthesized inflammatory factors, such as tumor necrosis factor (THF-α), interleukin 4, 5, 6, 8, 13, etc. These mediators play different roles in allergic rhinitis. The chemotactic effect of histamine on eosinophils plays an important role in the regulation of the body's allergy and inflammation and is directly related to the severity of the allergy. IL-4 is also called B cell stimulating factor, which activates B cells to secrete IgE; IL-5 mainly stimulates the proliferation, differentiation and activation of eosinophils.

[0025] Early-phase allergic reactions (EPR) occur within minutes of allergen challenge, such as sneezing and itching, followed by rhinorrhea and nasal congestion, which often disappear within 1 hour. This reaction results from the cross-linked complex of IgE-FcεRI and the allergen, leading to mast cell degranulation and the release of pre-synthesized mediators, such as histamine and the re-generation of mediators from membrane lipids, such as cysteinyl leukotrienes (leukotriene LTC4, LTD4 and LTE4) and prostaglandin (PD2). Histamine often acts on the H1 receptor of sensory nerve endings to cause itching, leading to a whole-body reflex, such as sneezing. Leukotrienes, prostaglandins and vascular endothelial growth factor cause plasma to leak from blood vessels, resulting in edema, blood pooling in wide venous sinuses, and increased secretion of glandular mucus. All of these can lead to a sense of nasal congestion.

[0026] Late-phase allergic reaction (LPR) is mainly manifested as persistent symptoms, with a peak decrease in nasal inspiratory flow occurring 4 - 12 hours after exposure. The released mediators are mainly histamine, platelet-derived growth factor 2, and leukotrienes, which induce the influx and activation of various inflammatory cells, thereby leading to the late-phase response. Adhesion molecules such as vascular cell adhesion molecule-1 promote the adhesion of circulating eosinophils to endothelial cells, thus facilitating the influx of inflammatory cells into the nasal mucosa. Chemotactic agents and cytokines such as IL-5 promote the infiltration of eosinophils, basophils, and T cells from the systemic circulation into the submucosa of the nasal mucosa. The subsequent release of mediators such as histamine and leukotrienes causes the late symptoms of allergic rhinitis, such as nasal congestion, nasal hyperemia, fatigue, and possible neurocognitive dysfunction.

[0027] Phage display peptide library technology uses the phage coat protein PⅢ or PⅧ gene as a vector, inserts a gene fragment encoding an exogenous short peptide, and the infectivity of the phage is not affected. Moreover, the inserted exogenous short peptide can form a certain spatial conformation at the N-terminus of the PⅢ or PⅧ protein on the phage surface. The basic principle of screening antigenic epitopes from a phage display random peptide library is biopanning.

[0028] The pharmaceutical composition for preventing or treating allergic diseases of the present invention may contain Lactobacillus rhamnosus. The Lactobacillus rhamnosus HN001 strain of the present invention can be recovered through processes such as centrifugation after cultivation, and then prepared in the form of a viable bacteria agent by drying, for example, by freeze-drying for use. Relative to the total weight of the composition, the pharmaceutical composition of the present invention may contain 10 8 CFU to 10 12 CFU of the Lactobacillus rhamnosus HN001 strain or a culture having an equivalent number of viable bacteria as an active ingredient.

[0029] In some examples of the present invention, the pharmaceutical composition containing the above-mentioned Lactobacillus rhamnosus HN001 strain and active polypeptide can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, but is not limited thereto.

[0030] The pharmaceutical composition of the present invention can be formulated into enteral or oral pharmaceutical products. Moreover, the pharmaceutical composition of the present invention can be formulated into enteric packaging using known methods to rapidly release the microorganism as an active substance in the intestine after passing through the gastrointestinal tract and reaching the small intestine.

[0031] In some examples of the present invention, the liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used water and liquid paraffin as simple diluents, excipients can also be included, for example, wetting agents, sweeteners, fragrances, preservatives, etc., but are not limited thereto.

[0032] In other embodiments, the pharmaceutical composition for preventing or treating allergic diseases of the present invention may further comprise at least one vitamin. The above-mentioned vitamin may be a fat-soluble or water-soluble vitamin. Suitable vitamins include, but are not limited to, vitamin D, vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, vitamin B2, niacin, vitamin B6, folic acid, pyridoxine, pantothenic acid, and biotin. Any suitable form of the above is a salt of a vitamin, a derivative of a vitamin, a compound of a vitamin having the same or similar activity, and a metabolite of a vitamin.

[0033] The pharmaceutical composition for preventing or treating allergic diseases of the present invention may further comprise a known additional therapeutic agent having the effect of preventing and treating allergic diseases. The acceptable additional therapeutic agents in the present invention are immunosuppressants, analgesics, steroids, non-steroidal anti-inflammatory drugs (NSAIDs), or cytokine antagonists and combinations thereof. Examples of the above immunosuppressants include, but are not limited to, glucocorticoids, cyclosporine, tacrolimus, pimecrolimus, calcineurin inhibitors including ISA(TX)247, rapamycin, phosphodiesterase 4 inhibitors, mycophenolate mofetil, dexamethasone, etc. All known immunosuppressants can be used together. Moreover, one immunosuppressant can be used alone, or two or more immunosuppressants can be used in combination. Preferably, the above immunosuppressant can be one or more selected from the group consisting of cyclosporine, tacrolimus, dexamethasone, and pimecrolimus. When the pharmaceutical composition of the present invention is administered in combination with other therapeutic agents, they can be administered sequentially or simultaneously, and can be administered once or multiple times.

[0034] In addition to the above active ingredients, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and / or excipient. In addition, it can be formulated together with various additives commonly used in pharmacy, such as binders, disintegrants, coating agents, lubricants, etc.

[0035] The acceptable excipients in the present invention include sugars such as sucrose, lactose, mannitol, glucose, etc., and starches such as corn starch, potato starch, rice starch, partially pre-gelatinized starch, etc. Binders include polysaccharides such as dextran, sodium alginate, carrageenan, guar gum, gum arabic, agar, etc., natural macromolecular substances such as tragacanth gum, gelatin, gluten, etc., cellulose derivatives such as hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, hydroxypropyl ethyl cellulose, sodium carboxymethyl cellulose, etc., and polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid, and vinyl acetate resin.

[0036] Acceptable disintegrants in the present invention may include cellulose derivatives such as carboxymethyl cellulose, calcium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, and starches such as sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch, and partially pregelatinized starch.

[0037] Examples of acceptable lubricants in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, hydrated silica, various waxes, and hydrogenated oils.

[0038] Coating agents include non-water-soluble polymers such as dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinyl acetal diethylaminoacetate, ethyl acrylate-methacrylic acid copolymer, ethyl acrylate-methyl methacrylate-ethyl methacrylate trimethylammonium chloride copolymer, ethyl cellulose, enteric polymers such as methacrylic acid-ethyl acrylate copolymer, hydroxypropyl cellulose phthalate, hydroxypropyl methylcellulose acetate succinate, and water-soluble polymers such as methyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, and polyethylene glycol, but are not limited thereto.

[0039] In the pharmaceutical composition for preventing or treating allergic diseases of the present invention, the dosage of the above-mentioned strain as an active ingredient can be determined according to factors including the type of various diseases, the age, weight, gender of the patient, the medical condition of the patient, the severity of the condition, the sensitivity to the drug, the administration time, the administration route and the metabolic ratio, the treatment period, the drugs used simultaneously, and other factors well known in the medical field. Therefore, although the dosage and treatment method can vary within a wide range, it is important to administer the minimum amount without side effects that can achieve the maximum effect after considering all the above factors, which can be easily determined by relevant practitioners using standard methods.

[0040] As used herein, "promote" or "increase" or "promoting" or "increasing" may be used interchangeably herein. These terms refer to an increase in a measured parameter of a treated cell, tissue, or individual compared to an untreated cell, tissue, or individual. The same cell or tissue or individual can also be compared before and after treatment. In some embodiments, the increase in the treated cell, or tissue, or individual is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or more compared to an untreated cell, or tissue, or individual.

[0041] As used herein, the terms "treatment" or "treating" may be used interchangeably herein. These terms refer to a course of action used to obtain a beneficial or desired result, including, but not limited to, a therapeutic benefit and / or a prophylactic benefit.

[0042] In this application, unless specifically stated otherwise, the use of the singular also includes the plural. In this application, unless otherwise indicated, the use of "or" means "and / or". Additionally, the use of the term "including" and other forms such as "includes" and "included" is not restrictive. Further, unless specifically stated otherwise, terms such as "element" or "component" cover both elements and components that include one unit and elements and components that include more than one subunit. Additionally, the use of the term "portion" may include a part of a portion or the whole portion. Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0043] The pharmaceutical composition of the present invention is formulated to have a pH of 5.5 to 7.5. In one embodiment, the pH of the aqueous medium can be adjusted by a suitable low concentration of biocompatible buffering components, non-limiting examples of which are tromethamine, sodium carbonate and sodium bicarbonate, and sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0044] The compositions of the present invention can be administered daily or intermittently, and the frequency of administration can be once a day or 2 to 3 times. If each of the two active ingredients is a single formulation, their frequencies of administration can be the same as or different from each other. Additionally, the compositions of the present invention can be used alone or in combination with other anti-allergy drugs. Considering all of the above factors, it is important to administer at the lowest dose without side effects to achieve the best therapeutic effect, which can be easily determined by those skilled in the art. In some embodiments, the dosing regimen is repeated, for example, once, twice, three times, or more; for example, repeated throughout the remaining life cycle of an individual in need.

[0045] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples, but the present invention is not limited in any way. Any transformation or improvement made based on the teachings of the present invention falls within the protection scope of the present invention.

[0046] Example 1 Screening of Polypeptides Binding to HR4

[0047] 1.1 Screening

[0048] Dissolve 50 μg / L HR4 protein (synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.) in 8.4 g / L NaHCO3, add it to the enzyme-linked immunosorbent assay (ELISA) plate respectively, and incubate overnight at 4°C. Add the blocking buffer, incubate at 37°C for 1.5 h, and wash 3 times with the buffer; dilute the phage display peptide library (New England Biolabs, US) with TBS (10 mmol / L Tris, pH 7.4), add the diluted phage to the ELISA plate, and incubate with shaking at 37°C for 2 h. Discard the supernatant, and wash the plate 10 times with 0.1% TBST. Add 100 μL of elution buffer (0.2 mol / L Glycine-HCl pH 2.2, 1 mg / mL BSA) to the corresponding wells, and neutralize with Tris-HCl until the pH reaches 7.0 after 15 min.

[0049] Culture Escherichia coli ER2738 until it reaches the logarithmic phase. Dilute the eluted phage, and then mix 10 μL of the dilution with 200 μL of Escherichia coli ER2738. Incubate at room temperature for 10 min. Mix the mixture with 3 mL of top agarose, then transfer it to an LB / X-Gal plate and invert it, and incubate overnight at 37°C.

[0050] Culture Escherichia coli ER2738 until it reaches the logarithmic phase, add the eluted phage to the Escherichia coli ER2738 culture, and incubate with shaking at 37°C at 220 rpm for 4.5 h. Centrifuge the culture, transfer the supernatant to a sterile tube, add 20% PEG / NaCl, and place it on ice for 1 - 2 h. Centrifuge the solution, suspend the precipitate with PBS, and transfer the supernatant to a sterile test tube after centrifugation for the next round of biopanning. Perform 4 rounds of biopanning in total. Randomly pick 30 blue plaques, namely phage monoclonal antibodies, from the plates titrated in the 4th round of screening. Culture Escherichia coli ER2738 overnight at 37°C at 220 rpm. Centrifuge the culture the next day and collect the supernatant for ELISA detection.

[0051] 1.2 Identification of candidate phages by ELISA

[0052] Coat the ELISA plate with 10 μg / L HR4 protein and 2 μg / L BSA overnight at 4°C, and wash the wells 3 times. Block the wells with 300 μL of blocking buffer and wash the plate 3 times. Add 100 μL of phage supernatant to the wells, incubate at 37°C for 1 h and then wash 3 times. Add 100 μL of anti-M13-HRP antibody (1:4000) to each well, incubate at 37°C for 1 h, and wash 3 times. Add 100 μL of TMB to each well and incubate at room temperature, and then add 100 μL of 2 M HCl to each well. Read the absorbance value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader.

[0053] 1.3 Phage single-stranded DNA sequence

[0054] According to the ELISA experiment results, phage monoclonal 3G2 was selected for sequencing. The phage monoclonal stock solution was added with PEG / NaCl, and the phages were precipitated at 4°C for 10 min. After high-speed centrifugation, the supernatant was removed. The precipitate was resuspended with 100 μL of iodide buffer (10 mmol / L Tris, 1 mmol / L EDTA, 4 mmol / L NaI), and then 250 μL of absolute ethanol was added to precipitate DNA at room temperature. The mixture was centrifuged at high speed for 10 min, the supernatant was removed, the precipitate was rinsed with 700 mL / L ethanol, and dried briefly under vacuum. Finally, the precipitate was resuspended with 30 μL of TE buffer (10 mmol / L Tris, 1 mmol / L EDTA). Take the above phage monoclonal DNA stock solution, send it to Shanghai Sangon Biological Engineering Co., Ltd., and use the -96g III sequencing primer for full-automatic sequencing. The phage sequencing results were read and translated according to the attached manual of the phage polypeptide display library. The sequence of polypeptide 3G2 is shown as SEQ ID NO: 1.

[0055] 1.4 Synthesis of polypeptide 3G2

[0056] Polypeptide 3G2 was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. according to SEQ ID NO: 1. After mass spectrometry and high performance liquid analysis, it was confirmed that the obtained product was the target polypeptide compound with a purity of ≥95%.

[0057] Example 2. Inhibition of histamine-induced activation of the HR4 signaling pathway by polypeptide 3G2

[0058] HR4 is coupled with the Gαi protein. After HR4 binds to histamine, it can inhibit the activity of intracellular adenylate cyclase and reduce the concentration of cyclic adenosine monophosphate (cAMP). Based on the above principle, the HEK293-CRE (Luc) reporter cell line (Aimix Biotechnology, ABS-T3203) was used to detect the inhibitory activity of polypeptide 3G2 in the HR4 pathway.

[0059] HEK293-CRE (Luc) cells transiently expressing Flag-HR4 were seeded in 96-well plates and cultured overnight. After changing the medium, polypeptide 3G2 was added to the wells of the treatment group and incubated at 37°C for 1 h. 100 μM Forskolin (Sigma, F3917) and different concentrations of histamine (Sigma, H7125) were added to each well and incubated at 37°C for 4 h. After discarding the culture medium, 50 μL of lysis buffer was added to each well, and the cells were shaken on a horizontal shaker at 4°C for 10 minutes to fully lyse the cells. After lysis, 30 μL of the lysed sample was mixed with 15 μL of the detection substrate (Beyotime, RG005), and quickly placed into a chemiluminescence detector to read the absorbance value of each well.

[0060] The results are as Figure 1As shown, polypeptide 3G2 can increase the expression level of the firefly luciferase gene regulated by the intracellular cAMP response element (CRE), indicating that polypeptide 3G2 can effectively inhibit the activation of the HR4 signaling pathway induced by histamine.

[0061] Example 3. Preparation of the mixed freeze-dried powder of polypeptide 3G2 and Lactobacillus rhamnosus probiotics

[0062] The Lactobacillus rhamnosus GG strain HN001 (deposit number: ATCC SD5675) stored at -80 °C was inoculated into MRS liquid medium and activated at 37 °C for 3 generations overnight. The activated Lactobacillus rhamnosus GG strain HN001 in the logarithmic growth phase was inoculated into MRS medium at an inoculation amount of 3% (volume fraction), and cultured statically and sealed at 37 °C for 24 h. The obtained bacterial liquid was reserved. Under sterile conditions, 2 mL of the bacterial liquid was inoculated into a fermenter containing optimized medium. The stirring speed was 100 rpm, the aeration volume was 0, and the fermentation was carried out at a constant temperature of 37 °C. After 3 h, the pH was maintained at 5.5 - 6.0 by adding food-grade NaOH solution, and the culture was continued for 15 h. The formula of the optimized medium is as follows: yeast powder 20.0 g / L, glucose 25.0 g / L, magnesium sulfate 0.5 g / L, Tween 80 0.5 g / L, sodium acetate 6.0 g / L, isomaltooligosaccharide 1.5 g / L, potassium dihydrogen phosphate 2.5 g / L, and the balance is sterile water, pH 6.80; the above components were weighed and mixed evenly, heated and dissolved, and the pH of the medium was adjusted to 6.8 with food-grade NaOH solution (1 mol / L), and sterilized at 121 °C for 15 min.

[0063] The fermentation broth was centrifuged at 12000 rpm for 10 min to obtain bacterial sludge. The bacterial sludge, freeze-drying protectant, and polypeptide 3G2 were mixed evenly in a ratio of 1:1:0.2 (volume ratio) to obtain a mixed solution containing probiotics and active polypeptide. The formula of the freeze-drying agent is: L-cysteine hydrochloride 0.15%, glycerol 1.2%, sodium glutamate 0.6%, trehalose 12%, skim milk 12%. The above mixed solution was frozen at -4 °C for 12 h and then placed in a vacuum freeze-dryer for freeze-drying for 24 h to obtain the freeze-dried powder containing probiotics and active polypeptide 3G2. By the above method, the freeze-dried powder of probiotics and the freeze-dried powder of polypeptide 3G2 were also prepared.

[0064] Example 4. Effects of polypeptide 3G2 and its composition on allergic diseases

[0065] Thirty male guinea pigs about 8 weeks old were randomly divided into groups of 6 each, namely the normal control group (G1), the model control group (G2), the polypeptide 3G2 treatment group (G3), the probiotic treatment group (G4), and the combined treatment group of polypeptide 3G2 and probiotics (G5).

[0066] Basic sensitization: Using 3 mg ovalbumin (OVA, Beijing Binuowei Biotechnology Co., Ltd., SLCH2414) as the antigen and 30 mg aluminum hydroxide gel (Beijing Binuowei Biotechnology Co., Ltd., VI311256) as the adjuvant, 1 mL of normal saline was drawn and prepared into an OVA suspension with a concentration of 0.3%. It was intraperitoneally injected once every 2 days for a total of 7 injections. The normal group was intraperitoneally injected with the same dose of normal saline.

[0067] Booster sensitization: After the intraperitoneal injection immunization stage ended, both nasal cavities were locally stimulated and boosted with 50 μL of a 5% ovalbumin - normal saline solution (a suspension prepared by adding 5 mg OVA to 10 mL of normal saline) dropped into the nose once a day for a total of 7 treatments. The normal group was dropped with the same dose of normal saline into the nose.

[0068] The normal group (G1) received no treatment; the model control group (G2) was animals sensitized with conventional OVA and did not receive treatment; groups G3 - G5 were treated with drug therapy by respectively administering the freeze-dried powder of polypeptide 3G2, the freeze-dried powder of probiotics, and the mixed freeze-dried powder of probiotics and polypeptide 3G2 after conventional OVA sensitization. The above-mentioned freeze-dried powders were respectively taken, and according to a dose of 1 mg / g, the freeze-dried powder was dissolved in sterile normal saline and administered by gavage once every 3 days for a total of 6 treatments.

[0069] After the animal model was successfully established, the animals would show symptoms such as sneezing, nose scratching, face scratching, and runny nose. The superposition quantitative score was used to analyze whether the allergy model was successfully established. After the last booster sensitization, each animal was observed for 30 minutes. Through observation records and combined with relevant behavioral indicators, it was verified that the allergy models of each group of animals were successfully constructed. During the experiment, it was observed that the nasal symptom behavioral scores of the G5 treatment group were at a relatively low level during the behavioral evaluation after 3 treatments, and the nasal symptom behavioral scores of the G3 and G4 treatment groups decreased during the behavioral evaluation after 4 treatments, indicating that the treatment groups had significant curative effects in relieving the symptoms of allergic rhinitis (data not shown).

[0070] Two days after the last administration for treatment, each group of guinea pigs was anesthetized with ether and fixed on the experimental table. After opening the chest to expose the heart with scissors, about 5 mL of blood was collected from the heart. After the collected blood was left standing at room temperature for 2 h, it was centrifuged at 1500 g for 15 min, and the serum was collected in a clean EB tube and stored for later use. The levels of IL4, IL6, and IFN-γ in the serum of each group of guinea pigs were detected using an ELISA detection kit. According to the OD values of the standard products in the experiment of IL4, IL6, and IFN-γ measured by the microplate reader, a standard curve was made. According to the OD values of each group of samples, the concentration of each well was calculated and then multiplied by the dilution factor to obtain the actual measured concentration value of the sample.

[0071] The results are as Figures 2A - 2CAs shown, compared with the model control group, after treatment with the freeze-dried powder of polypeptide 3G2 or the mixed freeze-dried powder of probiotics and polypeptide 3G2, the levels of IL4 and IL6 decreased to varying degrees, indicating that the freeze-dried powder of polypeptide 3G2 or the mixed freeze-dried powder of probiotics and polypeptide 3G2 can be effectively used for the effective treatment of allergic rhinitis.

Claims

1. A polypeptide that binds to HR4, which consists of the amino acid sequence shown in SEQ ID NO:

1.

2. A pharmaceutical composition comprising an effective amount of the polypeptide according to claim 1 and probiotics.

3. The pharmaceutical composition according to claim 2, wherein the probiotics are Lactobacillus rhamnosus.

4. The pharmaceutical composition according to claim 3, wherein the Lactobacillus rhamnosus is HN001.

5. The pharmaceutical composition according to any one of claims 2-4, which is administered in the form of a lyophilized powder.

6. The pharmaceutical composition according to any one of claims 2-4, which further comprises vitamins, immunosuppressants, analgesics, steroids, non-steroidal anti-inflammatory agents (NSAIDs) or cytokine antagonists and combinations thereof.

7. Use of the polypeptide according to claim 1 or the pharmaceutical composition according to any one of claims 2-6 for the preparation of a medicament for treating allergic diseases, wherein the allergic disease is allergic rhinitis.

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