Application of FTY720 in preparation of medicine for preventing and / or treating food allergy

Abstract

The invention provides application of FTY720 in preparation of a medicine for preventing and / or treating food allergy, and belongs to the technical field of medicine. The invention provides application of FTY720 in preparation of a medicine for preventing and / or treating food allergy. Animal experiments prove that the FTY720 can relieve the decrease of the body temperature of a mouse with food allergy; weight loss of mice with food allergy is relieved; the diarrhea occurrence rate of food-allergic mice can be reduced, and the possibility of diarrhea occurrence is reduced; the serum OVA specific IgE antibody level of a food allergy mouse can be reduced; the serum OVA specific IgG1 antibody level of the food allergy mouse is reduced; the jejunum tissue damage of a mouse with food allergy is relieved or relieved; the jejunum inflammatory factor level of food-allergic mice is reduced; the intestinal flora and the like of a mouse with food allergy are improved. The FTY720 provided by the invention can be used for preparing medicines for preventing and / or treating food allergy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of FTY720 in the preparation of drugs for preventing and / or treating food allergy. Background Art

[0002] Food allergy (FA) is a public health problem worldwide. It is an abnormal immune response of the body after susceptible individuals ingest food antigens, which can affect multiple systems of the body, mainly manifested in the digestive system, respiratory system, skin and mucous membrane system, etc. At present, there is no effective treatment method for FA. The only treatment measures are to avoid contact with allergens and treat allergic symptoms after accidental ingestion of allergens. Related therapeutic drugs, such as antihistamines, glucocorticoids, epinephrine, etc., can only temporarily relieve allergic symptoms and cannot solve the underlying immune disorder to fundamentally block the occurrence of food allergy.

[0003] FTY720 is an analogue of sphingosine and a regulator of sphingosine-1-phosphate receptor (S1PR), and it is the first oral drug approved by the US Food and Drug Administration (FDA) for the treatment of relapsing-remitting multiple sclerosis.

[0004] Currently, there is no relevant research on the treatment of food allergy with FTY720. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of FTY720 in the preparation of drugs for preventing and / or treating food allergy. Using FTY720 for food allergy can effectively relieve or treat food allergy symptoms.

[0006] The purpose of the present invention is achieved by the following technical solutions:

[0007] The present invention provides the application of FTY720 in the preparation of drugs for preventing and / or treating food allergy.

[0008] The present invention provides the application of intestinal flora in the preparation of drugs for preventing and / or treating food allergy; the intestinal flora includes Bacteroidota flora, Patescibacteria flora, and Desulfobacterota flora.

[0009] Preferably, the food allergy includes allergy induced by protein-containing foods.

[0010] Preferably, the protein-containing foods include foods containing ovalbumin.

[0011] Preferably, the disease condition of the food allergy includes allergic enteritis.

[0012] Preferably, the prevention and / or treatment of food allergy includes any one or more of the following (1) to (8):

[0013] (1) Alleviating the decrease in body temperature;

[0014] (2) Alleviating the decrease in body weight;

[0015] (3) Reducing the incidence of diarrhea and the likelihood of diarrhea occurrence;

[0016] (4) Reducing the level of serum OVA-specific IgE antibody;

[0017] (5) Reducing the level of serum OVA-specific IgG1 antibody;

[0018] (6) Alleviating or relieving jejunum tissue damage;

[0019] (7) Reducing the level of jejunum inflammatory factors;

[0020] (8) Improving the intestinal flora.

[0021] Preferably, the improvement of the intestinal flora includes any one or more of reducing the abundance of Firmicutes flora, reducing the abundance of Actinobacteria flora, increasing the abundance of Bacteroidota flora, increasing the abundance of patescibacteria flora, and increasing the abundance of Desulfobacterota flora.

[0022] Preferably, the jejunum inflammatory factors include any one or more of IL-4, IL-10, IL-13, IFN-γ, GRO-α, and TNF-α.

[0023] Preferably, the drug contains FTY720 and a pharmaceutically acceptable carrier.

[0024] Preferably, the drug contains FTY720 and pharmaceutical excipients.

[0025] Advantages of the present invention

[0026] The present invention provides the application of FTY720 in the preparation of drugs for preventing and / or treating food allergy. Through animal experiments, the present invention verifies that FTY720 can relieve the decrease in body temperature of food allergy mice; relieve the decrease in body weight of food allergy mice; reduce the incidence of diarrhea in food allergy mice and reduce the possibility of diarrhea occurrence; reduce the level of OVA-specific IgE antibody in the serum of food allergy mice; reduce the level of OVA-specific IgG1 antibody in the serum of food allergy mice; alleviate or relieve the jejunal tissue damage of food allergy mice; reduce the level of jejunal inflammatory factors in food allergy mice; and improve the intestinal flora of food allergy mice. FTY720 provided by the present invention can be used for preventing and / or treating food allergy. Brief Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 Flow chart for constructing mice in each experimental group of Example 1;

[0029] Figure 2 Detection result graphs of body temperature, body weight, diarrhea conditions and related indicators of H&E staining in the FA group and the control group in Example 1;

[0030] Figure 3 Detection result graphs of relative expression levels of OVA-specific antibodies in the sera of mice and cytokines mRNA in jejunal tissues in the FA group and the control group in Example 1;

[0031] Figure 4 Detection result graphs of changes in body temperature and body weight of mice in each experimental group of Example 1;

[0032] Figure 5 Detection result graphs of diarrhea and H&E staining of mice in each experimental group of Example 1;

[0033] Figure 6 Detection result graphs of relative expression levels of mRNA expression of jejunal inflammatory factors in mice in each experimental group of Example 1;

[0034] Figure 7 Detection result graphs of intestinal flora in the FA group, the control group and the FTY720 (i.m - immunize) group in Example 1;

[0035] Figure 8Graph showing the test results of relevant indicators such as body temperature, body weight, OVA-specific antibodies, and relative expression levels of cytokine mRNAs in the jejunal tissues of mice in each experimental group of Example 2;

[0036] Figure 9 Graph showing the test results of diarrhea and H&E staining-related indicators in mice in each experimental group of Example 2. Detailed implementation manners

[0037] The present invention provides the use of FTY720 in the preparation of drugs for preventing and / or treating food allergy.

[0038] FTY720 is an analogue of sphingosine and a regulator of sphingosine-1-phosphate receptor (S1PR). Through animal experiments, the present invention verifies that it can be used in the preparation of drugs for preventing and / or treating food allergy.

[0039] The present invention provides the use of gut microbiota in the preparation of drugs for preventing and / or treating food allergy; the gut microbiota includes Bacteroidota flora, Patescibacteria flora, and Desulfobacterota flora.

[0040] As an alternative implementation manner of the present invention, the source of the gut microbiota includes feces of FTY720-prevented food allergy mouse models and / or feces of normal mice; the food allergy mouse model is an OVA food allergy mouse model; the FTY720-prevented food allergy mouse model is a mouse model constructed by intramuscular injection of FTY720 while sensitizing with OVA food. The present invention has no special limitation on the method for obtaining the gut microbiota, and any conventional method in the art can be used. As an alternative implementation manner of the present invention, the method for obtaining the gut microbiota includes: collecting mouse feces. After collecting the mouse feces, the collected mouse feces are mixed with sterile PBS, and the mass-volume ratio of the feces to sterile PBS is 200 mg: 1 mL. As an alternative implementation manner of the present invention, 1 mL of sterile PBS is added to each 200 mg of mouse feces to obtain a gut microbiota suspension. After adding sterile PBS to the feces, the present invention preferably performs vortex mixing and then filters through a 70 μm sieve. During the filtering process, grinding and filtering are carried out simultaneously. After obtaining the gut microbiota suspension, the present invention preferably verifies the effect of the gut microbiota in preventing and / or treating food allergy through the gut microbiota suspension.

[0041] As an alternative embodiment of the present invention, the food allergy includes the allergy induced by protein-containing foods. In the present invention, the protein-containing foods include foods containing ovalbumin; the foods containing ovalbumin include eggs; the eggs include any one or more of chicken eggs, goose eggs, duck eggs and quail eggs. As an alternative embodiment of the present invention, the symptoms of the food allergy include allergic enteritis.

[0042] As an alternative embodiment of the present invention, the prevention and / or treatment of food allergy includes any one or more of the following (1) to (8):

[0043] (1) Alleviating the decrease in body temperature;

[0044] (2) Alleviating the decrease in body weight;

[0045] (3) Reducing the incidence of diarrhea and the likelihood of diarrhea;

[0046] (4) Reducing the level of serum OVA-specific IgE antibody;

[0047] (5) Reducing the level of serum OVA-specific IgG1 antibody;

[0048] (6) Alleviating or relieving jejunum tissue damage;

[0049] (7) Reducing the level of jejunum inflammatory factors;

[0050] (8) Improving the intestinal flora.

[0051] In the present invention, the FTY720 or the intestinal flora can alleviate the decrease in body temperature caused by food allergy. The results of the examples in the present invention show that the FTY720 or the intestinal flora can alleviate the decrease in body temperature of OVA-sensitized mice and maintain their body temperature at a normal level.

[0052] In the present invention, the FTY720 or the intestinal flora can alleviate the decrease in body weight caused by food allergy. The results of the examples in the present invention show that the FTY720 or the intestinal flora can alleviate the decrease in body weight of OVA-sensitized mice.

[0053] In the present invention, the FTY720 or the intestinal flora can reduce the incidence of diarrhea caused by food allergy and the likelihood of diarrhea. The results of the examples in the present invention show that the FTY720 or the intestinal flora can reduce the diarrhea score of OVA-sensitized mice.

[0054] In the present invention, the FTY720 or the intestinal flora can reduce the level of serum OVA-specific IgE antibody. The results of the examples of the present invention show that the FTY720 or the intestinal flora can reduce the level of serum OVA-specific IgE antibody in OVA-sensitized mice.

[0055] In the present invention, the FTY720 or the intestinal flora can reduce the level of serum OVA-specific IgG1 antibody. The results of the examples of the present invention show that the FTY720 or the intestinal flora can reduce the level of serum OVA-specific IgG1 antibody in OVA-sensitized mice.

[0056] In the present invention, the FTY720 or the intestinal flora can alleviate or relieve jejunal tissue damage caused by food allergy. The results of the examples of the present invention show that the FTY720 or the intestinal flora can alleviate or relieve inflammatory cell infiltration in the lamina propria of the jejunal mucosa, disorder of intestinal gland structure, and swelling of intestinal villi in OVA-sensitized mice, thereby alleviating or relieving jejunal tissue damage.

[0057] In the present invention, the FTY720 or the intestinal flora can reduce the level of jejunal inflammatory factors. In the present invention, the jejunal inflammatory factors include any one or more of IL-4, IL-10, IL-13, IFN-γ, GRO-α, and TNF-α. The results of the examples of the present invention show that the FTY720 or the intestinal flora can reduce the expression levels of jejunal inflammatory factors IL-4, IL-10, IL-13, IFN-γ, GRO-α, and TNF-α, thereby reducing the levels of the corresponding inflammatory factors.

[0058] In the present invention, the FTY720 or the intestinal flora can improve the intestinal flora. In the present invention, the improvement of the intestinal flora includes reducing the abundance of Firmicutes flora, reducing the abundance of Actinobacteria flora, increasing the abundance of Bacteroidota flora, increasing the abundance of Patescibacteria flora, and increasing the abundance of Desulfobacterota flora, any one or more of them. The results of the examples of the present invention show that the FTY720 or the intestinal flora can reduce the abundances of Firmicutes flora and Actinobacteria flora in OVA-sensitized mice, and can increase the abundances of Bacteroidota flora, Patescibacteria flora, and Desulfobacterota flora.

[0059] As an alternative embodiment of the present invention, the drug contains FTY720 and a pharmaceutically acceptable carrier, such as any one or more of microspheres or microcapsules, nanoparticles, and liposomes.

[0060] As an alternative embodiment of the present invention, the drug contains FTY720 and pharmaceutical excipients, such as any one or more of dimethyl sulfoxide, soybean oil, and glycerol.

[0061] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0062] Food allergy, full name: food allergy, abbreviation: FA. Ovalbumin, full name: ovalbumin, abbreviation: OVA; Intramuscular injection, abbreviation: i.m., full name: intramuscular; Gavage, abbreviation: i.g., full name: intragastric; Fecal microbiota transplantation, abbreviation: FMT, full name: Fecal Microbiota Transplantation.

[0063] FTY720 in the following examples was purchased from MCE Company.

[0064] Example 1

[0065] 1. Method for constructing a food allergy model of OVA-sensitized mice:

[0066] Thirty 6-week-old female BALB / c mice were randomly divided into 5 groups, with 6 mice in each group, namely: control group (Control group), food allergy group (Food Allergy, FA group), FTY720 prevention group (i.m-immunize), FTY720 intramuscular injection treatment group (i.m-stimulate), and FTY720 gavage treatment group (i.g-stimulate).

[0067] Among them, the FA group was intraperitoneally injected with 200 μL of phosphate buffer solution (PBS) containing 40 μg OVA and 0.4 mg Al(OH)3 on the 0th day, 7th day, and 14th day for basic sensitization and booster sensitization; the control group was intraperitoneally injected with the same dose of PBS containing 0.4 mg Al(OH)3 instead when the above treatment was carried out in the FA group. Starting from the 28th day (two weeks after the last sensitization), the FA group and the control group were respectively gavaged with 200 μL of normal saline containing 50 mg OVA every other day for gastrointestinal stimulation until the 38th day, with continuous excitation 6 times. Then, the corresponding indexes of food allergy were detected. The flowchart for constructing the FA group model is as shown in Figure 1 A in.

[0068] Treatment of food allergy with FTY720:

[0069] Experimental method for the FTY720 intramuscular injection treatment group (also denoted as FA+FTY720 (i.m-stimulate)): On days 0, 7, and 14, 200 μL of phosphate buffer solution (PBS) containing 40 μg of OVA and 0.4 mg of Al(OH)3 was intraperitoneally injected for basic sensitization and booster sensitization; starting from day 28 (two weeks after the last sensitization), 200 μL of normal saline containing 50 mg of OVA was intragastrically administered every other day for gastrointestinal stimulation until day 38, with a total of 6 consecutive challenges. Two weeks after the last sensitization, at the third OVA intragastric administration (i.e., day 32), FTY720 (100 μg / animal) was intramuscularly injected for three consecutive days. The flowchart for the construction of the FA+FTY720 (i.m-stimulate) model is as shown in Figure 1 Figure B in

[0070] Experimental method for the FTY720 intragastric administration treatment group (also denoted as FA+FTY720 (i.g-stimulate)): On days 0, 7, and 14, 200 μL of phosphate buffer solution (PBS) containing 40 μg of OVA and 0.4 mg of Al(OH)3 was intraperitoneally injected for basic sensitization and booster sensitization; starting from day 28 (two weeks after the last sensitization), 200 μL of normal saline containing 50 mg of OVA was intragastrically administered every other day for gastrointestinal stimulation until day 38, with a total of 6 consecutive challenges. Two weeks after the last sensitization, at the third OVA intragastric administration (i.e., day 32), FTY720 (100 μg / animal) was orally intragastrically administered for three consecutive days. The flowchart for the construction of the FA+FTY720 (i.g-stimulate) model is as shown in Figure 1 Figure B in

[0071] Experimental method for the FTY720 prevention group (also denoted as FA+FTY720 (i.m-immunize)): On days 0, 7, and 14, 200 μL of phosphate buffer solution (PBS) containing 40 μg of OVA and 0.4 mg of Al(OH)3 was intraperitoneally injected for basic sensitization and booster sensitization; on days 0, 7, and 14, FTY720 (100 μg / animal) was intramuscularly injected simultaneously with sensitization. On day 28 (two weeks after the last sensitization), 200 μL of normal saline containing 50 mg of OVA was intragastrically administered every other day for gastrointestinal stimulation until day 38, with a total of 6 consecutive challenges. The flowchart for the construction of the FA+FTY720 (i.m-immunize) model is as shown in Figure 1 Figure C in

[0072] 2. Detection of relevant indicators for the FA group and the control group

[0073] The body temperature changes of the FA group and the control group were measured. Before the 6th OVA gavage challenge and within 90 min after the 6th OVA gavage challenge, the rectal temperature of the mice was measured every 15 min, and the body temperature changes of the mice in the FA group and the control group at different times were calculated.

[0074] The body weight changes of the FA group and the control group were measured. Before the OVA gavage challenge and 24 h after each gavage challenge, the body weight of the mice was measured, and the body weight changes of the mice in the FA group and the control group with the number of OVA gavage challenges were calculated.

[0075] The diarrhea conditions of the FA group and the control group were scored: within 1 h after the 3rd to 6th OVA gavage challenges, the diarrhea conditions of the mice were observed, and the scoring criteria for the diarrhea degree were as follows: 0, normal feces; 1, a small amount of moist or unformed feces appeared; 2, some moist unformed feces appeared, accompanied by moderate staining of the perianal hair; 3, severe watery feces appeared, accompanied by severe staining of the perianal hair.

[0076] The jejunum pathology of the FA group and the control group: The mice were sacrificed 24 h after the 6th OVA challenge. Jejunum samples of 0.5 cm were taken 5 cm below the Treitz ligament, and the obtained specimens were fixed with 4% formaldehyde solution. After sectioning, baking, and hematoxylin-eosin (HE) staining, the morphology of the jejunum tissue and the inflammatory infiltration were observed under a microscope.

[0077] OVA-specific antibodies in the FA group and the control group: 24 h after the 6th OVA gavage challenge, the mouse sera were separated, and the titers of OVA-specific antibodies IgE and IgG1 were detected. The sera were diluted in different gradients for ELISA experiments. A ratio of the net OD value of the sample to be tested to the net OD value of the negative control ≥ 2.1 and an OD value ≥ 0.15 were considered positive, and the maximum antibody dilution factor with a positive result was the titer or potency of this serum antibody.

[0078] The expression of jejunum inflammatory factor mRNA in the FA group and the control group: The mice were sacrificed 24 h after the 6th OVA challenge. Jejunum samples were taken, total RNA was extracted, and the amount of the amplified product was detected by real-time quantitative PCR. Using β-actin as the internal reference gene, the relative expression of each gene was analyzed by the ΔΔCt method.

[0079] The detection results of the relevant indicators of the FA group and the control group are as Figures 2 - 3 shown. Among them, Figure 2 A is the rectal temperature change of the mice in the FA group and the control group within 90 min after the last challenge; B is the body weight change of the mice in the FA group and the control group before and after each challenge; C is the diarrhea condition that occurred within 2 h after the 3rd, 4th, 5th, and 6th OVA challenges in the FA group and the control group; D is the hematoxylin-eosin (H&E) staining result diagram of the jejunum of the mice in the FA group and the control group. Figure 3In A, the titers of OVA-specific IgE and IgG1 antibodies in the sera of mice in the FA group and the control group were detected by the last OVA challenge; in B, the relative expression levels of cytokines in the jejunal tissues of mice in the FA group and the control group were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Figures 2 - 3 *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 in it.

[0080] From Figures 2 - 3 it can be obtained that: the body temperature of the FA group was significantly lower than that of the control group; the body weight of the FA group was significantly lower than that of the control group; obvious diarrhea occurred in the FA group compared with the control group; there were inflammatory cell infiltrations, intestinal gland structure disorders, and intestinal villus swelling in the lamina propria of the jejunum in the FA group compared with the control group; the OVA-specific IgE and IgG1 antibodies in the sera of the FA group were significantly increased compared with the control group; the expression levels of jejunal cytokines in the FA group were significantly increased compared with the control group. The above results indicate that the food allergy model was successfully constructed.

[0081] 3. Compare the body temperature changes and body weight changes among the FA group, the FA + FTY720 (i.m-stimulate) group, and the control group; compare the relevant body temperature changes and body weight changes among the FA group, the FA + FTY720 (i.g-stimulate) group, and the control group; compare the relevant body temperature changes and body weight changes among the FA group, the FA + FTY720 (i.m-immunize), and the control group; the detection methods of body temperature and body weight in each experimental group are the same as those in step 2.

[0082] Compare the diarrhea scores of the FA group, the FA + FTY720 (i.m-stimulate) group, the FA + FTY720 (i.g-stimulate) group, the FA + FTY720 (i.m-immunize), and the control group, and the method is the same as that in step 2.

[0083] Compare the jejunal pathological conditions of the FA group, the FA + FTY720 (i.m-stimulate) group, the FA + FTY720 (i.g-stimulate) group, the FA + FTY720 (i.m-immunize), and the control group, and the method is the same as that in step 2.

[0084] The detection and comparison results of relevant indicators in each experimental group are as Figures 4 - 5 shown. Among them, Figure 4Panel A shows the comparison results of body temperatures of the FA group, the FA+FTY720 (i.m-stimulate) group, and the control group within 90 min after the last stimulation; Panel B shows the comparison results of the body weights of mice in the FA group, the FA+FTY720 (i.m-stimulate) group, and the control group measured before and after each stimulation; Panel C shows the comparison results of body temperatures of the FA group, the FA+FTY720 (i.m-immunize) group, and the control group within 90 min after the last stimulation; Panel D shows the comparison results of the body weights of mice in the FA group, the FA+FTY720 (i.m-immunize) group, and the control group measured before and after each stimulation; Panel E shows the comparison results of body temperatures of the FA group, the FA+FTY720 (i.g-stimulate) group, and the control group within 90 min after the last stimulation; Panel F shows the comparison results of the body weights of mice in the FA group, the FA+FTY720 (i.g-stimulate) group, and the control group measured before and after each stimulation. Figure 5 In Panel A, diarrhea occurred in mice of each experimental group within 2 h after the 3rd, 4th, 5th, and 6th OVA stimulations, as shown in Table 1; Panel B shows the hematoxylin-eosin (H&E) staining results of the jejunum of mice in each experimental group. Figures 4 - 5 *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Control is the control group; FA: food allergy group; FA+FTY720 (i.m-stimulate): FTY720 was intramuscularly injected during OVA stimulation; FA+FTY720 (i.m-immunize): FTY720 was intramuscularly injected during OVA sensitization; FA+FTY720 (i.g-stimulate): FTY720 was administered by gavage during OVA stimulation. The same applies hereinafter.

[0085] Table 1 Diarrhea scores of mice in each experimental group within 2 h after the 3rd, 4th, 5th, and 6th OVA stimulations

[0086]

[0087]

[0088] Figure 4 In Panels A, C, and E, it shows that FTY720 intervention can restore the phenotype of decreased body temperature in the FA group; Figure 4 In Panels B, D, and F, it shows that FTY720 intervention can restore the phenotype of decreased body weight in the FA group; Figure 5 In Panel A, it shows that FTY720 intervention can relieve the diarrhea phenotype in the FA group; Figure 5 In Panel B, it shows that in the FA group, there were inflammatory cell infiltrations in the lamina propria of the jejunum mucosa, disordered intestinal gland structures, and swollen intestinal villi, and the sensitization conditions of OVA in the three FTY720-treated groups were alleviated to varying degrees.

[0089] 4. The expression of jejunal inflammatory factors mRNA in each experimental group was detected by the same method as in step 2, and the results are as Figure 6 shown. It can be Figure 6 seen that FTY720 intervention can relieve the expression level of jejunal inflammatory factors in the FA group.

[0090] 5. The intestinal flora of the FA+FTY720 (i.m-immunize) group, FA group and control group were detected. The detection of intestinal flora was entrusted to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for 16S rDNA sequencing of intestinal flora.

[0091] Among them, the intestinal differential flora of the FA group and the control group are as Figure 7 shown in A therein, where C represents the control group; the intestinal differential flora of the FA group and the FTY720 group are as Figure 7 shown in B therein, where FTY720 represents the corresponding result of the FA+FTY720 (i.m-immunize) group, and the same below; the common intestinal differential flora of the FA group, the control group and the FTY720 group are as Figure 7 shown in C therein. Figure 7 A in Figure 7 shows that food allergy can cause a decrease in some intestinal flora, that is, the intestinal homeostasis is disrupted; Figure 7 B in

[0092] shows that compared with the FA group, FTY720 intervention will change the abundance of some flora;

[0093] Example 2 Fecal microbiota transplantation experiment

[0094] Preparation of fecal suspension and gavage method:

[0095] The mice were placed in a laminar flow hood, and feces were collected with a high-pressure sterile EP tube (the abdomen of the mice can be pressed appropriately). Add 1 mL of sterile PBS to every 200 mg of feces, vortex and mix well, filter through a 70 μm sieve, and filter while grinding with a sterile syringe core to ensure that during the grinding process, it is sufficient and the bacterial solution cannot be diluted to obtain a fecal suspension. Each mouse was gavaged with 200 μL of fecal suspension, and the volume of fecal suspension required for each gavage was calculated. The bacterial solution was aliquoted and stored at -80 °C in a refrigerator. The remaining bacterial solution after each gavage could not be frozen and used again. During gavage, the bacterial solution was melted in the palm of the hand and then quickly placed on ice, and the whole process was kept on ice. Gavage was carried out every other day for a total of 6 times, and the whole process should be rapid to avoid the death of anaerobic bacteria.

[0096] Twenty-four 6-week-old female BALB / c mice were randomly divided into 4 groups of 6 mice each, namely: control group (Control group), food allergy group (Food Allergy, FA group), fecal suspension group transplanted with FTY720 intervention group feces (FA+FMT(FTY720)), and fecal suspension group transplanted with control group feces (FA+FMT(Control)). Among them, the FTY720 intervention group was the group where, in Example 1, when FA+FTY720 (i.m-immunize) intraperitoneally injected the sensitizing substance on the 14th day and intramuscularly injected FTY720 at the same time, the feces of the corresponding mice were taken on the 28th day.

[0097] The treatment methods of the control group and the FA group were the same as those of the control group and the FA group in Example 1. Among them, the timing of taking the feces of the control group was the same as the timing of taking the feces of the FTY720 intervention group.

[0098] For the fecal suspension group transplanted with FTY720 intervention group feces, the FA modeling method was the same as that in Example 1. Starting from the day after the last sensitization (the 15th day), the fecal suspension of the FTY720 intervention group was intragastrically administered every other day for a total of 6 times. For the fecal suspension group transplanted with control group feces, the FA modeling method was the same as that in Example 1. Starting from the day after the last sensitization (the 15th day), the fecal suspension of the control group was intragastrically administered every other day for a total of 6 times. On the 28th day of each of the above experimental groups, OVA was used for intragastric challenge. The intragastric challenge method was the same as that in Example 1, and the corresponding indexes of food allergy were detected.

[0099] The body temperature changes of the mice in each experimental group were measured. Before the 6th OVA intragastric challenge and within 90 minutes after the challenge, the rectal temperature of the mice was measured every 15 minutes, and the body temperature changes of the mice in each experimental group at different times were calculated.

[0100] The body weight changes of the mice in each experimental group were measured. The body weight of the mice was measured before the OVA intragastric challenge and 24 hours after each intragastric challenge, and the body weight changes of the mice in each experimental group with the number of OVA intragastric challenges were calculated.

[0101] The diarrhea conditions of the mice in each experimental group were scored: within 1 hour after the 3rd to 6th OVA intragastric challenges, the diarrhea conditions of the mice were observed. The scoring criteria for the degree of diarrhea were as follows: 0, normal feces; 1, a small amount of moist or unformed feces appeared; 2, some moist unformed feces appeared, accompanied by moderate staining of the perianal hair; 3, severe watery feces appeared, accompanied by severe staining of the perianal hair.

[0102] The jejunum pathology of the mice in each experimental group: The mice were sacrificed 24 hours after the 6th OVA challenge. 0.5 cm of jejunum was taken 5 cm below the Tretiz ligament. The obtained specimens were fixed with 4% formaldehyde solution, and after sectioning, baking, and hematoxylin-eosin (HE) staining, the morphological changes and inflammatory infiltration of the jejunum tissue were observed under a microscope.

[0103] OVA-specific antibodies of mice in each experimental group: 24 h after the 6th oral administration of OVA for stimulation, mouse sera were separated and the titers of OVA-specific antibodies IgE and IgG1 were detected. The sera were diluted at different gradients for ELISA experiments. A sample was considered positive when the ratio of the net OD value of the sample to be tested to the net OD value of the negative control was ≥2.1 and the OD value was ≥0.15. The maximum antibody dilution factor with a positive result was the titer or potency of such serum antibody.

[0104] Expression of jejunal inflammatory factor mRNA in mice in each experimental group: The mice were sacrificed 24 h after the 6th OVA stimulation. The jejunum was taken, total RNA was extracted, and the amount of amplified product was detected by real-time quantitative PCR. Using β-actin as the internal reference gene, the relative expression of each gene was analyzed by the ΔΔCT method.

[0105] The detection results of related indicators of mice in each experimental group are as Figures 8 - 9 shown. Figure 8 In A, it is the graph of the change in rectal temperature detected in mice in each experimental group within 90 min after the last stimulation. Figure 8 In B, it is the change in body weight of mice in each experimental group before and after each stimulation. Figure 8 In C, it is the titers of OVA-specific IgE and IgG1 antibodies and the relative expression levels of IL-4 cytokine detected in the sera of mice in each experimental group after the last OVA stimulation. Figure 8 In D, it is the relative expression levels of cytokines detected in the jejunal tissues of mice in each experimental group by real-time fluorescence quantitative polymerase chain reaction. Figure 9 In A, it is the diarrhea situation of mice in each experimental group within 2 h after the 3rd, 4th, 5th, and 6th OVA stimulations. See Table 2 for details. Figure 9 In B, it is the graph of the hematoxylin-eosin (H&E) staining results of the jejunum of mice in each experimental group. Figures 8 - 9 In it, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Figure 9 In it, # indicates a significant difference between FA+FMT (FTY720) and FA+FMT (Control). Figures 8 - 9 In it, Control: control group; FA: food allergy group; FA+FMT (FTY720): fecal suspension of the FA group adoptively transferred with FTY7720 intervention; FA+FMT (Control): fecal suspension of the FA group adoptively transferred with the control group.

[0106] Table 2 Diarrhea scores of mice in each experimental group within 2 h after the 3rd, 4th, 5th, and 6th OVA stimulations

[0107]

[0108]

[0109] Figure 8 Explanation of A in [description]: The fecal microbiota transplantation experiment can alleviate the phenotype of body temperature decrease in the FA group. Figure 8 Explanation of B in [description]: The fecal microbiota transplantation experiment can alleviate the phenotype of body weight decrease in the FA group. Figure 8 Explanation of C - D in [description]: The fecal microbiota transplantation experiment can reduce the titers of serum - specific IgE and IgG1 in the FA group, and the fecal microbiota transplantation experiment can alleviate the expression levels of jejunal cytokines. Figure 9 Explanation of A in [description]: The fecal microbiota transplantation experiment can alleviate the diarrhea phenotype in the FA group to a certain extent. Figure 9 Explanation of B in [description]: H&E staining showed that in the FA group, there were inflammatory cell infiltrations, disordered intestinal gland structures, and swollen intestinal villi in the lamina propria of the jejunum. Fecal microbiota transplantation can restore the jejunal mucosal structure. The fecal microbiota transplantation experiment can alleviate the damage of jejunal tissues in the FA group to a certain extent. The above results indicate that fecal microbiota transplantation can alleviate the symptoms of food allergy.

[0110] Although the above - mentioned embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of FTY720 in the preparation of drugs for preventing and / or treating food allergies.

2. Application of intestinal flora in the preparation of drugs for the prevention and / or treatment of food allergies; The intestinal flora includes Bacteroidetes flora, Patellar flora and Desulfobacterium flora.

3. The use according to claim 1 or 2, characterized in that: The food allergy includes allergies induced by protein-containing foods.

4. The use according to claim 3, characterized in that: The protein-containing food includes an egg white protein-containing food.

5. The use according to claim 1 or 2, characterized in that: The food allergy condition includes allergic enteritis.

6. The use according to claim 1 or 2, characterized in that: The prevention and / or treatment of food allergy includes any one or more of the following (1) to (8): (1) Alleviate the drop in body temperature; (2) Alleviate weight loss; (3) Reduce the incidence of diarrhea and the likelihood of diarrhea; (4) reduce serum OVA-specific IgE antibody levels; (5) Reduce serum OVA-specific IgG1 antibody levels; (6) Reduce or alleviate jejunal tissue damage; (7) Reduce the level of jejunal inflammatory factors; (8) Improve intestinal flora.

7. The use according to claim 6, characterized in that: The improvement of intestinal flora includes any one or more of reducing the abundance of Firmicutes flora, reducing the abundance of Actinobacteria flora, increasing the abundance of Bacteroidetes flora, increasing the abundance of Patellar flora and increasing the abundance of Desulfobacterium flora.

8. The use according to claim 6, characterized in that: The jejunal inflammatory factors include any one or more of IL-4, IL-10, IL-13, IFN-γ, GRO-α and TNF-α.

9. The use according to claim 1 or 2, characterized in that: The medicament contains FTY720 and a pharmaceutically acceptable carrier.

10. The use according to claim 1 or 2, characterized in that: The medicine contains FTY720 and pharmaceutical excipients.

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