Preparation method of probiotics and application of probiotics in treatment of allergic rhinitis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of probiotics and application of the probiotics in treatment of allergic rhinitis. The product disclosed by the invention has a good treatment effect on allergic rhinitis, and is good in safety and remarkable in curative effect.

Description

Technical Field

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

[0002] Allergic rhinitis (AR), also known as allergic rhinitis, is a common non-infectious inflammatory disease of the nasal mucosa. When people with allergic constitution come into contact with different allergens, they will develop a type I allergic reaction mainly mediated by immunoglobulin E (IgE), and a variety of immune active cells and blood cytokines will participate in the development of the disease. In AR, the rapid reaction caused by IgE-mediated mast cell degranulation and mediator release leads to symptoms such as nasal congestion, itching, sneezing, and runny nose, sometimes accompanied by partial loss of sensation of the nasal mucosa, and eye symptoms may also occur at the same time, mainly including itchy eyes, red eyes, tears, burning sensation, etc. In the late stage, an inflammatory reaction with eosinophil infiltration may occur.

[0003] At present, the treatment of AR mainly includes drug therapy (① glucocorticoids, ② antihistamines, ③ anti-leukotriene drugs, ④ chromone drugs and ⑤ drug combination therapy), specific immunotherapy, surgery and other treatments (such as laser irradiation of nasal concha mucosa, radiofrequency and chemical cauterization, etc.). Nasal glucocorticoids and antihistamines and other drug treatments can better control AR symptoms, but cannot change the natural course of the disease. Commonly used antigen-specific immunotherapy has a long cycle and low safety, and there is a risk of aggravating AR 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 fail to control their symptoms well, and many patients need lifelong medication. Therefore, controlling the continued increase in the incidence of allergic rhinitis and finding new treatment strategies are still huge challenges we face.

[0004] In recent years, many new drugs and immunotherapies have been used in the clinical treatment of AR. Domestic and foreign survey results show that probiotics can play a good role in the prevention and control of respiratory allergic diseases. For example, probiotics work together to prevent and treat hypersensitivity reactions through multi-pathway and multi-level molecular biological mechanisms. The preventive and therapeutic effects of probiotics on hypersensitivity reactions are mainly manifested in: 1. Regulating immune cell subsets to balance the Th1 / Th2 cell ratio); 2. Promoting cell differentiation towards non-inflammatory or regulatory directions (such as the induction of regulatory T cells); 3. Inhibiting mast cell activation. These effects together help to alleviate and prevent hypersensitivity reactions. For example, studies have shown that supplementation with Bifidobacterium longum BB536 can regulate the Th1 / Th2 balance of patients with seasonal allergic rhinitis and reduce the severity of symptoms; supplementation with 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 receptors and histamine H4 receptors (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, a transmembrane G-protein-coupled receptor (GPCRs), which functions to inhibit adenylyl cyclase activity, activate phospholipase C and induce calcium ion flow. HR4 is preferentially distributed in immune organs and hematopoietic cells, and is involved in all immune cells of AR pathological immune response. Mast cells, CD4 + T, CD8 + The expression of HR4 can be detected in T cells, eosinophils, NK cells, dendritic cells, and monocytes, and has been shown to mediate immune regulation. HR4 antagonists inhibit cytokines and chemokines such as IL-6, KC, MIP-α, and IP-10 produced during the activation of dendritic cells induced by TLR ligands. HR4 antagonists and negative agonists have shown good anti-inflammatory prospects in in vitro experiments.

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

[0007] In view of the prior art, the purpose of the present invention is to provide an active polypeptide that binds to HR4, a composition thereof with probiotics, and use thereof 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 significant efficacy.

[0008] The present invention first provides an active polypeptide that binds to HR4, 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 capable of binding to HR4 is an HR4 antagonist polypeptide.

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

[0011] Another aspect of the present invention provides a pharmaceutical composition comprising probiotics as active ingredients and a polypeptide capable of binding to HR4, wherein the amino acid sequence of the polypeptide is 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 comprising the polypeptide is administered in the form of a lyophilized 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 use of the polypeptide or the pharmaceutical composition in preparing a drug for treating allergic diseases.

[0016] In some embodiments, the allergic disease is 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 excellent therapeutic effects on allergic diseases and has certain clinical application value. In addition, the polypeptide 3G2 provided by the present invention is easy to synthesize, low in cost, and easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results shown are that peptide 3G2 inhibits histamine-induced activation of the HR4 signaling pathway.

[0019] Figure 2A-2C The results shown are the changes of serum IL4( Figure 2A ), IL6( Figure 2B ) and IFN-γ( Figure 2C ) level. DETAILED DESCRIPTION

[0020] Unless defined otherwise, 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 in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” When used in conjunction with a percentage, the term “about” may mean ± 1%.

[0022] The term "probiotics" refers to active microorganisms that produce health benefits to the host when ingested in appropriate quantities. Usually, probiotics are mainly part of the intestinal microbiota, such as lactic acid bacteria and bifidobacteria. There are many types of probiotics, and the term probiotics was limited to lactic acid bacteria in the early days. 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, some Saccharomyces such as Saccharomyces boulardii are also included.

[0023] The term "atopic disease" refers to a group of diseases with a genetic tendency to develop allergic reactions. Examples of allergic diseases may be asthma, atopic dermatitis, allergic rhinitis, urticaria, allergic conjunctivitis, anaphylactic reactions, angioedema, food allergies, etc. Among them, the common allergens of allergic rhinitis are mainly: 1. Drug or chemical allergens, such as penicillin, sulfonamide, organic iodide and other molecules and their degradation products enter the body and bind to a certain protein to obtain immunogenicity and become allergens; 2. Inhalation allergens, such as pollen particles, dust mites, fungal hyphae, animal fur, etc.; 3. Food allergens such as food proteins such as milk, eggs, fish, and shrimp.

[0024] The occurrence mechanism of allergic diseases is divided into the induction stage and the effector stage. Induction stage: Allergens are phagocytosed by antigen presenting cells (APCs), and the major histocompatibility complex (MHC) class II molecules present antigen information to T cells. T cells are activated and secrete inflammatory factors such as IL-4, which activate B cells and drive IgE production. IgE binds to the receptor FcεRI on the membrane of mast cells in tissues and basophils in blood. Effector stage: When the allergen is contacted again, the multivalent antigen binds to the specific IgE on the cell surface, causing the cross-linking of the IgE-FcεRI complex, leading to mast cell degranulation and the release of a large number of inflammatory factors. Activated mast cells can release more than 40 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 chemotaxis of histamine on eosinophils plays an important role in regulating allergies and inflammation in the body and is directly related to the severity of allergies. IL-4, also known as B cell stimulating factor, activates B cells to secrete IgE; IL-5 mainly stimulates the proliferation, differentiation and activation of eosinophils.

[0025] The early allergic reaction (EPR) occurs within minutes of allergen attack, such as sneezing and itching, followed by rhinorrhea and nasal congestion, which often disappear within 1 hour. This reaction originates from the cross-linked complex of IgE-FcεRI and allergens, which leads to mast cell degranulation, release of pre-synthesized mediators such as histamine and de novo production of mediators from membrane lipids such as cysteinyl leukotrienes (leukotriene LTC4, LTD4 and LTE4) and prostaglandins (PD2). Histamine often acts on H1 receptors on sensory nerve endings to cause itching, leading to systemic reflexes such as sneezing. Leukotrienes, prostaglandins and vascular endothelial growth factor cause plasma to leak from blood vessels, resulting in edema, blood accumulation in wide venous sinuses, and increased glandular mucus secretion, all of which may lead to a feeling of nasal congestion.

[0026] Late phase hypersensitivity reactions (LPRs) are mainly characterized by persistent symptoms and a decrease in peak nasal inspiratory flow rate within 4-12 hours. The mediators released are mainly histamine, platelet-derived growth factor 2, and leukotrienes, which induce the influx and activation of various inflammatory cells, leading to a late phase response. Adhesion molecules such as vascular cell adhesion molecule-1 promote the adhesion of circulating eosinophils to endothelial cells, thereby promoting the influx of inflammatory cells into the nasal mucosa. Chemical inducers and cytokines such as IL-5 promote the infiltration of eosinophils, basophils, and T cells from the systemic circulation into the submucosal layer of the nasal mucosa. The subsequent release of mediators such as histamine and leukotrienes leads to the production of late symptoms of allergic rhinitis, such as nasal obstruction, nasal congestion, fatigue, and possible neurocognitive dysfunction.

[0027] Phage display peptide library technology uses the phage coat protein PⅢ or PⅧ gene as a vector to insert a gene fragment encoding an exogenous short peptide. The infection ability of the phage is not affected, and the inserted exogenous short peptide can also form a certain spatial conformation at the N-terminus of the PⅢ or PⅧ protein on the phage surface. The basic principle of screening antigen epitopes from phage display random peptide libraries 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 may be recovered by centrifugation or the like after culturing, and then dried, for example, by freeze drying, to prepare it in the form of a live bacterial agent 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 Lactobacillus rhamnosus HN001 strain or a culture with an equivalent number of live bacteria is used as the active ingredient.

[0029] In some examples of the present invention, the pharmaceutical composition comprising the above-mentioned Lactobacillus rhamnosus HN001 strain and the active polypeptide can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, 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 as a product for enteral or oral administration. In addition, the pharmaceutical composition of the present invention can be formulated into an enteric package using a known method to rapidly release the microorganisms as active substances in the intestine after passing through the gastrointestinal tract to reach the small intestine.

[0031] In some embodiments of the present invention, liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., which, in addition to water and liquid paraffin commonly used as simple diluents, may also contain excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., but are not limited to these.

[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 vitamins may be fat-soluble or water-soluble vitamins. Suitable vitamins include vitamin D, vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, vitamin B2, niacin, vitamin B6, folic acid, pyridoxine, pantothenic acid and biotin, but are not limited thereto. Any of the above suitable forms are salts of vitamins, derivatives of vitamins, compounds of vitamins having the same or similar activity, and metabolites of vitamins.

[0033] The pharmaceutical composition for preventing or treating allergic diseases of the present invention may also include a known additional therapeutic agent with the effect of preventing and treating allergic diseases. The acceptable additional therapeutic agent in the present invention is an immunosuppressant, analgesic, steroid, non-steroidal anti-inflammatory agent (NSAID) or cytokine antagonist and a combination thereof. The above-mentioned immunosuppressant can exemplify glucocorticoid, cyclosporin, tacrolimus, pimecrolimus, calcineurin inhibitors including ISA (TX) 247, rapamycin, phosphodiesterase 4 inhibitor, mycophenolate mofetil, dexamethasone, etc., but is not limited thereto, and all known immunosuppressants can be used together. In addition, one immunosuppressant can be used alone, or two or more immunosuppressants can be used in combination. Preferably, the above-mentioned immunosuppressant can use one or more selected from the group consisting of cyclosporin, tacrolimus, dexamethasone, and pimecrolimus. When the pharmaceutical composition of the present invention is administered in combination with other therapeutic agents, it can be administered sequentially or simultaneously, and can be administered once or multiple times.

[0034] In addition to the above-mentioned active ingredients, the pharmaceutical composition of the present invention may also contain pharmaceutically acceptable carriers and / or excipients, and may also be prepared by formulating with various additives commonly used in pharmacy, such as adhesives, disintegrators, coating agents, lubricants, etc.

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

[0036] Acceptable disintegrators in the present invention include cellulose derivatives such as carboxymethyl cellulose, calcium carboxymethyl cellulose, and 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 lubricants acceptable in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silicon dioxide, hydrated silicon dioxide, various waxes and hydrogenated oils.

[0038] The coating agent includes non-water-soluble polymers such as dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinyl acetaldehyde diethylamine acetate, ethyl acrylate-methacrylic acid copolymer, ethyl acrylate-methyl methacrylate-ethyl methacrylate trimethylammonium chloride copolymer, ethyl cellulose, methacrylic acid-ethyl acrylate copolymer, hydroxypropyl cellulose phthalate, hydroxypropyl methylcellulose acetate succinate and the like, and water-soluble polymers such as methylcellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, polyethylene glycol, etc., but is 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 based on factors including the type of various diseases, the patient's age, weight, sex, medical condition of the patient, severity of the condition, sensitivity to drugs, administration time, administration route and metabolic ratio, treatment period, 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 drug in an amount that can achieve the maximum effect in the minimum amount without side effects 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" are used interchangeably herein. These terms refer to an increase in a measured parameter in a treated cell, tissue, or individual compared to an untreated cell, tissue, or individual. The same cell, tissue, or individual can also be compared before and after treatment. In some embodiments, the increase in the treated cell, 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, tissue, or individual.

[0041] As used herein, "treatment," "treating," or "amelioration" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefits and / or prophylactic benefits.

[0042] In this application, unless otherwise specifically stated, the use of the singular also includes the plural. In this application, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "including" and other forms, such as "includes" and "included" is not restrictive. In addition, unless otherwise specifically stated, terms such as "element" or "component" cover both elements and components including one unit and elements and components including more than one subunit. In addition, the use of the term "part" may include a part of a part or an integral part. 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 specified 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 a pH of 5.5 to 7.5. In one embodiment, the pH of the aqueous medium can be adjusted by low concentrations of suitable biocompatible buffer components, non-limiting examples of which are tromethamine, sodium carbonate and sodium bicarbonate, and sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0044] The composition 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 preparation, the frequency of administration can be the same or different from each other. In addition, the composition of the present invention can be used alone or in combination with other antiallergic drugs. Taking all of the above factors into account, 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 for 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 embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0046] Example 1 Screening of polypeptides binding to HR4

[0047] 1.1 Screening

[0048] 50 μg / L HR4 protein (synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.) was dissolved in 8.4 g / L NaHCO3 and added to the ELISA plate, and incubated at 4°C overnight. Add blocking buffer, incubate at 37°C for 1.5 hours, and wash with buffer 3 times; 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 at 37°C with shaking for 2 hours. Discard the supernatant, add 0.1% TBST and wash the plate 10 times. Add 100 μL elution buffer (0.2 mol / LGlycine-HCL pH2.2, 1 mg / mL BSA) to the corresponding wells, and neutralize with Tris-HCl until the pH is 7.0 after 15 minutes.

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

[0050] E. coli ER2738 was cultured until it reached the logarithmic phase, and the eluted phage was added to the E. coli ER2738 culture and incubated at 37°C with shaking at 220 rpm for 4.5 h. The culture was centrifuged and the supernatant was transferred to a sterile tube, 20% PEG / NaCl was added, and it was placed on ice for 1-2 h. The solution was centrifuged and the precipitate was suspended with PBS. After centrifugation, the supernatant was transferred to a sterile test tube for the next bio-panning. A total of 4 pannings were performed, and 30 blue spots were randomly picked from the plates of the 4th round of screening titration, i.e., phage monoclones. Enterobacter ER2738 was cultured overnight at 37°C and 220 rpm. The culture was centrifuged the next day and the supernatant was collected for ELISA detection.

[0051] 1.2 ELISA identification of candidate phages

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

[0053] 1.3 Phage single-stranded DNA sequence

[0054] According to the results of the ELISA experiment, phage monoclonal clone 3G2 was selected for sequencing. PEG / NaCl was added to the phage monoclonal stock solution, and the phage was precipitated at 4°C for 10 minutes. The supernatant was removed after high-speed centrifugation. The precipitate was resuspended in 100μL iodide buffer (10mmol / L Tris, 1mmol / L EDTA, 4mmol / L NaI), and then 250μL anhydrous ethanol was added to precipitate DNA at room temperature. The mixture was centrifuged at high speed for 10 minutes, the supernatant was removed, the precipitate was rinsed with 700mL / L ethanol, and vacuum dried for a short time. Finally, the precipitate was resuspended in 30μL TE buffer (10mmol / L Tris, 1mmol / L EDTA). The above phage monoclonal DNA stock solution was taken and sent to Shanghai Shenggong Bioengineering Co., Ltd., and fully automatic sequencing was performed using -96g III sequencing primers. The phage sequencing results were read and translated according to the user manual attached to the phage peptide display library. The sequence of polypeptide 3G2 is shown in SEQ ID NO: 1.

[0055] 1.4 Synthesis of peptide 3G2

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

[0057] Example 2. Peptide 3G2 inhibits histamine-induced activation of the HR4 signaling pathway

[0058] HR4 is coupled to 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, HEK293-CRE (Luc) reporter cell line (Aimejie Technology, ABS-T3203) was used to detect the inhibitory activity of peptide 3G2 in the HR4 pathway.

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

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

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

[0062] The Lactobacillus rhamnosus GG strain HN001 (preservation number: ATCC SD5675) stored at -80°C was inoculated into MRS liquid culture medium, activated for 3 generations overnight at 37°C, and the Lactobacillus rhamnosus GG strain HN001 in the logarithmic growth phase after activation was inoculated into MRS culture medium with an inoculum amount of 3% (volume fraction), and cultured at 37°C for 24 hours in a sealed state, and the obtained bacterial solution was used for standby. Under sterile conditions, 2 mL of bacterial solution was inoculated into a fermentation tank equipped with an optimized culture medium, with a stirring speed of 100 rpm, a ventilation volume of 0, and a constant temperature fermentation at 37°C. After 3 hours, a food-grade NaOH solution was added to maintain the pH at 5.5-6.0, and the culture was carried out for 15 hours. The formula of the optimized culture 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; weigh the above ingredients, mix them evenly, heat them to dissolve, adjust the pH of the culture medium to 6.8 with food-grade NaOH solution (1 mol / L), and sterilize them at 121°C for 15 min.

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

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

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

[0066] Basic sensitization: 3 mg of ovalbumin (OVA, Beijing Bainuowei Biotechnology Co., Ltd., SLCH2414) was used as the antigen, 30 mg of aluminum hydroxide gel (Beijing Bainuowei Biotechnology Co., Ltd., VI311256) was used as the adjuvant, and 1 mL of normal saline was extracted to prepare a 0.3% OVA suspension. Intraperitoneal injection was performed once every 2 days for a total of 7 injections. The normal group was intraperitoneally injected with the same dose of normal saline.

[0067] Enhanced sensitization: After the intraperitoneal injection immunization phase, 50 μL of 5% ovalbumin-saline solution (5 mg OVA plus 10 mL saline solution to make a suspension) was dripped into the bilateral nasal cavities for local stimulation and enhanced sensitization stimulation, once a day, for a total of 7 times. The normal group received the same dose of saline in the nose.

[0068] The normal group (G1) was not treated; the model control group (G2) was a conventional OVA-sensitized animal; no treatment was performed; the G3-G5 groups were treated with peptide 3G2 freeze-dried powder, probiotic freeze-dried powder, and probiotic and peptide 3G2 mixed freeze-dried powder after conventional OVA sensitization. The above freeze-dried powders were taken separately, dissolved in sterile saline at a dose of 1 mg / g, and then gavaged once every 3 days, for a total of 6 times.

[0069] After the animal model is successfully constructed, the animal will have symptoms such as sneezing, scratching the nose, scratching the face, and runny nose. The superimposed quantitative score is used to analyze whether the allergic model is successfully constructed. After the last enhanced sensitization, each animal is observed for 30 minutes. The observation records are combined with relevant behavioral indicators to verify the successful construction of the animal allergy model in each group. During the experiment, it was observed that after three treatments, the behavioral scores of nasal symptoms in the G5 treatment group were at a low level during the behavioral evaluation. After four treatments, the behavioral scores of nasal symptoms in the G3 and G4 treatment groups decreased during the behavioral evaluation, indicating that the treatment groups have significant therapeutic effects in relieving the symptoms of allergic rhinitis (data not shown).

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

[0071] The results are as follows Figure 2A-2CAs shown, compared with the model control group, after treatment with polypeptide 3G2 freeze-dried powder or mixed freeze-dried powder of probiotics and polypeptide 3G2, the levels of IL4 and IL6 decreased to varying degrees, indicating that polypeptide 3G2 freeze-dried powder or 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. The polypeptide according to claim 1 , which is a HR4 antagonist.

3. A pharmaceutical composition comprising an effective amount of the polypeptide according to claim 1 or 2 and probiotics. The pharmaceutical composition according to claim 3 , wherein the probiotic is Bacillus rhamnosus. The pharmaceutical composition according to claim 4 , wherein the rhamnosus Bacillus is HN001.

6. The pharmaceutical composition according to any one of claims 3 to 5, which is administered in the form of a lyophilized powder.

7. The pharmaceutical composition according to any one of claims 3 to 6, further comprising vitamins, immunosuppressants, analgesics, steroids, non-steroidal anti-inflammatory agents (NSAIDs) or cytokine antagonists and combinations thereof.

8. Use of the polypeptide according to claim 1 or the pharmaceutical composition according to claims 3 to 7 for preparing a medicament for treating allergic diseases.

9. The use according to claim 8, wherein the allergic disease is food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria and allergic contact dermatitis.

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