Substance with anti-allergic activity and application thereof

Abstract

The invention discloses a substance with anti-allergic activity and application thereof, the substance with anti-allergic activity is a bitter receptor stimulant, and the bitter receptor stimulant is selected from at least one of denatonium benzoate, difenidol and quinine. The compound can reduce the expression level of IgE in serum, inhibit mast cell degranulation and regulate and control expression of cytokines at the downstream of Th1, Th2 and Dc cells, so that allergy is prevented and allergic symptoms are relieved, and the compound can be used for preparing medicines, health foods, functional foods and dietary supplements for preventing and relieving food allergy.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a substance with anti-allergic activity and application thereof. Background Art

[0002] In recent years, the incidence of food allergies has been increasing year by year worldwide. Food allergies have become a global food safety and public health issue, and are known as the "second wave" of allergy epidemics after asthma. Food allergy is a systemic immune response mediated by the immune system that is detrimental to the body. IgE-mediated food allergy is the most common allergic reaction. Patients will experience abdominal pain, diarrhea, nausea, vomiting, and even anaphylactic shock in severe cases, which has a certain risk of death. The number of people with food allergies has increased year by year, which not only imposes a serious economic burden on people, but also seriously affects their quality of life.

[0003] In the process of preventing and treating food allergies, the most important way is to avoid consuming allergenic foods, and the most important treatment method is chemical drug treatment. However, although current drug treatment can alleviate the condition to a certain extent, it cannot change the course of allergic diseases, cannot completely cure allergic diseases, and prevent the increase in global morbidity and mortality. Therefore, it is very necessary to explore an effective drug that can prevent and relieve allergies. Summary of the invention

[0004] To solve the above problems, in a first aspect, the present invention provides a substance with anti-allergic activity, which can prevent and alleviate allergies.

[0005] The technical solution of the present invention to solve the above problems is as follows:

[0006] A substance with anti-allergic activity, wherein the substance with anti-allergic activity is a bitter receptor agonist, and the bitter receptor agonist is selected from at least one of denatonium benzoate, diphenidol, and quinine.

[0007] Animals are usually very sensitive to bitter taste, and bitterness is considered a signal that is harmful to the body, which produces a disgust reaction and avoids the intake of toxic and harmful substances. However, not all bitter compounds are harmful. Although denatonium benzoate, diphenidol, and quinine have the sensory characteristics of bitterness, they have biological effects and can achieve the effect of allergy relief by activating the corresponding bitter receptors.

[0008] The bitter taste receptor agonists denatonium benzyl (DB), diphenidol (DH) and quinine (QH) can reduce the expression level of IgE in serum and alleviate IgE-mediated allergic reactions.

[0009] In a second aspect, the present invention also provides the use of the above-mentioned substance having anti-allergic activity, as follows:

[0010] The application of the substance with anti-allergic activity in the preparation of medicines for preventing and allergic relief, health foods, functional foods and dietary supplements.

[0011] Preferably, the allergy includes allergies caused by β-Lg and OVA allergens.

[0012] The substance with anti-allergic activity is used in the preparation of medicines, health foods, functional foods and dietary supplements for preventing and alleviating atopic dermatitis caused by β-Lg and OVA allergens.

[0013] The substance with anti-allergic activity is used in the preparation of a drug for reducing the expression level of IgE in serum and alleviating IgE-mediated allergic reactions.

[0014] The application of the substance with anti-allergic activity in the preparation of drugs for regulating the expression of cytokines downstream of Th1, Th2 and Dc cells.

[0015] The application of the substance with anti-allergic activity in the preparation of medicine for treating allergies caused by mast cell degranulation reaction.

[0016] Preferably, the mast cells include KU812 and RBL-H3.

[0017] The present invention also provides an antiallergic active compound preparation, wherein the effective ingredient of the preparation comprises the bitter taste receptor agonist mentioned above.

[0018] The preparation method of the antiallergic active compound preparation is as follows: the bitter receptor agonist is dissolved in a buffer solution to obtain the antiallergic active compound preparation.

[0019] Preferably, the buffer solution is phosphate buffered saline (PBS).

[0020] Preferably, the bitter taste receptor agonist is used at a concentration of 10 μM to 10 mM.

[0021] Preferably, the dosage forms of the preparation include: tablets, capsules, granules, powders, pills, solutions, injections, lyophilized powder injections, infusions, emulsions, ointments, lotions, aerosols, patches, cataplasms, etc.

[0022] The present invention also provides the application of the above antiallergic active compound preparation, which is as follows:

[0023] The preparation is used in preparing medicines, health foods, functional foods and dietary supplements for preventing and allergy relief.

[0024] The present invention has the following beneficial effects:

[0025] The present invention provides a substance with anti-allergic activity. The substance with anti-allergic activity is a bitter receptor agonist. The bitter receptor agonist is selected from at least one of denatonium benzoate, diphenidol, and quinine. The bitter receptor agonist can reduce the expression level of IgE in serum, inhibit mast cell degranulation, and regulate the expression of cytokines downstream of Th1, Th2, and Dc cells, thereby preventing allergies and relieving allergic symptoms. The substance can be used to prepare medicines, health foods, functional foods, and dietary supplements for preventing and relieving allergies. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the weight changes of mice during the OVA-sensitized mouse model experiment;

[0027] Figure 2 This is a graph showing changes in body temperature during the OVA-sensitized mouse model experiment;

[0028] Figure 3 This is the allergy score diagram of mice during the experiment in the OVA-sensitized mouse model;

[0029] Figure 4 This is a graph of mouse-specific IgE and IgG1 content;

[0030] Figure 5 To determine the ability of bitter taste receptor agonists to interfere with the degranulation of human mast cells KU812;

[0031] Figure 6 Determination of the ability of bitter taste receptor agonists to interfere with the degranulation of rat mast cells RBL-2H3;

[0032] Figure 7 Bitter taste receptor agonist DB interferes with the expression of IFN-γ cytokines in spleen cells of mice with type II immune response induced by LPS+IL-4;

[0033] Figure 8 Bitter taste receptor agonist DB interferes with the expression of IL-4 cytokine in spleen cells of mice with type II immune response induced by LPS+IL-4;

[0034] Fig. 9 Bitter taste receptor agonist DB interferes with the expression of FOXP3 transcription in spleen cells of mice with type II immune response induced by LPS+IL-4;

[0035] Fig.10 Bitter taste receptor agonist DB interferes with the expression of IL-10 cytokine in spleen cells of mice with type II immune response induced by LPS+IL-4;

[0036] Fig.11Bitter taste receptor agonist DB interferes with the expression of IL-6 cytokine in spleen cells of mice with LPS+IL-4-induced type II immune response. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0038] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0039] Example 1: Preparation of antiallergic bitter receptor agonist diphenidol (DH) preparation

[0040] The preparation method of diphenidol (DH) preparation with antiallergic activity bitter receptor agonist comprises the following steps:

[0041] (1) Weigh 17.30 mg of diphenhydramine (DH) on an electronic balance;

[0042] (2) Dissolve diphenhydramine (DH) in 10 ml of phosphate buffered saline (PBS), mix well until completely dissolved, and the concentration is 5 mM. Store at 4°C for later use.

[0043] Example 2: Preparation of antiallergic bitter receptor agonist denatonium benzyl (DB) preparation

[0044] The preparation method of the bitter receptor agonist denatonium benzyl (DB) preparation with antiallergic activity comprises the following steps:

[0045] (1) Weigh 22.32 mg of denatonium benzoate (DB) on an electronic balance;

[0046] (2) Dissolve denatonium benzoate (DB) in 10 ml of phosphate buffered saline (PBS), mix well until completely dissolved, and the concentration is 5 mM. Store at 4°C for later use.

[0047] Example 3: Application of bitter receptor agonist preparation in alleviating allergies in mice

[0048] 1. Establishment of OVA ear sensitization model in C57BL / 6 mice

[0049] The animal model constructed in this study used MC903-ethanol as an adjuvant to induce skin damage, OVA as the allergen protein, and bitter receptor agonists for in vivo intervention. It was divided into three stages: pre-feeding, sensitization, and oral gavage stimulation. The specific operation of the mouse immunization program is as follows:

[0050] (1) The mice were randomly divided into 5 groups, namely, adjuvant group (control group), OVA sensitization group, denatonium benzyl (DB) intervention sensitization group, diphenidol (DH) intervention sensitization group, and quinine (QH) intervention sensitization group. The bitter taste receptor agonist dosage for mice was 20 mg / kg.

[0051] (2) Mice were pre-fed with normal feed for one week and had free access to water.

[0052] (3) The sensitization phase begins in the second week.

[0053] ① Adjuvant group: 2 nmol MC903 was applied to both ears every day after 7 days of pre-feeding.

[0054] ②OVA sensitization group: After 7 days of pre-feeding, OVA was applied to the ears every day for sensitization.

[0055] ③ Denatonium Benzyl (DB) intervention sensitization group: After 7 days of pre-feeding, OVA was applied to the ears and denatonium benzoate solution was gavaged every day.

[0056] ④ Difenidol (DH) intervention sensitization group: After 7 days of pre-feeding, OVA was applied to the ears for sensitization and difenidol solution was gavaged every day.

[0057] ⑤ Quinine (QH) intervention sensitization group: After 7 days of pre-feeding, OVA was applied to the ears for sensitization and quinine solution was gavaged every day.

[0058] (4) Day 22 was the stimulation day: the control group mice were gavaged with 200 μL PBS solution, the sensitized group mice were gavaged with 200 μL OVA solution (100 mg / mL) dissolved in PBS, and each bitter receptor agonist intervention group was gavaged with 200 μL OVA solution dissolved in PBS.

[0059] In the pre-feeding and sensitization stages, the weight of the mice was weighed and recorded on days 1, 7, 14, and 21, and the weight of the mice was weighed and recorded before and after the high-dose OVA gavage challenge on day 22. The mice were challenged with high-dose OVA by gavage, and the changes in the rectal body temperature of the mice were measured and recorded before and after the challenge. The allergic symptoms of the mice were observed within half an hour after the challenge, and the mice in each group were scored and recorded according to the clinical allergic symptom scoring criteria for mice shown in Table 1.

[0060] Table 1 Scoring criteria for clinical allergic symptoms in mice

[0061]

[0062] Within 1 hour after the challenge, blood samples were collected from the eye sockets of mice by venous blood sampling and placed in a coagulant tube. The samples were allowed to stand for 1 hour and centrifuged at 4500 rpm for 20 minutes. The upper transparent serum was collected and stored in aliquots at -80°C. Blood was collected again by eye removal within 24 hours after the challenge, and serum was obtained and stored according to the above method.

[0063] 2. Determination of specific IgE in mouse serum

[0064] The double antibody sandwich indirect ELISA method was used for relative quantitative detection of OVA protein-specific IgE antibody response in mouse serum.

[0065] (1) Material preparation:

[0066] 1) Coating solution: weigh 1.59 g Na 2 CO 3 , 2.93 g NaHCO 3 , dilute to 1L with ultrapure water and store in a refrigerator at 4℃;

[0067] 2) Washing solution: Add 0.05% Tween-20 to 1L PBS buffer, mix thoroughly and wait for bubbles to disappear before use;

[0068] 3) Blocking solution and antibody diluent: Accurately weigh 5 g of BSA powder and dissolve it in 100 mL of PBS buffer. Mix thoroughly and store in a refrigerator at 4°C.

[0069] 4) Stop solution: Prepare 1 M H2O with concentrated sulfuric acid in a fume hood. 2 SO 4 .

[0070] (2) Coating: Dissolve OVA to 10 μg / mL using coating solution, add 100 μL to each well of the ELISA plate, and coat overnight at 4°C;

[0071] (3) Washing and blocking: discard the coating solution and pat dry on filter paper; add 250 μL of washing solution to each well, let stand for 2 min, discard the washing solution and pat dry, repeat washing three times; add 200 μL of blocking solution to each well and incubate at 37°C for 90 min;

[0072] (4) Washing and sample incubation: discard the blocking solution and pat dry on filter paper; add 250 μL of washing solution to each well, let stand for 2 min, discard the washing solution and pat dry, and repeat washing 3 times; dilute the serum with antibody diluent at a certain ratio (IgE = 1:6), add 100 μL of diluted serum to each well, and incubate at 37°C for 2 h;

[0073] (5) Washing and secondary antibody incubation: discard serum and pat dry on filter paper; add 250 μL of washing solution to each well, let stand for 2 min, discard the washing solution and pat dry, repeat washing 3 times; dilute GoatAnti-MouseIgE-HRP with antibody diluent at 1:1000, add 100 μL to each well, and incubate at 37°C for 90 min;

[0074] (6) Washing and TMB incubation: discard the secondary antibody and pat dry on filter paper; add 250 μL of washing solution to each well, let stand for 5 min, discard the washing solution and pat dry, repeat washing 5 times; add 100 μL of TMB solution to each well and incubate at 37°C in the dark for 15 min;

[0075] (7) Stop the reaction and measure the OD value: add 50 μL of stop solution to each well and measure the OD value at a wavelength of 450 nm using an enzyme reader.

[0076] 3. Bitter receptor agonists significantly improve allergy symptoms

[0077] 3.1 Changes in mouse body weight

[0078] like Figure 1 As shown in the figure, overall, the weight of all mice increased during the pre-feeding stage, indicating that the mice were in good growth condition; the weight of mice began to decrease during the sensitization stage, indicating that the adjuvant and allergen stimulation of mice during the sensitization stage affected the weight of mice. Compared with the control group, the weight of mice in the sensitization group continued to decrease during the sensitization stage, and the weight decreased significantly; the weight loss level of mice in the intervention groups of bitter receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) was between the OVA sensitization group and the control group. Through the weekly weight changes of mice, the results showed that the weight of mice would change after the allergen OVA sensitized the body. It is speculated that OVA as a food allergen stimulated the body to produce a food allergy reaction. Supplementation of bitter receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) can alleviate the weight loss of mice caused by food allergy. Overall, the intervention of bitter receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) alleviated the weight loss of mice caused by allergy.

[0079] 3.2 Changes in rectal temperature of mice

[0080] like Figure 2As shown, the changes in rectal body temperature of mice in each group before and after allergen OVA stimulation. For the control group, it can be seen that there is no significant change in the rectal body temperature of mice before and after stimulation; for the OVA sensitization group, the rectal body temperature of mice decreased significantly after stimulation; for the bitter taste receptor agonist denatonium benzoate (DB), diphenidol (DH), and quinine (QH) intervention group, the rectal body temperature of mice also decreased after stimulation, but the degree was weaker than that of the OVA sensitization group. The results show that the allergen OVA can affect the changes in the rectal body temperature of mice, and the bitter taste receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) have a mitigating effect on the degree of rectal temperature reduction after intervention.

[0081] 3.3 Allergy symptom scoring in mice

[0082] After mice develop allergic symptoms, they generally experience gastrointestinal and respiratory reactions. Symptom score is one of the important indicators for evaluating and reflecting the degree of allergy in the body, such as Figure 3 As shown in the figure, within 2 hours after the high-dose allergen OVA was stimulated, the mice in each group showed different degrees of allergic symptoms compared with the control group. The allergic symptoms of the mice in the OVA sensitization group and the bitter receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) intervention groups showed significantly more severe allergic symptoms, while the allergic symptoms of the mice in the bitter receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) intervention groups were milder. The results of the mouse allergy symptom score showed that OVA can significantly stimulate mice to produce food allergic reactions, and the bitter receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) intervention can effectively alleviate the food allergy symptoms of mice.

[0083] 4. Changes in OVA-specific IgE and IgG1 levels in mouse serum

[0084] like Figure 4 As shown, the changes in the content of OVA-specific IgE in the serum of mice one week after sensitization and the changes in the content of OVA-specific IgE, IgG1 and in the serum of mice after oral gavage were determined by indirect enzyme-linked immunosorbent assay. During food allergic reactions, Th2 cells stimulate B cells to produce antibodies IgE and IgG1 by secreting cytokines.

[0085] After one week of sensitization, the IgE content in the serum of mice in the OVA-sensitized group was significantly upregulated compared with that in the control group, and the OVA-specific IgE content was significantly downregulated after intervention with bitter receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH). After intragastric stimulation, compared with mice sensitized for one week, the content level of OVA-specific IgE was generally upregulated, and the trend of the change of OVA-specific IgE content was similar to that of mice sensitized for one week, indicating that bitter receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) alleviated the sensitization effect of OVA.

[0086] After intragastric administration, the levels of IgG1 and IgG2a in the serum of mice were significantly upregulated by the allergen OVA compared with the control group, and intervention with bitter taste receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) could reduce the level of IgG1. The results showed that intervention with bitter taste receptor agonists denatonium benzoate (DB), diphenidol (DH), and quinine (QH) alleviated food allergic reactions in mice.

[0087] In summary, 20 mg / kg of bitter receptor agonists denatonium benzyl (DB), diphenidol (DH), and quinine (QH) can significantly alleviate the allergic symptoms of mice.

[0088] Example 4: Application of bitter receptor agonist preparations to alleviate mast cell degranulation

[0089] Mast cells are important cells in the effector stage of food allergy. When degranulation occurs, they release inflammatory mediators such as IL-4 and β-hexosaminidase. These inflammatory mediators act on the skin, respiratory tract and other parts to induce food allergies, and then produce corresponding allergic symptoms.

[0090] Determination of β-hexosaminidase release rate

[0091] Here, human KU812 and rat RBL-2H3 mast cells were used, respectively, and the ability of the complexes of β-Lg and the bitter receptor agonists denatonium benzyl (DB) and diphenidol (DH) to induce mast cell degranulation was determined by measuring the release rate of β-hexosaminidase.

[0092] The experimental operation is as follows: Cell recovery: Take out the frozen cells and quickly place them in a 37°C water bath. After waiting for them to completely thaw, quickly transfer them to the clean bench workbench, add the corresponding cell complete culture medium to resuspend, centrifuge at 4°C, 500g for 5 minutes, aspirate the supernatant, retain the cell pellet, and then add sterile PBS solution to resuspend, centrifuge according to the above conditions, discard the supernatant, and gently resuspend the cell pellet with complete culture medium, transfer them all to the cell culture dish, and then culture them in a 37°C cell culture incubator.

[0093] After mast cells were revived, when the cells were in good growth condition, the cells were inoculated into 96-well cell plates at 200 μL / well, 20 μL of allergic serum was added to each well, and then placed in an incubator for 24 hours. After the incubation was completed, the centrifuge was set to 500 rpm, and then centrifuged for 5 minutes, the supernatant was aspirated, and rinsed with PBS 3 times. Denatonium benzoate (DB), and 50 μM and 150 μM diphenidol (DH) were accurately weighed, dissolved in DMSO to 50 mM stock solution, and then diluted to 100 μM and 500 μM denatonium benzoate (DB), and 50 μM and 150 μM diphenidol (DH), respectively. Divided into β-Lg treatment group (allergic group) and an equal amount of PBS group (blank control group) and β-Lg and 100μM and 500μM denatonium benzoate (DB), and 50μM and 150μM diphenidol (DH), and supplemented with PBS to equal amounts in each group. Add 20μL of human milk oligosaccharide and β-Lg complex sample solution to the corresponding wells and incubate in an incubator for 6h. Centrifuge again according to the centrifugation conditions set above, then take the supernatant and add it to a new 96-well plate, add the same volume of PNAG to each well, and incubate in an incubator for 6h. Then lyse the cells with 0.1% TritonX-100, take the cell lysate and add it to the corresponding wells, then add the same volume of PANG to each well and incubate in an incubator for 6h. Add Na 2 CO 3 / NaHCO 3 Solution, stop the color reaction, put into the microplate reader to measure the absorbance at 405nm, read the data, and calculate according to β-hexosaminidase release rate (%) = (OD test group supernatant - OD control group supernatant) / (OD test group supernatant - OD control group supernatant + OD test group lysate - OD control group lysate) × 100%.

[0094] Mast cell degranulation capacity analysis (determination of mast cell release of β-hexosaminidase)

[0095] Compared with ELISA, mast cell degranulation is a more direct and effective immunological method. Therefore, this part explored and evaluated the ability of β-Lg and the bitter receptor agonists denatonium benzyl (DB) and diphenidol (DH) to induce mast cell degranulation by measuring the release rate of β-hexosaminidase.

[0096] like Figure 5As shown, for human peripheral blood basophilic leukemia cells KU812, the β-hexosaminidase released by the blank control group was 21.30%, and the β-hexosaminidase released by the β-Lg treatment group without bitter receptor agonists reached 35.71%. Compared with the βLG group, the β-hexosaminidase release rate of β-Lg combined with bitter receptor agonists denatonium benzoate (DB) and diphenidol (DH) was reduced to 26.47%, 26.98%, 27.68% and 27.50%, respectively.

[0097] like Figure 6 As shown, for rat basophilic leukemia cells RBL-2H3, the β-hexosaminidase released by the blank control group was 20.49%, and the β-hexosaminidase released by the β-Lg treatment group without the bitter receptor agonist reached 35.45%. Compared with the β-Lg group, the β-hexosaminidase release rate of β-Lg combined with the bitter receptor agonist was reduced to 26.12%, 23.37%, 23.48%, and 28.58%, respectively. The results showing that the two mast cells release β-hexosaminidase indicate that the interaction between the bitter receptor agonists denatonium benzyl (DB) and diphenidol (DH) and β-Lg significantly reduces the release of β-hexosaminidase and reduces the in vitro sensitization of β-Lg.

[0098] In summary, 100 μM and 500 μM denatonium benzoate (DB), and 50 μM and 150 μM diphenidol (DH) could significantly alleviate the symptoms of mast cell degranulation.

[0099] Example 5: Bitter receptor agonist preparations regulate Th1 Th2 Treg cytokine balance

[0100] 1. Preparation of Mouse Spleen Single Cell Suspension

[0101] The mice were killed by cervical dislocation and then soaked in 70% alcohol for 3 minutes for disinfection and transferred to a clean bench. After wiping the abdominal cavity with alcohol cotton, the abdominal cavity was opened and the spleen tissue was carefully removed. The spleen was cut into pieces with sterile scissors, placed on a 70μm sieve, and gently ground with the inner core of a syringe until the spleen turned white, and sterile PBS buffer was continuously added to the tissue. The cells were collected in a 15mL centrifuge tube and centrifuged at 500g and 4℃ for 5min. After discarding the supernatant, 5ml of red blood cell lysis buffer was added for lysis, gently blown, mixed, and lysed at room temperature for 5min until the red blood cells were completely broken. 500g, 4℃ centrifuged for 5min. Discard the supernatant, continue to add 10mL PBS buffer to resuspend the cell pellet, and centrifuge at 500g at 4℃ for 5min. Discard the supernatant and repeat the washing twice. Dilute the number of cells in the 6-well plate to 106 / mL; place it in a cell culture incubator for culture. The groups were divided into a blank group, an LPS+IL-4 group, and an LPS+IL-4+denatonium benzoate (DB) group (DB concentration was 100 μM, and the preparation method was the same as in Example 4). The cells were stimulated for three days, and then RNA was extracted.

[0102] 2. Extraction of Total RNA from Cells

[0103] The extraction steps of total cell RNA were based on the instructions of the RNA extraction kit. The centrifuge tubes and reagents used in the operation were enzyme-free. The extraction steps were as follows:

[0104] (1) Prepare RNA lysis buffer in a fume hood. Add 20 μL of β-mercaptoethanol to the reagent.

[0105] (2) For adherent cells, carefully remove the culture medium with a waste pump and rinse with PBS solution. Add 1 mL of the RNA lysis solution prepared above, and transfer the cells to a centrifuge tube after careful pipetting. For suspended cells, collect the cells in a centrifuge tube, centrifuge (500g, 4°C) for 5 minutes, add PBS for washing, centrifuge (500g, 4°C) for 5 minutes, add 1 mL of the RNA lysis solution prepared above, and mix carefully by pipetting.

[0106] (3) Add chloroform to each tube, vortex to mix, and centrifuge (12000g, 4°C) for 15 min.

[0107] (4) After centrifugation, aspirate the supernatant into a centrifuge tube, add the same volume of 70% ethanol as the supernatant, and vortex to mix;

[0108] (5) Transfer the sample from (4) to an RNA extraction column and centrifuge for 1 min (10,000 g, 4°C).

[0109] (6) Add 500 μL of washing solution I and centrifuge (10,000 g, 4°C) for 30 s; add 500 μL of washing solution II and centrifuge (10,000 g, 4°C) for 1 min. Repeat the step of adding washing solution II twice.

[0110] (7) Centrifuge (12000g, 4°C) for 2 min to completely dry the tube; insert the column into the centrifuge tube, add DEPC (preheated to 70°C), and finally centrifuge (12000g, 4°C) for 2 min to collect the total RNA in the cells.

[0111] 3. RNA concentration and purity determination

[0112] The purity and concentration of RNA were determined using the Nucleic Acid mode of the NanoDrop 2000 nucleic acid microanalyzer.

[0113] 4. Reverse transcription and cDNA synthesis

[0114] The RNA samples were reverse transcribed, and the specific steps were as follows:

[0115] (1) In an enzyme-free centrifuge tube, mix by pipetting according to the system shown in Table 2, and incubate at 42°C for 2 min:

[0116] Table 2. First step reverse transcription reaction system

[0117]

[0118] (2) Add the corresponding reagents to the PCR reaction tube as shown in Table 3, gently pipette to mix, and quickly centrifuge to the bottom of the reaction tube.

[0119] (3) The prepared reaction system is reverse transcribed according to the reaction program set in Table 4.

[0120] (4) Aliquoting and storage: After reverse transcription, the cDNA is aliquoted and stored in a -80°C refrigerator.

[0121] Table 3 Second step reverse transcription reaction system

[0122]

[0123] Table 4 Reverse transcription reaction procedure

[0124]

[0125] 5. RNA Real-time Fluorescence Quantitative PCR

[0126] According to the instructions of the qPCR kit, β-actin was used as the internal reference gene and the relative expression of the target gene was calculated according to 2 -ΔΔCtThe calculation method is as follows: add the reagents in Table 5 to the 96-well PCR microplate and centrifuge to make the reagents gather at the bottom of the 96-well plate and mix evenly. The whole experiment is protected from light. The program for the machine is detailed in Table 6, and the primers are detailed in Table 7.

[0127] Table 5 qPCR reaction system

[0128]

[0129] Table 6 qPCR reaction procedure

[0130]

[0131] Table 7 qPCR upstream and downstream primer sequences

[0132]

[0133] In summary, if Figure 7-11 As shown in the results, the addition of DB can significantly inhibit the gene expression of Th2 cytokines (IL-4), while promoting the gene expression of Th1 (IFN-γ) and Treg cytokines (IL-10 and transcription factor Foxp3). This shows that the addition of DB helps to rebalance the immune response, thereby alleviating allergic symptoms. By enhancing the activity of Th1 and Treg, DB not only inhibits the occurrence of allergies, but also may help maintain immune homeostasis, providing a potential intervention strategy for the treatment of allergies. It can be concluded that 100 μM denatonium benzoate (DB) can significantly alleviate and inhibit Th2-related inflammatory responses in mice at the gene expression level, promote the differentiation of Th0 cells into Treg cells at the gene expression level, induce the body to produce immune tolerance, and thus alleviate food allergies.

Claims

1. A substance having antiallergic activity, characterized in that: The substance with anti-allergic activity is a bitter receptor agonist, and the bitter receptor agonist is selected from at least one of denatonium benzoate, diphenidol, and quinine.

2. Use of the substance with anti-allergic activity according to claim 1 in the preparation of drugs, health foods, functional foods and dietary supplements for preventing and allergic reactions.

3. The use according to claim 2, characterized in that: The allergies include those caused by β-Lg and OVA allergens.

4. Use of the substance with antiallergic activity according to claim 1 in the preparation of medicines, health foods, functional foods and dietary supplements for preventing and alleviating atopic dermatitis caused by β-Lg and OVA allergens.

5. Use of the substance with antiallergic activity according to claim 1 in the preparation of a drug for reducing the expression level of IgE in serum and alleviating IgE-mediated allergic reactions.

6. Use of the substance with antiallergic activity according to claim 1 in the preparation of a drug for regulating the expression of cytokines downstream of Th1, Th2 and Dc cells.

7. Use of the substance with antiallergic activity according to claim 1 in the preparation of a drug for treating allergies caused by mast cell degranulation reaction.

8. An antiallergic active compound preparation, characterized in that: The active ingredient of the preparation comprises the bitter taste receptor agonist according to claim 1.

9. The preparation according to claim 8, characterized in that The bitter taste receptor agonist is used at a concentration of 10 μM to 10 mM.

10. Use of the preparation according to claim 8 in the preparation of medicines, health foods, functional foods and dietary supplements for preventing and allergy relief.

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