Application of bacteroides fragilis in preparation of composition for treating allergic skin diseases

Abstract

The invention discloses application of bacteroides fragilis in preparation of a composition for treating allergic skin diseases. The bacteroides fragilis ZY-312 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.10685 or inactivated bacteria and an extract capsular polysaccharide A of the bacteroides fragilis ZY-312 can be used for promoting the content of IFN-gamma (Interferon-gamma) in serum by inhibiting the content of IL-4 and IgE (Immunoglobulin E) in the serum and skin lesion tissues in vivo, inhibiting allergic inflammation reaction caused by Th2 cell deflection and reducing back skin inflammation score, ear swelling, ear allergy reaction and the like. The traditional Chinese medicine composition can effectively prevent and treat allergic dermatosis, obviously improve comprehensive curative effect, effectively prevent occurrence and development of allergic dermatosis, and improve life quality of patients.

Description

[0001] The microbial strains used in the implementation of the present invention have been deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Committee (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​April 2, 2015. Classification name: Bacteroides fragilis ZY-312, deposit number CGMCC No.10685. Bacteroides fragilis ZY-312 was isolated and obtained by the applicant of the present invention, and has been protected by a patent (patent number 201510459408.X). According to the provisions of the Patent Examination Guidelines, the public can buy it from commercial channels or it has been authorized, and no deposit is required, that is, no deposit certificate is required. Technical Field

[0002] The invention relates to the field of biomedicine, and in particular to an application of Bacteroides fragilis in preparing a composition for treating allergic skin diseases. Background Art

[0003] Allergic skin disease (ASD) refers to a type of chronic inflammatory skin disease caused by contact with allergens, mainly including atopic dermatitis (AD), eczema and chronic urticaria. The main clinical symptoms of ASD are unbearable itching, repeated attacks, and easy development into chronic inflammatory skin diseases. It can also cause a series of mental illnesses such as anxiety, depression, and autism, affecting the physical and mental health of patients. Although the dermatitis symptoms of some patients will gradually disappear with age, about 1 / 2 of children with atopic dermatitis may develop allergic asthma in the future, and 2 / 3 of children are at risk of allergic rhinitis.

[0004] Allergic skin diseases can occur at any age, in any part of the body, and in any season, but often recur or worsen in winter. AD affects 15%-30% of children and 3% of adults worldwide. With the development of urbanization, the incidence rate is increasing year by year, bringing a huge burden to patients and their families.

[0005] At present, Western medicine for the treatment of allergic skin diseases mostly adopts the methods of finding and eliminating allergens, desensitization therapy, and non-specific drug treatment such as antihistamines and glucocorticoids. However, the drug has a single action link, large adverse reactions, and is very easy to relapse. For example, glucocorticoids can significantly inhibit type IV allergic reactions and are currently commonly used drugs for the treatment of acute and chronic ASD. They have also achieved certain clinical results, but whether they are used locally or systemically, long-term use shows obvious adverse reactions, such as pigmentation, skin atrophy, reduced resistance of gastric mucosa, centripetal obesity, etc. In addition, in clinical treatment, skin lesions often worsen after drug withdrawal or hormone-dependent dermatitis. The antihistamines currently used are all drugs for anti-type I allergic reactions, and they only have the effect of temporarily relieving itching. In addition, side effects such as drowsiness, dry mouth, fatigue, dizziness and cardiac toxicity are prone to occur during treatment, and long-term use may also cause tolerance. Therefore, the above-mentioned drugs are greatly restricted when used in chronic ASD.

[0006] The etiology and pathogenesis of ASD are quite complex, involving multiple factors both inside and outside the body. The internal factors are currently believed to be mainly caused by T cell-mediated cellular immune disorders. Under normal circumstances, the body's T helper cell 1 (T helper cell 1, Th1) and Th2 cell functions are in a dynamic equilibrium state, and Th1 cells and Th2 cells regulate and inhibit each other by secreting different cytokines, thereby maintaining the body's normal cellular and humoral immune functions; under the stimulation of certain factors, the function of one subset of Th1 and Th2 cells increases, while the function of the other subset decreases. This phenomenon is called Th1 / Th2 drift. Research results have shown that the Th1 / Th2 cells in the peripheral blood and skin lesions of ASD patients are unbalanced and tend to be Th2. For example, the levels of interleukin-4 (IL-4) and IL-5 expressed or secreted by Th2 cells in skin lesions and peripheral blood are increased, while the levels of interferon gamma (IFN-γ) and IL-2 expressed or secreted by Th1 cells are reduced.

[0007] The occurrence and development of ASD is not only closely related to changes in the skin microecology, but with the development of sequencing technology, the role of intestinal microecology in the occurrence and development of ASD is also increasingly valued. Compared with healthy individuals, the diversity of intestinal microorganisms in ASD patients is significantly reduced, and the composition of intestinal microorganisms has also changed significantly. The relative abundance of microorganisms that are beneficial to human health in the intestinal flora of infants, children and adult patients is significantly reduced, and the abundance of harmful intestinal bacteria such as Escherichia coli, Clostridium difficile and Staphylococcus aureus is significantly increased.

[0008] Bacteroides fragilis (B. fragilis) is a Gram-negative, rod-shaped bacterium with blunt and densely stained ends, capsule, no spores, and no motility. It is divided into enterotoxigenic (ETBF) and non-enterotoxigenic (NTBF). It is part of the normal intestinal flora of humans and animals, mainly existing in the colon, and can also colonize and grow in the mucosa of the respiratory tract, gastrointestinal tract, and urogenital tract. Studies have found that non-enterotoxigenic Bacteroides fragilis (NTBF) has an important prebiotic effect and maintains the balance of Th1 / Th2. The development of Bacteroides fragilis for the preparation of drugs for the treatment of allergic skin diseases is promising and necessary. Summary of the invention

[0009] In order to overcome the above-mentioned defects existing in the prior art, the purpose of the present invention is to provide an application of Bacteroides fragilis in the treatment of allergic skin diseases. The present invention has proved through a large number of experiments that Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685 can inhibit the content of IL-4 and IgE in serum and skin lesions in vivo, promote the content of IFN-γ in serum, inhibit the allergic inflammatory response caused by Th2 cell deviation, reduce the back skin inflammation score, ear swelling and ear allergic reaction, etc. It can effectively prevent and treat allergic skin diseases and improve the quality of life of patients.

[0010] In order to achieve the above object, the present invention adopts the following technical solution:

[0011] In a first aspect, a use of Bacteroides fragilis in preparing a composition for treating allergic skin diseases is provided, wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No.10685.

[0012] In some embodiments, the Bacteroides fragilis is one or more of the following: live Bacteroides fragilis, inactivated, genetically recombinant, transformed or modified, attenuated, chemically treated, physically treated or inactivated Bacteroides fragilis, and liquid culture supernatant of Bacteroides fragilis.

[0013] Exemplarily, the inactivated Bacteroides fragilis is an inactivated bacterium with a complete morphological structure or an inactivated bacterium with an incomplete morphological structure; preferably, it is an inactivated bacterium with a complete morphological structure.

[0014] Exemplarily, the Bacteroides fragilis is inactivated by any one or more of dry heat, moist heat, filtration, organic solvents, chemical reagents, ultraviolet or infrared rays, fermentation, freeze-drying, genetic recombination, genetic modification or transformation;

[0015] In some of the embodiments, in the above applications, the composition can be any one of a pharmaceutical composition, a food, a health product or a food additive.

[0016] Illustratively, the pharmaceutical composition contains a pharmaceutically effective dose of inactivated Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685; and / or, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0017] Illustratively, the dosage form of the pharmaceutical composition includes pills, tablets, granules, capsules, powders, suspensions, oral liquids, tube feeding preparations, enemas, liniments, and gels.

[0018] Illustratively, the administration of the pharmaceutical composition includes transdermal administration, oral administration, enema, and application.

[0019] Exemplarily, the administration cycle of the pharmaceutical composition is intermittent administration, periodic administration, continuous administration or long-term administration.

[0020] Exemplarily, the pharmaceutical composition includes a human medicine or an animal medicine.

[0021] Exemplarily, the food comprises milk powder, cheese, curd, yogurt, ice cream or fermented cereal food. The food can also be animal food, such as feed etc.

[0022] In some embodiments, the allergic skin diseases include atopic dermatitis, eczema and chronic urticaria.

[0023] In a second aspect, a use of a Bacteroides fragilis extract in preparing a composition for treating eczema or chronic urticaria is provided, wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685, and the Bacteroides fragilis extract contains capsular polysaccharide A;

[0024] In some embodiments, the content of capsular polysaccharide A lipid is less than 0.02 wt%, the protein residue is less than 1%, and the nucleic acid residue is less than 0.05%;

[0025] In some embodiments, the structure of the capsular polysaccharide A is shown below:

[0026]

[0027] Exemplarily, the weight average molecular weight of the capsular polysaccharide A is 80-90 kD, and the ratio of weight average molecular weight to number average molecular weight (Mw / Mn) is 1.0-1.2.

[0028] In some embodiments, the composition is any one of a pharmaceutical composition, a food, a health product or a food additive.

[0029] Illustratively, the pharmaceutical composition contains a pharmaceutically effective dose of capsular polysaccharide A of Bacteroides fragilis ZY-312 extract with a deposit number of CGMCC No. 10685; and / or, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0030] Illustratively, the dosage form of the pharmaceutical composition includes pills, tablets, granules, capsules, powders, suspensions, oral liquids, tube feeding preparations, enemas, liniments, and gels.

[0031] Illustratively, the administration of the pharmaceutical composition includes transdermal administration, oral administration, enema, and application.

[0032] Exemplarily, the administration cycle of the pharmaceutical composition is intermittent administration, periodic administration, continuous administration or long-term administration.

[0033] Exemplarily, the pharmaceutical composition is for human or animal administration.

[0034] Exemplarily, the food is milk powder, cheese, curd, yogurt, ice cream or fermented cereal food; preferably, the food can also be animal food, such as feed.

[0035] Beneficial effects of the present invention:

[0036] The present invention proves through a large number of experiments that the live or inactivated bacteria of Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685 and its extract capsular polysaccharide A can inhibit the IL-4 and IgE content in serum and skin lesions in vivo, promote the IFN-γ content in serum, inhibit the allergic inflammatory reaction caused by Th2 cell deviation, reduce the back skin inflammation score, ear swelling and ear allergy reaction, etc. Its effect is better than that of the standard strain NCTC-9343, and there is a statistical difference. It can effectively prevent and treat allergic skin diseases including atopic dermatitis, eczema and chronic urticaria, significantly improve the comprehensive therapeutic effect, effectively prevent the occurrence and development of allergic skin diseases, and improve the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a colony characteristic diagram of Bacteroides fragilis ZY-312 of Example 1 of the present invention;

[0038] Figure 2 This is a microscopic observation picture of Bacteroides fragilis ZY-312 after Gram staining in Example 1 of the present invention;

[0039] Figure 3 This is a transmission electron micrograph of the inactivated Bacteroides fragilis ZY-312 powder of Example 1 of the present invention;

[0040] Figure 4is the chemical structural formula of the structural unit of the Bacteroides fragilis extract of Example 2 of the present invention. DETAILED DESCRIPTION

[0041] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0042] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available. All experimental animals were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; or they can be prepared by known methods. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0043] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0044] Example 1: Fermentation culture of Bacteroides fragilis

[0045] The Bacteroides fragilis ZY-312 strain was streaked onto a blood plate and cultured anaerobically for 48 hours. The colony morphology, staining characteristics, size, club shape, and distribution were observed. Colony characteristics: After culturing Bacteroides fragilis ZY-312 on a blood plate for 48 hours, the colony was round, slightly convex, translucent, white, with a smooth surface and no hemolysis. The colony diameter was between 1-3 mm. Figure 1 .

[0046] Microscopic morphology: Gram staining of Bacteroides fragilis ZY-312 showed that it was a Gram-negative bacterium with a typical rod shape, blunt and darkly stained ends, and a non-stained part in the middle of the bacteria that looked like a vacuole. Figure 2 .

[0047] Example 2: Bacteroides fragilis sample preparation

[0048] 1. Preparation of live bacterial suspension of Bacteroides fragilis

[0049] A single colony cultured in Example 1 was selected and inoculated into a plant-derived peptone liquid culture medium for fermentation culture for 8 hours (temperature was 37° C.) to obtain a live bacterial liquid of Bacteroides fragilis.

[0050] 2. Preparation of inactivated Bacteroides fragilis solution

[0051] Conventional heat inactivation was used to prepare the inactivated bacterial solution of Bacteroides fragilis. The live bacterial solution of Bacteroides fragilis ZY-312 was placed in a beaker, and the beaker was placed in a constant temperature water bath at 100° C. for 20 to 30 minutes to obtain the inactivated bacterial solution of Bacteroides fragilis.

[0052] 3. Preparation of inactivated Bacteroides fragilis slurry and inactivated powder

[0053] The inactivated bacterial liquid of Bacteroides fragilis ZY-312 was centrifuged for precipitation at a speed of 3000 r / min for 15 min, the supernatant was removed, and the precipitate was collected to obtain the inactivated bacterial sludge of Bacteroides fragilis ZY-312.

[0054] Take the inactivated Bacteroides fragilis ZY-312 bacterial sludge and add the excipient, stir and completely dissolve to obtain the inactivated bacterial powder stock solution, and vacuum freeze-dry the inactivated bacterial powder stock solution to obtain the inactivated bacterial powder. After pre-freezing at -40±2℃ for 1 to 3 hours, pre-freeze at -20±2℃ for 0.5 to 1 hour, and finally pre-freeze at -40±2℃ for 0.5 to 2 hours, dry once (-5±2℃ and 0±2℃) and desorb and dry (35±2℃) under a vacuum degree of 0.25mbar, the bacterial count of the bacterial powder reaches 1×10 11 Cells / g or more. The inactivated bacteria powder prepared in the above steps has complete bacterial morphology, see Figure 3 .

[0055] 4. Preparation of Bacteroides fragilis Inactivation Gel

[0056] (1) 1×10 8 For every 500 g preparation of Bacteroides fragilis inactivated gel with a specification of cells / g, the inactivated Bacteroides fragilis powder prepared above (calculated based on a bacterial content of 1×1011 cells / g, 0.5 g), carbomer homopolymer (type B) (2.5 g), triethanolamine (2.5 g), glycerol (25 g), ethylparaben (0.5 g), polysorbate 80 (0.5 g), and purified water (468.5 g) need to be weighed and set aside.

[0057] (2) Take ethylparaben and place it in a tilting vacuum emulsifier, add 300 g of purified water, start stirring, heat to 80°C and keep warm for 30-60 minutes, cool the room temperature water to below 40°C, add glycerin, stir evenly and stop stirring, add carbomer homopolymer (type B), soak for at least 5 hours until there is no white core. If white core is observed, the soaking time should be extended.

[0058] (3) Take 20 g of purified water, add it to triethanolamine, stir evenly, and set aside.

[0059] (4) Take 30 g of purified water and add it to the inactivated Bacteroides fragilis powder, stir to disperse it, and set aside.

[0060] (5) Take 15 g of purified water, heat it to 80°C, add polysorbate 80, stir until completely dissolved, cool to room temperature, and set aside.

[0061] (6) Set the stirring speed of the tilting vacuum emulsifier to 50 Hz, start stirring, and keep it for 15 minutes.

[0062] (7) Add the inactivated Bacteroides fragilis powder solution into a tiltable vacuum emulsifier and keep stirring for 15 minutes.

[0063] (8) Take the inactivated Bacteroides fragilis ZY-312 inactivated bacterial powder and put it into a tiltable vacuum emulsifier. Keep stirring for 15-20 minutes, slowly drop about 1 / 6 of the triethanolamine solution into the gel solution, stir for 5 minutes, drop about 1 / 6 of the triethanolamine solution into the gel solution again, stir for 5 minutes, slowly drop the remaining triethanolamine solution at one time, add the polysorbate 80 solution and the remaining purified water, keep stirring, and start the vacuum pump after stirring for 45 minutes, keep the vacuum degree at -90Kpa or above, and evacuate for 15 minutes. Transfer to the filling machine, fill according to the specification of 10g / tube, and prepare ZY-312 gel.

[0064] 5. Preparation of Capsular Polysaccharide A from Bacteroides fragilis Extract

[0065] (1) The bacterial sludge obtained by centrifuging the bacterial liquid of Bacteroides fragilis ZY-312 was resuspended in purified water at a weight ratio of 1 g:6 g of bacterial sludge, and the pH was adjusted to 3.5 with 1 mol / L hydrochloric acid solution. The mixture was extracted at 100°C for 1.5 h, cooled to room temperature, and centrifuged at 12000 g for 10 min at room temperature. The supernatant was collected to obtain a crude sugar solution.

[0066] (2) The crude sugar solution was ultrafiltered and concentrated through a 10KD ultrafiltration membrane to remove small molecular impurities until the conductivity was stable, and the reflux liquid was collected; (3) An equal volume of 40mmol / L Tris-HCl (pH8.5) was added to the reflux liquid for salt conversion; DEAE Sepharose FastFlow ion exchange column chromatography (16mm×200mm) was performed at a flow rate of 20mL / min, and 20mmol / L Tris-HCl (pH8.5, containing 0.2mol / L NaCl) was used for gradient elution for 25 column volumes, and the fractions were collected in sections, 100mL / bottle (components), and SEC-HPLC tracking monitoring was performed. The fractions with a single, symmetrical absorption peak at 206nm were combined, ultrafiltered through a 10KD ultrafiltration membrane, and purified water was added for repeated ultrafiltration until the conductivity was stable. The reflux liquid was collected and freeze-dried to obtain the Bacteroides fragilis ZY-312 extract. The extract was analyzed by nuclear magnetic resonance spectrometer to be capsular polysaccharide A, with a bound lipid content of less than 0.02%, a protein residue of less than 1%, and a nucleic acid residue of less than 0.05%. The obtained capsular polysaccharide A has a weight average molecular weight of 80-90 kDa and a Mw / Mn of 1.0-1.2 according to GPC (gel permeation chromatography) analysis. The chemical structure is shown in the attached Figure 4 Capsular polysaccharide A from Bacteroides fragilis ZY-312 extract was used in the following examples.

[0067] Example 3: Efficacy test of oral administration of Bacteroides fragilis in treating 2,4-dinitrofluorobenzene (DNFB)-induced dermatitis in mice

[0068] 100 BALB / c male mice of SPF grade 18-20g were purchased. After animal quarantine and adaptive feeding for 5 days, group experiments were performed. Except for 10 animals in the normal group, the remaining animals were randomly divided into model group, ZY-312 live bacteria low and high dose groups, ZY-312 inactivated bacteria low and high dose groups, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, Bacteroides fragilis ZY-312 extract PSA group, ketotifen group, 10 animals in each group, and the model was constructed on the first day of the experiment. The abdominal hair of each group of mice was shaved, and the abdomen of the mice was sensitized with 0.5% DNFB solution (Sigma-Aldrich, D1529). On the 5th, 8th, 11th, and 14th days, 0.5% DNFB solution was evenly applied to the back of the model group and each drug group for attack. From the 5th to the 15th day, each drug group was orally administered with the corresponding test drug once a day.

[0069] The inflammation of the back skin of each group was observed with the naked eye and scored before drug administration on the 6th, 9th, 12th and 15th day. About 1 hour after drug administration on the 15th day, blood samples of mice in each group were collected, and serum was frozen in a -80℃ refrigerator, and IgE and IL-4 in serum were measured by ELISA kits.

[0070] 1. Construction of DNFB-induced dermatitis model

[0071] Weigh 30 mg of 2,4-dinitrofluorobenzene (DNFB) into a 10 mL EP tube, add 3 mL of olive oil and 3 mL of acetone, mix well, and prepare a 0.5% DNFB solution for immediate use. Shave the hair of about 2 cm × 2 cm on the abdomen of each group of mice, and evenly apply 50 μL of 0.5% DNFB solution for sensitization. On the 5th day, shave the hair of about 2 cm × 2 cm on the back of each group, and remove the hair with depilatory cream. On the 5th, 8th, 11th, and 14th days, evenly apply 50 μL of 0.5% DNFB solution on the back of the mice in the model group and each drug group for attack.

[0072] 2. Grouping and Dosing

[0073] Except for 10 animals in the normal group, the rest of the modeling group mice were randomly divided into 9 groups on the 32nd day, with 10 mice in each group: model group, ZY-312 low viable bacteria (10 8 CFU / mL), high (10 10 CFU / mL), ZY-312 low inactivated bacteria (10 8 / mL), high (10 10 / mL), NCTC-9343 live bacteria group (10 10 CFU / mL), NCTC-9343 inactivated bacteria group (10 10 / mL), PSA group (5mg / kg), and ketotifen group (20μg / mouse).

[0074] Model group: mice were orally administered 0.2 mL of normal saline once a day for 5 to 15 days;

[0075] The low-dose and high-dose ZY-312 live bacteria groups and the NCTC-9343 live bacteria group were prepared with 5×10 8 CFU / mL, 5×10 10 CFU / mL, 0.2 mL / animal was administered by intragastric administration once a day for 5 to 15 days.

[0076] The low-dose and high-dose groups of ZY-312 inactivated bacteria and the NCTC-9343 inactivated bacteria group were prepared with 5×10 8 CFU / mL, 5×10 10 CFU / mL, 0.2 mL / animal was administered by intragastric administration once a day for 5 to 15 days.

[0077] Bacteroides fragilis ZY-312PSA group: Capsular polysaccharide A (PSA) extracted from Bacteroides fragilis was prepared with normal saline to 5 mg / mL and administered by oral gavage at a rate of 0.2 mL / mouse, once a day for 5 to 15 consecutive days.

[0078] Ketotifen group: The positive control drug was ketotifen, an antihistamine purchased from Sigma-Aldrich, USA. Ketotifen was prepared into a 100 μg / mL solution with normal saline and administered by gavage at 0.2 mL / mouse, once a day, for 5 to 15 consecutive days;

[0079] 3. Testing indicators and methods

[0080] (1) Back skin injury score

[0081] The inflammation of the back skin of each group was observed and scored with the naked eye before drug treatment on the 6th, 9th, 12th and 15th day. The severity of skin lesions was judged with reference to the Chinese Guidelines for the Diagnosis and Treatment of Atopic Dermatitis (2020 Edition) (Chinese Journal of Dermatology, 2020, 053(002):81-88). The following four clinical symptoms were scored separately: 1) hemorrhage / erythema, 2) scarring / dryness, 3) edema, 4) epidermal exfoliation / erosion and lichenification; each clinical symptom was scored according to the grades of 0 (none), 1 (mild), 2 (moderate) and 3 (severe) (Study on the effect and mechanism of terpenoid acetic acid on atopic dermatitis in NC / Nga mice, Wang Yiteng, Master's degree thesis, Issue 4, 2017). The total score of each animal was calculated separately.

[0082] (2) Cytokine detection

[0083] ELISA was used to detect the levels of IgE (ThermoFisher, EMIGHEX5) and IL-4 (ThermoFisher, BMS613) cytokines in mouse serum.

[0084] 4. Test results

[0085] The back skin injury scores of mice in the model group gradually increased from 9 to 15 days, reaching a peak on 15 days. As shown in Table 1, compared with the model group, the back skin injury scores of oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, ZY-312 inactivated bacteria high, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, and Bacteroides fragilis ZY-312PSA group were all reduced, and ZY-312 live bacteria and inactivated bacteria had statistical differences at 12 and 15 days; NCTC-9343 live bacteria and inactivated bacteria, PSA group had statistical differences at 15 days. Compared with the standard strain, oral ZY-312 live bacteria or inactivated bacteria were superior to the standard strain NCTC-9343, with statistical differences at 12 and 15 days. The positive drug ketotifen is a commonly used drug for the treatment of allergic dermatitis. The results showed that ketotifen can inhibit the back skin injury scores of mice induced by DNFB. Oral administration of live and inactivated ZY-312 bacteria effectively inhibited DNFB-induced skin injury scores in mice, and the effect was better than that in the PSA group (P<0.05), effectively alleviating inflammation in the skin on the back of mice.

[0086] (1) Back skin injury score

[0087] Table 1 Effect of drug administration on back skin injury score (Mean±SD)

[0088]

[0089] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P<0.05; ** indicates extremely significant difference P<0.01. Compared with the NCTC-9343 live bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. # indicates significant difference P<0.05. Compared with the NCTC-9343 inactivated bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. @ indicates significant difference P<0.05. Compared with the PSA group, ■ indicates significant difference P<0.05.

[0090] (2) Cytokines

[0091] Table 2 Effects of drug administration on serum cytokines in mice (Mean±SD)

[0092]

[0093] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P<0.05; ** indicates extremely significant difference P<0.01. Compared with the NCTC-9343 live bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. ## indicates extremely significant difference P<0.01. Compared with the NCTC-9343 inactivated bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. @@ indicates extremely significant difference P<0.01. Compared with the PSA group, ■ indicates significant difference P<0.05; ■■ indicates extremely significant difference P<0.01.

[0094] IgE is a type of immunoglobulin, which is positively correlated with allergic-related diseases to a certain extent, thus reflecting the degree of inflammatory activity. IL-4 is a cytokine secreted by Th2 cells. The Th1 / Th2 cells in the peripheral blood and skin lesions of patients with allergic dermatitis are unbalanced and tend to be in the Th2 direction. IL-4 is also closely related to the onset of allergic dermatitis. As shown in Table 2, the serum IgE and IL-4 levels in the model group increased significantly. Compared with the model group, the serum IgE and IL-4 levels of oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, ZY-312 inactivated bacteria high, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, PSA group and ketotifen group were all reduced, and there were statistical differences. Compared with the standard strain, oral ZY-312 live bacteria high or inactivated bacteria high were superior to the standard strain NCTC-9343, and there were statistical differences. Compared with the PSA group, oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, and ZY-312 inactivated bacteria high were superior to the PSA group, and the difference was statistically significant.

[0095] In conclusion, oral administration of Bacteroides fragilis can effectively treat the DNFB-induced dermatitis mouse model.

[0096] Example 4: Efficacy test of Bacteroides fragilis smear administration in treating calcipotriol (MC903)-induced dermatitis in mice

[0097] 1. Experimental design and process

[0098] BALB / c female mice were purchased and after passing the quarantine, 70 mice were randomly divided into 7 groups, with 10 mice in each group: normal group, model group, ZY-312 gel low (10 8 Pieces / pieces), high (10 10 NCTC-9343 gel group (10 10Each group had 10 animals. The abdominal hair of mice in each group was shaved the day before. Except for the animals in the normal group, 45μM MC903 (APExBIO, A3271) prepared with anhydrous ethanol was applied to the abdomen and left ears of mice on the first day of the experiment, with 75μL and 25μL respectively for sensitization. The application was continued for 5 days from the 1st to the 5th day, and stimulated again after an interval of 2 days, for a total of 12 days, and the modeling lasted for 16 days. From the 12th to the 22nd day, each drug group was smeared with the corresponding test drug, once a day.

[0099] The inflammation of the back skin of each group was observed with the naked eye and clinical scores were performed before drug administration on the 12th, 18th and 23rd days. After the 23rd day, the back skin samples of each group of mice were collected, and the IgE and IL-4 in the back skin were measured by ELISA kits. The left ears of each group of mice were collected to test the ear thickness and weigh the ear weight of the mice.

[0100] 2. Grouping and Dosing

[0101] Seventy rats were randomly divided into 7 groups, with 10 rats in each group: normal group, model group, ZY-312 gel low (10 8 Pieces / pieces), high (10 10 NCTC-9343 gel group (10 10 / vial), pimecrolimus group (MEDA, 60 mg / animal), and PSA group (5 mg / kg).

[0102] Normal group and model group: no treatment;

[0103] ZY-312 gel low-dose and high-dose groups: Low-dose and high-dose Bacteroides fragilis ZY312 gel was administered at 25 mg / mouse on the left ear and 80 mg / mouse on the back skin, respectively, once a day for 12 to 22 consecutive days.

[0104] NCTC-9343 gel group: NCTC-9343 gel was applied to the left ear at 25 mg / mouse and to the back skin at 80 mg / mouse, once a day for 12 to 22 consecutive days.

[0105] PSA group: Bacteroides fragilis extract capsular polysaccharide A (PSA) was prepared with normal saline to 0.5 mg / mL, and applied at 10 mL / kg, once a day, for 5 to 15 days. Pimecrolimus group: The positive control drug was pimecrolimus. 20 mg / mouse on the left ear and 70 mg / mouse on the back skin, applied once a day, for 12 to 22 days.

[0106] 3. Testing indicators and methods

[0107] (1) Clinical scoring

[0108] The inflammation of the back skin of each group was observed and scored with the naked eye before drug treatment on the 12th, 18th and 23rd days. The severity of skin lesions was judged with reference to the Chinese Guidelines for the Diagnosis and Treatment of Atopic Dermatitis (2020 Edition) (Chinese Journal of Dermatology, 2020, 053(002):81-88). The following four clinical symptoms were scored separately: 1) bleeding / erythema, 2) scarring / dryness, 3) edema, 4) epidermal exfoliation / erosion and lichenification; each clinical symptom was scored according to the grades of 0 (none), 1 (mild), 2 (moderate) and 3 (severe) (Study on the effect and mechanism of terpenoid acetic acid on atopic dermatitis in NC / Nga mice, Wang Yiteng, Master's degree thesis, Issue 4, 2017). The total score of each animal was calculated separately.

[0109] (2) Skin thickness measurement

[0110] The thickness of the back skin was measured with a micrometer on the 12th, 18th and 23rd day.

[0111] (3) Measurement of mouse ear thickness and weight

[0112] After the mice were killed, specially trained personnel used an electronic vernier caliper to measure the thickness of the middle part of the right ear of the mice and recorded it. An ear piece with a diameter of 8 mm in the middle of the ear was removed with a punch and weighed.

[0113] (4) Cytokine detection

[0114] ELISA was used to detect the levels of cytokines such as IgE (ThermoFisher, EMIGHEX5) and IL-4 (ThermoFisher, BMS613) in the back skin.

[0115] 4. Test results

[0116] (1) Clinical scoring of back skin

[0117] Table 3 Effects of drug administration on back clinical scores (Mean±SD)

[0118]

[0119] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P<0.05; ** indicates extremely significant difference P<0.01. Compared with the NCTC-9343 gel group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. ## indicates extremely significant difference P<0.01. Compared with the PSA group, ■ indicates significant difference P<0.05; ■■ indicates extremely significant difference P<0.01.

[0120] (2) Back skin thickness measurement

[0121] Table 4 Effect of drug administration on back skin thickness (Mean±SD)

[0122]

[0123] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P<0.05; ** indicates extremely significant difference P<0.01. Compared with the NCTC-9343 gel group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. # indicates significant difference P<0.05, ## indicates significant difference P<0.01. Compared with the PSA group, ■ indicates significant difference P<0.05; ■■ indicates extremely significant difference P<0.01.

[0124] As shown in Tables 3 and 4, compared with the model group, the back skin injury scores and back skin thickness of the ZY-312 gel low, ZY-312 gel high, NCTC-9343 gel, and PSA groups were reduced, and the ZY-312 gel application was statistically different at 18 and 23 days; the NCTC-9343 gel group was only statistically different at 23 days. Compared with the standard strain, the ZY-312 gel application was better than the standard strain NCTC-9343 gel, with statistical differences at 18 and 23 days. Compared with the PSA group, the ZY-312 gel high group was better than the PSA group, with statistical differences at 18 and 23 days. The positive drug pimecrolimus cream is a commonly used drug for the treatment of allergic dermatitis. The results showed that pimecrolimus cream can inhibit the increase in the back skin injury score and the increase in the back skin thickness of mice induced by DNFB. Applying low or high doses of ZY-312 gel effectively inhibited MC903-induced back skin inflammation in mice.

[0125] (3) Measurement of mouse ear thickness and weight

[0126] Table 5 Effect of ear swelling after drug administration (Mean±SD)

[0127]

[0128] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P<0.05; ** indicates extremely significant difference P<0.01. Compared with the NCTC-9343 gel group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. # indicates significant difference P<0.05, ## indicates extremely significant difference P<0.01. Compared with the PSA group, ■ indicates significant difference P<0.05; ■■ indicates extremely significant difference P<0.01.

[0129] As can be seen from Table 5, after the modeling of mice in the model group, the ears were swollen and the ear weight was increased. Compared with the model group, the ear thickness and ear weight of the mice smeared with ZY-312 gel low, ZY-312 gel high and pimecrolimus cream were reduced, and all were statistically significant; the NCTC-9343 gel group and the PSA group had statistically significant differences in the ear weight test index compared with the model group. Compared with the standard strain NCTC-9343 gel group, the ZY-312 gel high group had a better effect, and the ear thickness and ear weight were statistically significant. Compared with the PSA group, the ear weight of the mice smeared with ZY-312 gel high was reduced, with statistical significance. Applying ZY-312 low or high dose gel effectively inhibited MC903-induced mouse ear skin inflammation.

[0130] (4) Skin tissue cytokines

[0131] Table 6 Cytokine levels in skin tissue of mice in each group (Mean±SD)

[0132]

[0133] Note: Compared with the model group, * indicates significant difference (P < 0.05); ** indicates extremely significant difference (P < 0.01). Compared with the NCTC-9343 gel group, the P value between the two groups was calculated using the unpaired t-test (two-tailed) method. # indicates significant difference (P < 0.05); ## indicates extremely significant difference (P < 0.01). Compared with the PSA group, ■ indicates significant difference (P < 0.05).

[0134] IgE reflects the degree of inflammatory activity. IL-4 is a cytokine secreted by Th2 cells. The skin lesions of patients with allergic dermatitis tend to be Th2. IL-4 is also closely related to the onset of allergic dermatitis. The results of mouse skin ELISA showed (see Table 6) that the levels of IgE and IL-4 in the skin tissue of the model group were significantly upregulated. Compared with the model group, the levels of IgE and IL-4 in the skin of the ZY-312 gel low, ZY-312 gel high, NCTC-9343 gel, PSA group and pimecrolimus cream were reduced, and there were statistical differences. Compared with the NCTC-9343 gel group, the levels of IgE and IL-4 in the ZY-312 gel high group were reduced, and there were statistical differences. Compared with the PSA group, the IL-4 content in the ZY-312 gel high group was reduced, and there were statistical differences.

[0135] In summary, topical administration of Bacteroides fragilis ZY-312 can effectively treat the calcipotriol-induced dermatitis mouse model.

[0136] Example 5: Efficacy test of oral administration of Bacteroides fragilis in treating dermatitis in mice induced by dust mites and Staphylococcus aureus superantigen

[0137] 1. Experimental design and process

[0138] Six-week-old male C57BL / 6N mice were purchased and passed the quarantine. Except for 10 animals in the normal group, the rest of the animals were used to construct the model. A sensitizer containing 100 μL of dust mites (Dref) and 50 μL of Staphylococcus aureus superantigen (SEB) was applied to the hair removal area on the back of the mice for continuous stimulation for 3 days. On the 4th day after the sensitizer was administered, the stimulation was removed. A new round of stimulation was performed every 2 days, and the stimulation lasted for 6 times until the 27th day. After the modeling was completed, the modeled animals were divided into the model group, the ZY-312 viable low (10 7 CFU / mL), high (10 9 CFU / mL), ZY-312 low inactivated bacteria (10 7 / mL), high (10 9 / mL), NCTC-9343 live bacteria group (10 9 CFU / mL), NCTC-9343 inactivated bacteria group (10 9 / mL), PSA group, ketotifen (20μg / mouse). From 28th to 38th day, the designed dose was orally administered once a day.

[0139] All animals were scored for dermatitis symptoms on the day of grouping (28th day) and on days 32, 35, and 38. Blood samples were collected from animals in each group on day 38, and ELISA was used to detect IFN-γ and IL-4 in mouse serum.

[0140] 2. Grouping and Dosing

[0141] Except for 10 normal group animals, the rest of the model group mice were randomly divided into 9 groups according to the dermatitis symptom scores on the 28th day, with 10 mice in each group: model group, ZY-312 low viable bacteria (10 7 CFU / mL), high (10 9 CFU / mL), ZY-312 low inactivated bacteria (10 7 / mL), high (10 9 / mL), NCTC-9343 live bacteria group (10 9 CFU / mL), NCTC-9343 inactivated bacteria group (10 9 / mL), PSA group (5mg / kg), and ketotifen group (20μg / mouse).

[0142] Model group: Mice were orally administered 0.2 mL of normal saline once a day for 28 to 38 days.

[0143] The low-dose and high-dose ZY-312 live bacteria groups and the NCTC-9343 live bacteria group were prepared with 5×10 7 CFU / mL, 5×10 9 CFU / mL, 0.2 mL / animal was administered by intragastric administration once a day for 28 to 38 days.

[0144] The low-dose and high-dose groups of ZY-312 inactivated bacteria and the NCTC-9343 inactivated bacteria group were prepared with 5×10 7 CFU / mL, 5×10 9 CFU / mL, 0.2 mL / animal was administered by intragastric administration once a day for 28 to 38 days.

[0145] PSA group: Capsular polysaccharide A (PSA) extracted from Bacteroides fragilis was prepared with normal saline to 5 mg / mL and administered by oral gavage at a rate of 0.2 mL / mouse, once a day for 5 to 15 consecutive days.

[0146] Ketotifen group: The positive control drug was ketotifen, an antihistamine purchased from Sigma-Aldrich, USA. Ketotifen was prepared into a 100 μg / mL solution with normal saline and administered by gavage at 0.2 mL / mouse, once a day, for 28 to 38 days.

[0147] 3. Testing indicators and methods

[0148] (1) Back skin inflammation score

[0149] The inflammation of the back skin of each group was observed and scored with the naked eye before drug treatment on the 28th, 32nd, and 35th day and before dissection (38th day). The severity of skin lesions was judged with reference to the Chinese Guidelines for the Diagnosis and Treatment of Atopic Dermatitis (2020 Edition) (Chinese Journal of Dermatology, 2020, 053(002):81-88). The following four clinical symptoms were scored separately: 1) hemorrhage / erythema, 2) scarring / dryness, 3) edema, 4) epidermal exfoliation / erosion and lichenification; each clinical symptom was scored according to the grades of 0 (none), 1 (mild), 2 (moderate) and 3 (severe) (Study on the effect and mechanism of terpenoid acetic acid on atopic dermatitis in NC / Nga mice, Wang Yiteng, Master's degree thesis, Issue 4, 2017). The total score of each animal was calculated separately.

[0150] (2) Cytokine detection

[0151] Elisa was used to detect the levels of IFN-γ (R&D Systems) and IL-4 (R&D Systems) cytokines in mouse serum.

[0152] IV. Experimental Results

[0153] (1) Back skin inflammation score

[0154] Table 7 Effect of drug administration on back skin inflammation score (Mean±SD)

[0155]

[0156] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P<0.05; ** indicates extremely significant difference P<0.01. Compared with the NCTC-9343 live bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. # indicates significant difference P<0.05, ## indicates extremely significant difference P<0.01. Compared with the NCTC-9343 inactivated bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. @ indicates significant difference P<0.05, @@ indicates extremely significant difference P<0.01. Compared with the PSA group, ■ indicates significant difference P<0.05; ■■ indicates extremely significant difference P<0.01.

[0157] The results showed (see Table 7) that after the model was established, the back skin inflammation score was significantly increased and there was no difference among the groups. Compared with the model group, the back skin injury scores of the oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, ZY-312 inactivated bacteria high, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, PSA group and ketotifen group were all reduced, and ZY-312 live bacteria and inactivated bacteria had statistical differences at 32, 35, and 38 days; NCTC-9343 live bacteria and inactivated bacteria had statistical differences at 35 and 38 days. Compared with the standard strain, oral ZY-312 live bacteria or inactivated bacteria were superior to the standard strain NCTC-9343, with statistical differences at 32, 35, and 38 days. Compared with the PSA group, the oral ZY-312 live bacteria high group and ZY-312 inactivated bacteria high group had better effects at 32, 35, and 38 days, with statistically significant differences. Oral ZY-312 live bacteria and inactivated bacteria both effectively inhibited the skin injury scores on the back of mice induced by dust mites combined with Staphylococcus aureus superantigen, and effectively alleviated inflammation on the back skin of mice.

[0158] (2) Cytokines

[0159] Table 8 Cytokine levels in mice in each group (Mean±SD)

[0160]

[0161] Note: Compared with the model group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01. Compared with the NCTC-9343 live bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. # indicates significant difference p<0.05, ## indicates extremely significant difference p<0.01. Compared with the NCTC-9343 inactivated bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. @ indicates significant difference p<0.05, @@ indicates extremely significant difference p<0.01. Compared with the PSA group, ■ indicates significant difference p<0.05; ■■ indicates extremely significant difference p<0.01.

[0162] IFN-γ is a typical Th1-type cytokine that has the function of inhibiting Th2 cell differentiation. The Th1 / Th2 cells in the peripheral blood and skin lesions of patients with atopic dermatitis are out of balance and tend to be Th2. IFN-γ secretion is closely related to the onset of atopic dermatitis. IL-4 is a cytokine secreted by Th2 cells, and IL-4 is also closely related to the onset of atopic dermatitis. The results in Table 8 show that the serum IFN-γ content in the model group was down-regulated. Compared with the model group, the serum of oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, ZY-312 inactivated bacteria high, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, PSA group and ketotifen group restored IFN-γ secretion; the serum IL-4 content in the model group increased significantly. Compared with the model group, the serum IL-4 content of oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, ZY-312 inactivated bacteria high, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, PSA group and ketotifen group decreased. Compared with the standard strain, oral ZY-312 live bacteria high or inactivated bacteria high were superior to the standard strain NCTC-9343, and there was a statistical difference. Compared with the PSA group, the oral ZY-312 live bacteria high group and the ZY-312 inactivated bacteria high group had better effects, and the difference was statistically significant.

[0163] In summary, oral administration of Bacteroides fragilis ZY-312 can effectively treat the dermatitis mouse model induced by dust mites and Staphylococcus aureus superantigen.

[0164] Example 6: Efficacy test of Bacteroides fragilis in treating 2,4-dinitrochlorobenzene (DNCB)-induced dermatitis-eczema in mice

[0165] 1. Experimental design and process

[0166] 100 BALB / c male mice of SPF grade 18-20g were purchased. Animal quarantine and adaptive feeding were performed for 5 days, and group experiments were performed. Except for 10 animals in the normal group, the remaining animals were randomly divided into model group, ZY-312 live bacteria low and high dose groups, ZY-312 inactivated bacteria low and high dose groups, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, PSA group, and prednisone group, with 10 animals in each group, and the model was constructed on the first day of the experiment. The abdominal hair of each group of mice was shaved, and 1% DNCB solution (Sigma-Aldrich) prepared with acetone olive oil solution was applied to the abdomen and both sides of the left ear of the mice, 100μL and 20μL respectively for sensitization. The same operation was performed on the second day to strengthen sensitization. On the fifth day, 1% DNCB solution was evenly applied to both sides of the left ears of the mice in the model group and each drug group for attack. On the 5th day, medication was started 3 hours after stimulation, and each drug group was orally administered with the corresponding test drug once a day.

[0167] The ear allergy scores of each group were observed with the naked eye and the ear thickness was measured before the treatment on the 1st and 2nd day after the challenge. One hour after the last administration, blood samples of mice in each group were collected, and serum was frozen in a -80℃ refrigerator. The IgE and IL-4 in the serum were determined by ELISA kits. The left ears of mice in each group were collected to measure the ear weight of mice.

[0168] 2. Grouping and Dosing

[0169] Except for 10 normal group animals, the remaining 50 mice in the model group were randomly divided into 5 groups, with 10 mice in each group: model group, ZY-312 low viable bacteria (10 8 CFU / mL), high (10 10 CFU / mL), ZY-312 low inactivated bacteria (10 8 / mL), high (10 10 / mL), NCTC-9343 live bacteria group (10 10 CFU / mL), NCTC-9343 inactivated bacteria group (10 10 / mL), PSA group (5mg / kg), and prednisone group (0.04mg / mouse).

[0170] Model group: mice were orally administered 0.2 mL of normal saline once a day for 5 to 7 days;

[0171] The low-dose and high-dose ZY-312 live bacteria groups and the NCTC-9343 live bacteria group were prepared with 5×10 8 CFU / mL, 5×10 10 CFU / mL, 0.2 mL / animal was administered by intragastric administration once a day for 5 to 7 consecutive days.

[0172] The low-dose and high-dose groups of ZY-312 inactivated bacteria and the NCTC-9343 inactivated bacteria group were prepared with 5×10 8 CFU / mL, 5×10 10 CFU / mL, 0.2 mL / animal was administered by intragastric administration once a day for 5 to 7 consecutive days.

[0173] PSA group: Capsular polysaccharide A (PSA) extracted from Bacteroides fragilis was prepared with normal saline to 5 mg / mL and administered by oral gavage at a rate of 0.2 mL / mouse, once a day for 5 to 15 consecutive days.

[0174] Prednisone group: The positive control drug was prednisone, a glucocorticoid purchased from Zhejiang Xianju Pharmaceutical Co., Ltd. During the experiment, 0.5% sodium carboxycellulose was made into a 0.2 mg / mL suspension, and 0.2 mL / mouse was administered by gavage, once a day, for 5 to 7 consecutive days;

[0175] 3. Testing indicators and methods

[0176] (1) Otoallergic reaction score

[0177] Before drug administration on the 6th and 7th day, the ear allergic reaction of mice in each group was observed with naked eyes to score. The scoring criteria are as follows:

[0178] Erythema: no erythema (0), mild erythema (1), moderate erythema without crusting (2), moderate to severe erythema with mild crusting (3), severe erythema with severe crusting (4);

[0179] Edema: no edema (0), mild edema (1), moderate edema (2), severe edema (3);

[0180] Scratches: None (0), Yes (1)

[0181] The maximum score is 8 points, and the total score of each animal is calculated.

[0182] (2) Detection of mouse ear thickness and weight

[0183] After the mice were killed, specially trained personnel used an electronic vernier caliper to measure the thickness of the middle part of the right ear of the mice and recorded it. An ear piece with a diameter of 8 mm in the middle of the ear was removed with a punch and weighed.

[0184] (3) EASI scoring The clinical symptoms of the skin of mice in each group were evaluated using the EASI scoring standard (reference: Eczema Area and Severity Index Scoring Method, Chinese Journal of Dermatology, 2004 (1): 3-4), which evaluates the four indicators of erythema, edema / infiltration / papule, scaling, and lichenification, with a score of 0 to 3: 0 = no symptoms; 1 = mild; 2 = moderate; 3 = severe, and the total score was obtained by adding up the scores of each indicator. The observer scored before and after the drug administration, and took photos to record the results.

[0185] (4) Cytokine detection

[0186] ELISA was used to detect the levels of IgE (ThermoFisher, EMIGHEX5) and IL-4 (ThermoFisher, BMS613) cytokines in mouse serum.

[0187] 4. Test results

[0188] (1) Otoallergic reaction score

[0189] Table 9 Effect of drug administration on ear allergy score (Mean±SD)

[0190]

[0191] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P<0.05; ** indicates extremely significant difference P<0.01. Compared with the NCTC-9343 live bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. # indicates significant difference P<0.05. Compared with the NCTC-9343 inactivated bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. @ indicates significant difference P<0.05. Compared with the PSA group, ■ indicates significant difference P<0.05; ■■ indicates extremely significant difference P<0.01.

[0192] (2) Mouse ear weight measurement

[0193] Table 10 Effects of drug administration on ear thickness and weight of mice (Mean±SD)

[0194]

[0195]

[0196] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P<0.05; ** indicates extremely significant difference P<0.01. Compared with the NCTC-9343 live bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. ## indicates extremely significant difference P<0.01. Compared with the NCTC-9343 inactivated bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. @ indicates significant difference P<0.05, @@ indicates extremely significant difference P<0.01. Compared with the PSA group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method, ■ indicates significant difference P<0.05; ■■ indicates extremely significant difference P<0.01.

[0197] The results showed (see Tables 9 and 10) that the ear allergy scores of the model group were increased after stimulation, and the ears were swollen. Compared with the model group, the ear allergy scores of the oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, ZY-312 inactivated bacteria high, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, PSA group and prednisone group were all reduced, and there were statistical differences at 6 and 7 days. At the same time, the ear thickness and ear weight of mice in each administration group were also reduced. Compared with the standard strain, oral ZY-312 live bacteria high or inactivated bacteria high inhibited DNCB-induced mouse ear allergy, which was better than the standard strain NCTC-9343, and there were statistical differences at 6 and 7 days. The results of mouse ear thickness and ear weight were also better than those of the standard strain NCTC-9343. Compared with the PSA group, the oral ZY-312 live bacteria high group and ZY-312 inactivated bacteria high group had better effects on the downregulation of ear allergy scores and ear thickness and weight of mice at 6 and 7 days, and there was a statistical difference. Oral ZY-312 live bacteria and inactivated bacteria can effectively inhibit DNCB-induced ear allergy in mice and effectively relieve skin inflammation in mice.

[0198] (3) EASI score

[0199] Table 11 Effects of drug administration on EASI scores of mice (Mean±SD)

[0200]

[0201] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. *** indicates extremely significant difference P<0.001. Compared with the NCTC-9343 live bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. ### indicates extremely significant difference P<0.001. Compared with the NCTC-9343 inactivated bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. @ indicates significant difference P<0.05, @@@ indicates extremely significant difference P<0.001. Compared with the PSA group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method, ■■ indicates extremely significant difference P<0.01, and ■■■ indicates extremely significant difference P<0.001. Compared with the ZY-312 inactivated bacteria low group, the P value between the two groups was calculated using the unpaired t-test (two-tailed) method, and + indicated a very significant difference, P<0.05.

[0202] The results showed (see Table 11) that there was no statistical difference in the EASI scores of the modeling groups before administration. After the end of administration, the EASI scores of each administration group were reduced compared with the model group (P < 0.001). Compared with the NCTC-9343 live bacteria group, the EASI scores of the oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, ZY-312 inactivated bacteria high, PSA group and prednisone group were significantly reduced (P < 0.001). Compared with the NCTC-9343 inactivated bacteria group, the EASI scores of the oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, ZY-312 inactivated bacteria high, PSA group were significantly reduced (at least P < 0.05). Compared with the PSA group, oral administration of ZY-312 live bacteria high, ZY-312 inactivated bacteria low, and ZY-312 inactivated bacteria high reduced EASI scores (at least P<0.01). Compared with the ZY-312 inactivated bacteria low group, the ZY-312 inactivated bacteria high group reduced EASI scores (P<0.05). In summary, oral administration of ZY-312 live bacteria and inactivated bacteria can reduce the eczema area and severity index of mice, among which the ZY-312 inactivated bacteria high group has the best effect.

[0203] (4) Cytokines

[0204] Table 12 Effects of administration on mouse serum cytokines (Mean±SD)

[0205]

[0206] Note: Compared with the model group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P<0.05; ** indicates extremely significant difference P<0.01. Compared with the NCTC-9343 live bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. # indicates significant difference P<0.05. Compared with the NCTC-9343 inactivated bacteria group, the P values ​​between the two groups were calculated according to the unpaired t-test (two-tailed) method. @ indicates significant difference P<0.05. Compared with the PSA group, ■ indicates significant difference P<0.05.

[0207] IgE is a kind of immunoglobulin, which is positively correlated with allergic-related diseases to reflect the activity of inflammation. IL-4 is a representative cytokine of Th2 cells. Th1 / Th2 cells in the peripheral blood of patients with allergic dermatitis are unbalanced and tend to be in the direction of Th2. IL-4 is also closely related to the onset of allergic dermatitis. The results show (Table 12) that the serum IgE and IL-4 levels in the model group increased significantly. Compared with the model group, the serum IgE and IL-4 levels of oral ZY-312 live bacteria low, ZY-312 live bacteria high, ZY-312 inactivated bacteria low, ZY-312 inactivated bacteria high, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, PSA group and prednisone group were all reduced, and there were statistical differences. Compared with the standard strain, oral ZY-312 live bacteria high or inactivated bacteria high were better than the standard strain NCTC-9343, and there were statistical differences. Compared with the PSA group, the IgE and IL-4 levels in the oral ZY-312 live bacteria high group, ZY-312 inactivated bacteria low group and ZY-312 inactivated bacteria high group were significantly reduced, and the difference was statistically significant.

[0208] In conclusion, oral administration of Bacteroides fragilis ZY-312 can effectively treat the DNCB-induced dermatitis-eczema model in mice.

[0209] Example 7: Efficacy test of Bacteroides fragilis in treating immune contact urticaria

[0210] 1. Experimental design and process

[0211] 80 SPF-grade 20±2g BALB / c mice were purchased, half male and half female. After animal quarantine and adaptive feeding for 7 days, group experiments were performed. Except for 10 animals in the normal group, the remaining animals were randomly divided into model group, ZY-312 live bacteria group, ZY-312 inactivated bacteria group, NCTC-9343 live bacteria group, NCTC-9343 inactivated bacteria group, PSA group, and loratadine group, with 10 animals in each group, and the model was constructed on the first day of the experiment. The back of the mice in each model group was shaved in advance, and 0.5mL of anti-dinitrophenol (anti-DNP) IgE monoclonal antibody (1:5000) was injected into the tail vein for sensitization. 24h later, 2,4-dinitrofluorobenzene (DNFB) was applied to both ears of the mice for stimulation.

[0212] The general condition and scratching behavior of mice in each group were observed with naked eyes before administration of treatment on the 1st to 3rd day after the challenge, and the wheal condition was recorded. One hour after the last administration, blood samples of mice in each group were collected, and serum was frozen in a -80℃ refrigerator. The IgE, IFN-γ and IL-4 in the serum were determined by ELISA kits. The left ears of mice in each group were collected to measure the ear weight of mice.

[0213] 2. Grouping and Dosing

[0214] Except for 10 normal group animals, the remaining 70 mice in the model group were randomly divided into 7 groups, with 10 mice in each group: model group, ZY-312 live bacteria group (10 9 CFU / mL), ZY-312 inactivated bacteria group (10 9 / mL), NCTC-9343 live bacteria group (10 9 CFU / mL), NCTC-9343 inactivated bacteria group (10 9 / mL), PSA group (5mg / kg), and loratadine group (0.05mg / mouse).

[0215] Model group: mice were orally administered 0.2 mL of normal saline once a day for 7 consecutive days;

[0216] ZY-312 live bacteria group and NCTC-9343 live bacteria group: 5×10 9 CFU / mL, 0.2 mL / animal was administered by intragastric administration once a day for 7 consecutive days.

[0217] ZY-312 inactivated bacteria group and NCTC-9343 inactivated bacteria group: 5×10 9 CFU / mL, 0.2 mL / animal was administered by intragastric administration once a day for 7 consecutive days.

[0218] PSA group: Capsular polysaccharide A (PSA) extracted from Bacteroides fragilis was prepared with normal saline to 5 mg / mL and administered by oral gavage at a rate of 0.2 mL / mouse, once a day for 7 consecutive days.

[0219] Loratadine group: The positive control drug was loratadine, an antihistamine purchased from Shanghai Schering-Plough Pharmaceutical Co., Ltd. During the experiment, 0.5% sodium carboxycellulose was made into a 0.25 mg / mL suspension, and 0.2 mL / mouse was administered by gavage, once a day, for 7 consecutive days;

[0220] 3. Testing indicators and methods

[0221] (1) Observation of general condition and scratching behavior

[0222] The general behavior of the mice was recorded and observed every day. One hour after stimulation and every day thereafter, the mice's activities were filmed for 15 minutes, the video was played back, and the number of scratching times was counted.

[0223] (2) Wheal detection

[0224] After the tail vein injection of Evans blue, the automatic timer was used to take pictures every 30 minutes to record the number, frequency, size and disappearance time of the wheals on the exposed part of the back of the mouse.

[0225] (3) Cytokine detection

[0226] ELISA was used to detect the levels of IgE (ThermoFisher, EMIGHEX5), IFN-γ (ThermoFisher, 88-7314-88) and IL-4 (ThermoFisher, BMS613) cytokines in mouse serum.

[0227] 4. Test results

[0228] (1) 24 hours after stimulation, the model group mice had a poor mental state, slow movements, significantly reduced activity, messy hair, poor appetite, slowed sleep time, and significantly slowed or even decreased weight gain. 3 days after administration, the drug-treated group mice had a better mental state, significantly increased activity, increased appetite, prolonged sleep time, and gradually returned to normal weight gain, with the ZY312 inactivated bacteria group having the most significant effect.

[0229] Table 13 Changes in the number of scratching behaviors of mice after administration (Mean±SD)

[0230]

[0231] Note: Compared with the model group, *** indicates an extremely significant difference P<0.001. Compared with the NCTC-9343 live bacteria group, ### indicates an extremely significant difference P<0.001. Compared with the NCTC-9343 inactivated bacteria group, + indicates a significant difference P<0.05; +++ indicates an extremely significant difference P<0.001. Compared with the PSA group, ■ indicates a significant difference P<0.05; ■■■ indicates an extremely significant difference P<0.001.

[0232] (2) Wheal detection

[0233] Table 14 Changes in the number of wheal attacks in mice after administration (Mean±SD)

[0234]

[0235]

[0236] Note: Compared with the model group, *** indicates extremely significant difference P<0.001. Compared with the NCTC-9343 live bacteria group, # indicates significant difference P<0.05, ### indicates extremely significant difference P<0.001. Compared with the PSA group, + indicates significant difference P<0.05.

[0237] The results showed (see Tables 13 and 14) that the number of scratching of mice in the model group increased after stimulation, and wheals appeared successively in the depilatory area on the back. Compared with the model group, the number of scratching and wheal attacks of mice in each medication group showed a decreasing trend, and there was a statistical difference. Compared with the live bacteria of the standard strain, the oral administration of the inactivated bacteria of the standard strain had the same efficacy, and the live bacteria of ZY-312, the inactivated bacteria of ZY-312, and PSA had better inhibitory effects on immune contact urticaria reactions. Compared with the PSA group, the oral administration of live and inactivated bacteria of ZY-312 had better effects, among which the inactivated bacteria of ZY-312 had the best effect.

[0238] (3) Cytokines

[0239] Table 15 Effects of administration on mouse serum cytokines (Mean±SD)

[0240]

[0241] Note: Compared with the model group, *** indicates extremely significant difference P<0.001. Compared with the NCTC-9343 live bacteria group, # indicates significant difference P<0.05, ### indicates significant difference P<0.001. Compared with the PSA group, + indicates significant difference P<0.05.

[0242] IgE is a type of immunoglobulin, which is positively correlated with allergic-related diseases and thus reflects the degree of inflammatory activity. TNF-α is a type of proinflammatory factor. TNF-α can quickly recruit inflammatory cytokines, induce the expression of sticky factors, and accelerate the release of neutrophils and platelet activation factors. IL-4 is a representative cytokine of Th2 cells. The Th1 / Th2 cells in the peripheral blood of patients with urticaria are unbalanced and tend to be in the direction of Th2. IL-4 is closely related to the onset of urticaria. The results showed (Table 15) that the serum IgE, TNF-α and IL-4 levels in the model group increased significantly. Compared with the model group, the serum IgE, TNF-α and IL-4 levels in the medication group were all reduced, and there were statistical differences. Compared with the standard strain live bacteria, the oral standard strain inactivated bacteria have equivalent efficacy, and the ZY-312 live bacteria, inactivated bacteria or PSA have better effects, and there are statistical differences. Compared with the PSA group, oral administration of live and inactivated ZY-312 bacteria had better effects, among which inactivated ZY-312 bacteria had the best effect.

[0243] In summary, oral administration of Bacteroides fragilis ZY-312 can effectively treat the immune contact urticaria model, among which the inactivated ZY-312 bacteria has the best effect.

[0244] Of course, the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art may make various corresponding changes and modifications based on the present invention, but these corresponding changes and modifications should all fall within the scope of protection of the claims attached to the present invention.

Claims

1. A use of Bacteroides fragilis in preparing a composition for treating allergic skin diseases, characterized in that: The Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No.10685.

2. The use according to claim 1, characterized in that: The Bacteroides fragilis is one or more of the following: live Bacteroides fragilis, Bacteroides fragilis that has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, physically treated or inactivated, and liquid culture supernatant of Bacteroides fragilis.

3. The use according to claim 2, characterized in that: The inactivated Bacteroides fragilis is an inactivated bacterium with complete morphological structure or an inactivated bacterium with incomplete morphological structure; preferably, the inactivated bacterium has a complete morphological structure; Further preferably, the Bacteroides fragilis is inactivated by any one or more of dry heat, wet heat, filtration, organic solvents, chemical reagents, ultraviolet or infrared rays, fermentation, freeze-drying, genetic recombination, genetic modification or transformation; preferably, the inactivated bacterial powder is obtained by freeze-drying.

4. The use according to any one of claims 1 to 3, characterized in that: The composition is any one of a pharmaceutical composition, a food, a health product or a food additive.

5. The use according to claim 4, characterized in that: The pharmaceutical composition contains a pharmaceutically effective dose of inactivated Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685; and / or, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the dosage form of the pharmaceutical composition includes pills, tablets, granules, capsules, powders, suspensions, oral liquids, tube feeding preparations, enemas, liniments, and gels; Preferably, the administration of the pharmaceutical composition includes transdermal administration, oral administration, enema, and application; Preferably, the administration cycle of the pharmaceutical composition is intermittent administration, periodic administration, continuous administration or long-term administration. Preferably, the pharmaceutical composition is for human or animal administration.

6. The use according to claim 4, characterized in that: The food is milk powder, cheese, curd, yogurt, ice cream or fermented cereal food; preferably, the food can also be animal food, such as feed.

7. The use according to any one of claims 1 to 6, characterized in that: The allergic skin diseases include atopic dermatitis, eczema or chronic urticaria.

8. Use of a Bacteroides fragilis extract in preparing a composition for treating eczema or chronic urticaria, characterized in that: The Bacteroides fragilis is Bacteroides fragilis ZY-312 with a deposit number of CGMCC No. 10685, and the Bacteroides fragilis extract contains capsular polysaccharide A; Preferably, the content of lipid in the capsular polysaccharide A is less than 0.02 wt%, the protein residue is less than 1%, and the nucleic acid residue is less than 0.05%; Preferably, the structure of the capsular polysaccharide A is as follows: Further preferably, the weight average molecular weight of the capsular polysaccharide A is 80-90 kD, and the ratio of weight average molecular weight to number average molecular weight (Mw / Mn) is 1.0-1.

2.

9. The use according to claim 8, characterized in that: The composition is any one of a pharmaceutical composition, a food, a health product or a food additive.

10. The use according to claim 9, characterized in that: The pharmaceutical composition contains a pharmaceutically effective dose of capsular polysaccharide A of Bacteroides fragilis ZY-312 extract with a deposit number of CGMCC No. 10685; and / or, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the dosage form of the pharmaceutical composition includes pills, tablets, granules, capsules, powders, suspensions, oral liquids, tube feeding preparations, enemas, liniments, and gels; Preferably, the administration of the pharmaceutical composition includes transdermal administration, oral administration, enema, and application; Preferably, the administration cycle of the pharmaceutical composition is intermittent administration, periodic administration, continuous administration or long-term administration. Preferably, the pharmaceutical composition is for human or animal administration.

11. The use according to claim 9, characterized in that: The food is milk powder, cheese, curd, yogurt, ice cream or fermented cereal food; preferably, the food can also be animal food, such as feed.

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