New application of bifidobacterium breve CCFM1078 in relieving atopic dermatitis

Abstract

The invention discloses a novel application of bifidobacterium breve CCFM1078 in relieving atopic dermatitis, and belongs to the technical field of microorganisms. The bifidobacterium breve CCFM1078 provided by the invention can relieve atopic dermatitis, and is specifically reflected in that (1) ear swelling and back skin thickening of mice are relieved, dermatological symptoms are relieved, and mastocyte infiltration is reduced; (2) the relative expression quantity of mRNA (messenger Ribonucleic Acid) of mouse skin barrier function related genes is increased; (3) reducing the total IgE level of mouse serum; (4) reducing the levels of IL-4, IL-13 and TSLP in mouse skin tissues, improving the levels of IL-10 and IFN-gamma, recovering Th1 / Th2 immune balance, and maintaining immune homeostasis; (5) activating the expression of AHR and CYP1A1 in the skin tissue of the mouse; (6) improving IgA and sIgA secretion of mouse colon tissue; and (7) compared with a representative high-yield ILA strain, the ILA yield is higher.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology and the field of medicine, and specifically relates to a new application of Bifidobacterium breve CCFM1078 in relieving atopic dermatitis. BACKGROUND

[0002] Atopic dermatitis (AD) is a common chronic relapsing inflammatory skin disease, and its main clinical features are severe skin itching, dryness, edema and recurrent eczema-like skin rash, and it is often accompanied by asthma, allergic rhinitis and other allergic diseases. As a non-infectious disease, AD has a particularly high incidence in the child population, affecting about 20% of children, and 2% to 8% of adults, and the incidence is increasing year by year worldwide, seriously affecting the physical and mental health of patients.

[0003] The pathogenesis of AD involves the interaction of multiple factors such as genetics, immune disorders and environment. In terms of immune mechanisms, AD patients usually show immune bias dominated by helper T cell type 2 (Th2) response, which leads to the release of large amounts of inflammatory factors such as IL-4 and IL-13, induces the increase of serum IgE level and infiltration of eosinophils and mast cells, and thus aggravates skin inflammation. In addition, the dysfunction of regulatory T cells (Treg) and the overactivation of antigen-presenting cells such as Langerhans cells and dendritic cells also promote the development of chronic AD. Clinical data shows that the total serum IgE and allergen-specific IgE of most patients are significantly increased, and most of them are related to environmental or food allergens.

[0004] According to the laboratory examination characteristics and skin inflammation patterns, AD can be divided into several types: 1) According to the total IgE level and whether there is specific IgE, it is divided into endogenous and exogenous. Endogenous refers to normal serum total IgE level (<200 KU / L), no history of atopic disease, and lack of allergen-specific IgE. Exogenous refers to high levels of IgE, personal or family history of atopic disease, and elevated levels of food and / or inhaled allergen-specific IgE. 2) According to the skin inflammation pattern, it is divided into Th2, Th22, Th17 and Th1 dominated, or mixed inflammation patterns, such as Th2 type inflammation in childhood AD, Th2 / Th22 mixed inflammation in adult AD, and Th2 / Th17 mixed inflammation in Asian. Currently, the clinical intervention treatment strategy for AD is mainly aimed at relieving symptoms and controlling recurrence. Common methods include topical glucocorticoids, calcineurin inhibitors, oral antihistamines, immunosuppressants, and anti-inflammatory drugs. These drugs can improve inflammation and relieve itching in the short term, but long-term use often accompanies skin barrier damage, increased risk of infection, and drug resistance. At the same time, some patients have poor tolerance or low compliance to existing drugs, which limits their clinical application effect. Therefore, it is of great significance to develop safe, effective and well-tolerated treatment methods.

[0005] In recent years, more and more studies have revealed that the early life is a critical window period for the development and maturation of the immune system. The development of immune homeostasis and the composition and diversity of gut microbiota at this stage play a decisive role in training the host immune system and inducing immune tolerance. Early life gut microbiota imbalance is considered as one of the important environmental triggers of AD occurrence, which may provide a solid theoretical basis for early life probiotic intervention from the perspective of gut microbiota. Therefore, in this highly plastic stage of early life, shaping a healthy gut microecology through probiotic intervention may be a potential strategy to prevent and relieve AD by correcting immune bias and strengthening skin barrier. At the same time, due to the importance of genetic factors in the occurrence of AD, how to reduce the risk of offspring of atopic dermatitis patients is also a necessary factor for early life intervention.

[0006] Although the potential of probiotic intervention in AD has been widely recognized, existing research and technology are mainly focused on the adjuvant therapy of individuals with the disease or the general application in non-specific window period. Currently, there is no research that explicitly proposes and verifies the application of specific Bifidobacterium breve strains in early life, a specific critical period, for the prevention and relief of atopic dermatitis.

[0007] Given the profound impact of early life immune homeostasis and gut microbiota establishment on the development of atopic dermatitis, and considering the limitations of existing therapies, coupled with the current lack of known technologies for early life intervention using Bifidobacterium breve, developing a novel, safe, effective, and postnatally modulating microecological treatment based on specific probiotic strains, targeting this critical early life window, would be of great significance for fundamentally reducing the incidence of atopic dermatitis, alleviating patient symptoms, improving quality of life, and reducing the risk of disease in patients and their offspring.

[0008] Repeated DNFB (2,4-dinitrofluorobenzene) sensitization is a single-chemical-induced, contact sensitization reaction, distinct from the multifactorial and complex etiology of clinically diagnosed atopic dermatitis (AD). It often mimics the acute prodromal stage of AD, manifesting as skin erythema, edema, scaling, even erosion, and intense itching, accompanied by elevated serum IgE levels and excessive activation of the Th2 response. Screening for a strain capable of treating DNFB-induced acute prodromal stage symptoms of AD is of great significance for the treatment of atopic dermatitis. Summary of the Invention

[0009] [Purpose of the Invention] The purpose of this invention is to provide a strain of Bifidobacterium breve with novel applications in alleviating atopic dermatitis through in vivo studies on newborn animals. Bifidobacterium breve ), to develop safe and effective probiotic prevention and treatment methods for patients with atopic dermatitis.

[0010] [Technical Solution] To achieve the above objectives, this invention provides a strain of Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium breve The use of CCFM1078 in the preparation of medicines or foods for the prevention and / or treatment of atopic dermatitis.

[0011] The Bifidobacterium breve described in this invention ( Bifidobacterium breve CCFM1078 was isolated from infant fecal samples from Wuxi City, Jiangsu Province. After isolation and screening, the strain was identified as Bifidobacterium breve FJSWX17M1 and named Bifidobacterium breve CCFM1078. The Bifidobacterium breve CCFM1078 has been disclosed in patent CN112111424A and has the accession number GDMCC No: 61011.

[0012] In one embodiment of the present invention, the *Bifidobacterium breve* CCFM1078 colonies on mMRS solid medium are white and round with regular and smooth edges, raised in the middle, and are sticky, soft, and moist, indicating that it is a Gram-positive bacterium.

[0013] In one embodiment of the present application, the product comprises a pharmaceutical product or a food product.

[0014] In one embodiment of the present application, the food product comprises a health food product comprising the Bifidobacterium breve CCFM1078 described above.

[0015] In one embodiment of the present application, the number of bacterial cells of the Bifidobacterium breve CCFM1078 described above in the pharmaceutical product is not less than 1 x 10 9 CFU / mL or 1 x 10 9 CFU / g.

[0016] In one embodiment of the present application, the method for collecting the bacterial cells of the Bifidobacterium breve CCFM1078 comprises inoculating the Bifidobacterium breve CCFM1078 described above into a culture medium at an inoculation amount of 2 to 4% of the total mass of the culture medium, culturing at 37°C for 24 to 48 hours to obtain a culture solution, and centrifuging the culture solution to obtain the bacterial cells.

[0017] In one embodiment of the present application, the culture medium is an mMRS culture medium.

[0018] In one embodiment of the present application, the atopic dermatitis is atopic dermatitis induced by 2,4-dinitrofluorobenzene (DNFB).

[0019] In one embodiment of the present application, the pharmaceutical product or the food product comprises the Bifidobacterium breve CCFM1078, a pharmaceutical carrier, and / or a pharmaceutical excipient.

[0020] In one embodiment of the present application, the pharmaceutical carrier comprises one or more of a filler, a binder, a wetting agent, a disintegrant, a lubricant, and a flavoring agent commonly used in medicine.

[0021] In one embodiment of the present application, the pharmaceutical excipient comprises an excipient and / or an additional agent.

[0022] In one embodiment of the present application, the pharmaceutical product comprises live bacterial cells, freeze-dried bacterial cells, a fermentation product, or a lysate of the Bifidobacterium breve CCFM1078.

[0023] In one embodiment of the present application, the pharmaceutical product is a microbial preparation of the Bifidobacterium breve CCFM1078.

[0024] In one embodiment of the present application, the dosage form of the pharmaceutical product comprises a liquid preparation, a solid preparation, or a semi-solid preparation.

[0025] In one embodiment of the present application, the solid preparation comprises a tablet, a granule, a powder, or a block.

[0026] In an embodiment of the present application, the liquid preparation comprises an aqueous solution, a suspension, an emulsion.

[0027] [Advantages] 1. The present application screens a strain of Bifidobacterium breve (B. breve) CCFM1078, which has the effect of relieving the symptoms of atopic dermatitis caused by 2,4-dinitrofluorobenzene (DNFB) when intervened in the early life, specifically in: Bifidobacterium breve (1) improving the degree of ear swelling in atopic dermatitis mice; Bifidobacterium breve (2) improving the pathological symptoms of atopic dermatitis mice; (3) improving the degranulation of mast cells in mice and significantly reducing the inflammation of the skin of atopic dermatitis mice; (4) increasing the relative expression of mRNA of skin barrier function-related genes; (5) reducing the total IgE level in the serum of atopic dermatitis mice; (6) reducing the levels of Th2 cell-related cytokines (IL-4, IL-13) in the skin tissue of mice and increasing the levels of Th1 cell-related cytokines (IFN-γ) in the skin tissue of mice, thereby restoring the immune balance between Th1 and Th2 in the skin tissue of atopic dermatitis mice; (7) increasing the level of cytokine IL-10 in the skin tissue of atopic dermatitis mice to maintain immune homeostasis; (8) reducing the level of thymic stromal lymphopoietin (TSLP) in the skin tissue of mice; (9) activating the expression of AHR and CYP1A1 in the skin tissue of atopic dermatitis mice; (10) increasing the secretion of IgA and sIgA in the colon tissue of atopic dermatitis mice; (11) producing high levels of ILA in vitro compared with other representative strains. Therefore, B. breve CCFM1078 has a wide application prospect in the preparation of products for preventing and / or treating atopic dermatitis, such as probiotic food, health products or drugs.

[0028] 2. B. breve is a kind of probiotic bacteria, which has been included in the "List of Bacteria that Can be Used in Food" issued by the Ministry of Health, so the B. breve CCFM1078 screened by the present application does not pose any potential safety hazards to patients with atopic dermatitis. Bifidobacterium breve

[0029] 2. B. breve is a kind of probiotic bacteria, which has been included in the "List of Bacteria that Can be Used in Food" issued by the Ministry of Health, so the B. breve CCFM1078 screened by the present application does not pose any potential safety hazards to patients with atopic dermatitis. Bifidobacterium breve

[0030] ​​​3. The cultivation process of B. breve only needs culture medium and some control of culture conditions, and the cost is relatively low, which will not bring too much economic burden to patients with atopic dermatitis. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 : Comparison of the effects of different groups on reducing the ear thickness of atopic dermatitis mice.

[0032] Figure 2 : Comparison of the effects of different groups on relieving the pathological symptoms (such as epidermal thickness and inflammatory cell infiltration) of atopic dermatitis mice.

[0033] Figure 3 : Comparison of the effects of different groups on reducing the degranulation of mast cells in the skin tissue sections of atopic dermatitis mice; black arrows indicate mast cells.

[0034] Figure 4 : Comparison of the effects of different groups on reducing the number of mast cells in the skin tissue sections of atopic dermatitis mice.

[0035] Figure 5 : Comparison of the effects of different groups on increasing the mRNA expression of skin barrier function-related genes Spink5 in atopic dermatitis mice.

[0036] Figure 6 : Comparison of the effects of different groups on increasing the mRNA expression of skin barrier function-related genes Lor in atopic dermatitis mice.

[0037] Figure 7 : Comparison of the effects of different groups on reducing the total IgE level in the serum of atopic dermatitis mice.

[0038] Figure 8 : Comparison of the effects of different groups on reducing the IL-4 level in the skin tissue of atopic dermatitis mice.

[0039] Figure 9 : Comparison of the effects of different groups on reducing the IL-13 level in the skin tissue of atopic dermatitis mice.

[0040] Figure 10 : Comparison of the effects of different groups on increasing the IL-10 level in the skin tissue of atopic dermatitis mice.

[0041] Figure 11 : Comparison of the effects of different groups on increasing the IFN-γ level in the skin tissue of atopic dermatitis mice.

[0042] Figure 12 : Comparison of the effects of different groups on reducing the TSLP level in the skin tissue of atopic dermatitis mice.

[0043] Figure 13 : Comparison of the effects of different groups on the expression of AHR and CYP1A1 in the skin tissue sections of atopic dermatitis mice.

[0044] Figure 14 : Effects of different groups on the relative expression of mRNA in the skin tissue of atopic dermatitis mice. Ahr

[0045] Figure 15 : Effects of different groups on the relative expression of mRNA in the skin tissue of atopic dermatitis mice. Cyp1a1

[0046] Figure 16 : Comparison of the effects of different groups on the IgA secretion in the colon tissue of atopic dermatitis mice.

[0047] Figure 17 : Comparison of the effects of different groups on the sIgA secretion in the colon tissue of atopic dermatitis mice.

[0048] Figure 18 : Comparison of the content of tryptophan metabolite indole-3-lactic acid (ILA) in the 48h fermentation supernatant of different strains. DETAILED DESCRIPTION

[0049] The application will be further described in combination with the following specific examples.

[0050] The BALB / c mice involved in the following examples were purchased from Zhejiang Weitong Lihua Company; the Bifidobacterium breve (B. breve) CCFM1078 involved in the following examples was isolated by the Biotechnology Center of the Food College of Jiangnan University and has been disclosed in patent CN112111424A, with the preservation number of GDMCC No: 61011; the ELISA kits for detecting IgE, IL-4, IL-13, IL-10, IFN-γ, TSLP, IgA and sIgA in the following examples were all purchased from Lianke Biological. Bifidobacterium breve The application relates to the following culture medium:

[0051] mMRS liquid culture medium: 10 g / L of tryptone, 10 g / L of beef extract, 5 g / L of yeast powder, 20 g / L of glucose, 2 g / L of diammonium hydrogen citrate, 5 g / L of sodium acetate, 2.6 g / L of dipotassium hydrogen phosphate, 0.5 g / L of magnesium sulfate heptahydrate, 0.25 g / L of manganese sulfate monohydrate, 1 mL / L of Tween 80, 0.5 g / L of cysteine hydrochloride, and water to 1000 mL. The mMRS solid culture medium is obtained by adding 1.5% (v / w) agar powder to the above basis liquid culture medium.

[0052] ​​​

[0053] Example 1: Preparation of B. breve CCFM1078 bacterial suspension In all examples, the B. breve CCFM1078 bacterial suspension was prepared as follows: The B. breve CCFM1078 bacterial liquid was streaked on mMRS solid medium and incubated anaerobically at 37°C for 48 h to obtain single colonies. The single colonies were picked and inoculated into mMRS liquid medium and incubated anaerobically at 37°C for 24 h to obtain an activation liquid. The activation liquid was inoculated into mMRS liquid medium at an inoculation amount of 1% (v / v) and incubated anaerobically at 37°C for 24 h to obtain a first-stage seed liquid. The first-stage seed liquid was inoculated into mMRS liquid medium at an inoculation amount of 1% (v / v) and incubated at 37°C for 24 h to obtain a second-stage seed liquid. The second-stage seed liquid was inoculated into mMRS liquid medium at an inoculation amount of 1% (v / v) and incubated at 37°C for 24 h to obtain a bacterial liquid. The bacterial liquid was centrifuged at 6000 g for 15 min to collect the precipitate. The precipitate was washed twice with a physiological saline buffer and then centrifuged at 6000 g for 10 min to obtain bacterial cells. The B. breve bacterial cells were resuspended in physiological saline to a cell concentration of 1×10 9 CFU / mL to obtain the B. breve CCFM1078 bacterial suspension.

[0054] Example 2: Effect of B. breve CCFM1078 on ear thickness of mice with atopic dermatitis The animal experiment was designed as follows: Female BALB / c pregnant mice, 7-8 weeks old, specific pathogen free (SPF). All mice were raised in the SPF barrier facility of the Jiangnan University Animal Experiment Center, at a temperature of 22-26°C, a relative humidity of 40-70%, a 12 h light-dark cycle, and with ad libitum access to sterile water and standard feed. After the pregnant mice gave birth, the female offspring were intervened. From the 7th day after birth, the female offspring were orally administered physiological saline (blank group and DNFB group), B. breve CCFM1078 (1×10 9CFU / day (CCFM1078 group) and ketotifen (Keto) solution (1 mg / kg body weight, drug group) were administered until the end of the experiment. All female pups from the same mother were assigned to the same group. At three weeks of age, the pups were weaned and separated into different cages, and the fur on their backs was shaved to create a model with an area of ​​approximately 2.5 cm long × 1.5 cm wide. A basal solution was prepared using acetone and olive oil in a 4:1 ratio, and then 0.5% and 0.2% DNFB sensitization drugs were prepared using DNFB and the basal solution. On day 1 of modeling, 20 µL of 0.5% DNFB drug was applied to the back skin and right ear of mice in the DNFB group, CCFM1078 group, and drug group to induce sensitization. Then, on days 5, 8, 11, and 14, 20 µL of 0.2% DNFB solution was applied to the back skin and right ear of mice to induce atopic dermatitis; mice in the control group were treated with the same amount of basal solution at the same time. Until the pups were 5 weeks old (day 35), their feces were collected and stored at -80°C, and then they were euthanized. The thickness of their ears was measured and their blood, skin tissue, colon tissue, etc., were collected.

[0055] Appendix Figure 1 The results showed that, compared with the blank group (0.21 mm), the model group (0.58 mm) exhibited significantly increased ear tissue edema, and the CCFM1078 intervention group (0.29 mm) significantly reduced ear thickness after DNFB application (p<0.05), with an effect comparable to that of the drug group in alleviating ear thickness. These experimental results indicate that CCFM1078 intervention effectively alleviated local inflammatory infiltration in the mouse ear, with an effect comparable to that of drug treatment.

[0056] Example 3: Effects of Bifidobacterium breve CCFM1078 on dermatological symptoms in mice with atopic dermatitis See Example 1 and Example 2 for strain culture and animal experiments.

[0057] After euthanizing the mice, skin samples from the hairless area on the back of the mice were taken for histopathological analysis. The histopathological sections of the mouse back skin were stained with hematoxylin and eosin, and then evaluated histopathologically by professional technicians (results are shown below). Figure 2 ).

[0058] Depend on Figure 2As can be seen, the skin structure of the blank group is complete, the epidermis is uniform in thickness and the stratum corneum is clear, and there is no inflammatory infiltration in the subcutaneous tissue. In contrast, the skin tissue of the model group shows obvious pathological changes, manifested as focal epidermal necrosis, karyopyknosis, fragmentation or dissolution, extensive severe thickening of the epidermis, uneven thickness, elongation of dermal papilla reduction; a large number of necrotic cell fragments can be seen in the stratum corneum, accompanied by focal parakeratosis. Compared with the model group, the skin tissue of the CCFM1078 intervention group was significantly improved, the degree of epidermal thickening was reduced, and only moderate mild thickening was observed, with occasional elongation of the dermal papilla, accompanied by a small amount of lymphocyte, macrophage and granulocyte infiltration, and the degree of hyperkeratosis was reduced, and the overall pathological characteristics tended to be mild. And the relief of the drug group is equivalent, both reduce the epidermal thickening and hyperkeratosis. The above experimental results show that B. breve CCFM1078 has the effect of improving the pathological damage of skin tissue in atopic dermatitis mice, and the relief degree is equivalent to that of the drug group, which relieves the symptoms of atopic dermatitis to some extent from the surface level.

[0059] Example 4: Effect of B. breve CCFM1078 on mast cell infiltration in the skin of atopic dermatitis mice Strain culture and animal experiments are shown in Example 1 and Example 2.

[0060] After the mice were sacrificed, the skin of the depilated area on the back of the mice was taken for histopathological analysis. The histopathological sections of the skin of the back of the mice were stained with toluidine blue, and then the histopathological evaluation was performed by professional technicians (results as shown in Figure 3 , Figure 4 ).

[0061] As shown in Figure 3 The distribution of mast cells was observed, and it was found that compared with the blank group, the mast cell infiltration of the model group was significantly increased and the distribution range was wide. As shown in Figure 4 Compared with the blank group (25 / mm 2 ), the number of mast cells in the skin of the back of the model group mice was significantly increased (107 / mm 2 ) (p<0.05), suggesting that mast cells were abnormally activated and infiltrated in large numbers into the skin lesion area under the condition of atopic dermatitis. Compared with the model group, the number of mast cells in the skin of the back of the mice in the CCFM1078 intervention group was significantly reduced (55 / mm 2 ) (p<0.05), and the number of mast cells in the skin of the back of the mice in the drug group was also significantly reduced (34 / mm 2 ) (p<0.05). It is shown that B. breve CCFM1078 can significantly reduce the infiltration of mast cells in atopic dermatitis mice, thereby possibly relieving the inflammatory reaction mediated by mast cells, and can reach a level close to that of atopic dermatitis treatment drugs, and improve the skin barrier function.

[0062] Example 5: Effects of Bifidobacterium breve CCFM1078 on the mRNA expression of skin barrier function-related genes in atopic dermatitis mice See Example 1 and Example 2 for strain culture and animal experiments.

[0063] After euthanizing the mice, a portion of the skin from the bald area on the back of the mice was collected, and total RNA was extracted using a column chromatography method. After reverse transcription into cDNA, quantitative PCR (qPCR) was used to detect the serine protease inhibitor Kazal5, a gene related to skin barrier function. Spink5 ), acetoprotein ( Lor The mRNA expression level of ). The serine protease inhibitor Kazal5 ( Spink5 Decreased gene expression levels lead to increased protease activity in the epidermis, subsequently causing damage and peeling of the stratum corneum structure. Nailin is a major structural protein constituting the keratinized capsule; decreased expression levels of its encoded gene impair the integrity and resilience of the keratinized capsule, resulting in a significant weakening of the epidermal permeability barrier function. Furthermore... Lor Gene expression levels are suppressed by cytokines such as IL-4 and IL-13 in atopic dermatitis.

[0064] The results are as follows Figure 5 , 6 As shown, compared with the control group, DNFB modeling suppressed the expression of the above-mentioned genes and had a significant effect on... Lor Gene expression was significantly suppressed (p<0.05) in the CCFM1078 intervention group. Spink5 and Lor The relative expression level of the mRNA in the control group was significantly increased (p<0.05), which was comparable to the increase in relative mRNA expression level in the drug group (p<0.05). This suggests that Bifidobacterium breve CCFM1078 and the drug may slow down the pathological progression of atopic dermatitis by restoring the integrity of the skin barrier.

[0065] Example 6: Effect of Bifidobacterium breve CCFM1078 on serum total IgE levels in mice with atopic dermatitis See Example 1 and Example 2 for strain culture and animal experiments.

[0066] After euthanizing the mice, serum samples were collected, and the total serum IgE level was measured using an enzyme-linked immunosorbent assay (ELISA) kit (results are shown below). Figure 7 ).

[0067] The occurrence of atopic dermatitis is mainly related to impaired skin barrier function caused by elevated IgE and decreased filaggrin expression; approximately 80% of atopic dermatitis patients exhibit elevated IgE levels. Figure 7As shown, compared with the control group (7.79 μg / mL), the serum IgE level in the DNFB-induced mouse model was significantly increased (15.34 μg / mL) (p<0.05), while after CCFM1078 intervention, the IgE level was significantly decreased (6.98 μg / mL) (p<0.05). After drug intervention, the IgE level also decreased significantly (8.99 μg / mL) (p<0.05), indicating that CCFM1078 intervention was more effective than drug intervention in reducing IgE levels.

[0068] The above results indicate that Bifidobacterium breve CCFM1078 significantly reduced the total IgE level in mouse serum, and the effect was better than that of the drug group, which is beneficial to alleviating the pathological inflammation of atopic dermatitis.

[0069] Example 7: Effects of Bifidobacterium breve CCFM1078 on inflammatory factors in the skin of mice with atopic dermatitis See Example 1 and Example 2 for strain culture and animal experiments.

[0070] After euthanizing the mice, skin tissue samples were collected. The supernatant was obtained after high-throughput grinding, and the levels of cytokines IL-4, IL-13, IL-10, IFN-γ, and thymic stromal lymphopoietin (TSLP) in the skin tissue were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Simultaneously, the total protein content in the skin tissue supernatant was determined using the BCA method. The contents of each indicator were calculated (results are shown in the figure). Figures 8-12 ).

[0071] like Figure 8 As shown, compared with the model group (106.46 pg / mg protein), intervention with Bifidobacterium breve CCFM1078 significantly reduced IL-4 levels in mouse skin tissue (68.46 pg / mg protein) (p<0.05), and was comparable to the blank group (59.53 pg / mg protein). The drug group also achieved a similar effect to CCFM1078, significantly reducing IL-4 levels (76.7 pg / mg protein) (p<0.05), but CCFM1078 was more effective.

[0072] like Figure 9As shown, compared with the control group (80.31 pg / mg protein), the model group significantly increased the IL-13 level in mouse skin tissue (139.27 pg / mg protein) (p<0.05). Compared with the model group, intervention with Bifidobacterium breve CCFM1078 significantly reduced the IL-13 level in mouse skin tissue (92.9 pg / mg protein) (p<0.05), restoring it to a level comparable to the control group. Drug treatment also significantly reduced the IL-13 level (88.46 pg / mg protein) (p<0.05) to a level comparable to the control group. These findings indicate that Bifidobacterium breve CCFM1078 inhibits the Th2 immune response, thereby helping to alleviate the pathological inflammatory state caused by abnormal Th2 immune responses.

[0073] like Figure 10 As shown, compared with the model group (227.95 pg / mg protein), drug treatment significantly upregulated IL-10 expression (395.77 pg / mg protein) (p<0.05), and restored it to a level comparable to the blank group (413.84 pg / mg protein). CCFM1078 intervention upregulated IL-10 levels (305.58 pg / mg protein), but the effect was not statistically significant. This may be achieved by promoting immune tolerance and thus restoring immune homeostasis.

[0074] like Figure 11 As shown, compared with the blank group (6.1 pg / mg protein), the model group significantly reduced the IFN-γ level in mouse skin tissue (1.8 pg / mg protein) (p<0.05). Compared with the model group, intervention with Bifidobacterium breve CCFM1078 significantly increased the IFN-γ level in mouse skin tissue (4.56 pg / mg protein) (p<0.05). Drug treatment also significantly increased the IFN-γ level (4.05 pg / mg protein) (p<0.05), but CCFM1078 was more effective. These findings indicate that Bifidobacterium breve CCFM1078 activates the Th1 immune response and restores the Th1 / Th2 immune balance, thereby helping to alleviate the pathological inflammatory state caused by the Th1 / Th2 immune balance shifting towards a Th2 immune bias.

[0075] like Figure 12 As shown, compared with the model group (48.48 pg / mg protein), drug treatment significantly downregulated the level of epidermal cytokine TSLP (24.89 pg / mg protein) (p<0.05). Similarly, intervention with Bifidobacterium breve CCFM1078 significantly downregulated the level of epidermal cytokine TSLP (24.78 pg / mg protein) (p<0.05), comparable to the control group (31.06 pg / mg protein), thereby alleviating the excessive immune activation induced by epidermal barrier damage and further blocking the amplification effect of upstream pro-inflammatory signals.

[0076] Example 8: Effects of Bifidobacterium breve CCFM1078 on the expression of AHR and CYP1A1 proteins in the skin tissue of mice with atopic dermatitis See Example 1 and Example 2 for strain culture and animal experiments.

[0077] After euthanizing the mice, skin sections from the hairless area on the back of the mice were subjected to immunofluorescence staining to label AHR and CYP1A1 proteins. Separately, total RNA was extracted from a portion of the hairless skin section on the back of the mice using a column chromatography method. After reverse transcription into cDNA, the relative mRNA expression levels of AHR and CYP1A1 were detected using quantitative PCR (qPCR) (results are shown in Figure 1). Figures 13-15 ).

[0078] like Figure 13 The expression levels of AHR and CYP1A1 proteins were comparable in the control group and the model group. Figure 14 , 15 The relative expression level of its mRNA also indirectly confirms this point. Figure 13 In the study, intervention with Bifidobacterium breve CCFM1078 significantly activated [the system]. Ahr and its downstream target genes Cyp1a1 Drug intervention also increased the expression levels of the two proteins mentioned above, but did not achieve the same effect as CCFM1078. Furthermore, as... Figure 14 , 15 As shown, Bifidobacterium breve CCFM1078 significantly improved Ahr and Cyp1a1 The relative expression level of mRNA (p<0.05), which is consistent with... Figure 13 The appearance was consistent. The drug group also improved. Ahr The relative expression level of mRNA was reduced, but not significantly increased, while its expression level was significantly increased. Cyp1a1 The relative mRNA expression level (p<0.05) suggests that it may affect the pathological symptoms of atopic dermatitis through other pathways. This indicates that *Bifidobacterium breve* CCFM1078 may alleviate the pathological symptoms of atopic dermatitis in mice by activating the expression of AHR and its downstream target gene CYP1A1, thereby increasing AHR expression and causing it to bind to the TSLP promoter, thus blocking TSLP expression in keratinocytes, inhibiting the Th2 immune response, and ultimately alleviating the pathological symptoms of atopic dermatitis in mice.

[0079] Example 9: Effects of Bifidobacterium breve CCFM1078 on IgA and sIgA secretion in colonic tissue of atopic dermatitis mice See Example 1 and Example 2 for strain culture and animal experiments.

[0080] After euthanizing the mice, colon tissue samples were taken from some mice, and the secretion levels of IgA and sIgA in the colon tissue were measured using an enzyme-linked immunosorbent assay (ELISA) kit (results are shown in Figure 1). Figures 16-17 ).

[0081] sIgA is the first line of defense in the intestinal mucosal immune barrier, which plays a role in neutralizing pathogenic bacteria, inhibiting the growth of pathogenic bacteria and binding bacteria to the mucus layer, so as to protect the intestinal epithelium from damage caused by intestinal toxins and pathogenic microorganisms. As shown in Table 2, compared with the blank group (1.65 μg / mg protein), the model group (1.16 μg / mg protein) significantly reduced the IgA level in the colon of mice (p<0.05). Compared with the model group, the short bifidobacterium CCFM1078 intervention significantly increased the IgA level in the colon of mice (1.74 μg / mg protein) (p<0.05), and the drug intervention also restored the IgA content to the level comparable to the blank group, which was significantly increased compared with the model group (1.62 μg / mg protein) (p<0.05). Figure 16 In the middle, compared with the blank group (126.98 μg / mg protein), the model group (94.75 μg / mg protein) reduced the sIgA content in the colon of mice. The short bifidobacterium CCFM1078 (174.15 μg / mg protein) and drug (151.86 μg / mg protein) intervention restored the sIgA level, and the sIgA content was increased compared with the blank group, but there was no significant difference. Figure 17

[0082] Example 10: Fermentation of short bifidobacterium CCFM1078 to produce high ILA The specific steps are as follows: The short bifidobacterium CCFM1078, long bifidobacterium infantis CCFM1269 and short bifidobacterium CCFM1025 were inoculated into mMRS solid medium added with 10 mmol / L tryptophan and cultured at 37°C in an anaerobic environment for 48 h. In the liquid culture stage, 5 mL of mMRS liquid medium containing tryptophan (10 mmol / L) was used, and the culture was carried out at 37°C in an anaerobic environment for 24 h. After two passages, the same initial bacterial concentration (1×10 9 CFU / mL) was inoculated into mMRS medium containing tryptophan (10 mmol / L), and cultured for 48 h. After 48 h of fermentation, the detection was carried out. After the culture was completed, it was vortexed for 10 s, centrifuged at 7000 g at 4°C for 10 min to collect 1 mL of supernatant, transferred to a 1.5 mL centrifuge tube, vortexed for 10 s, and then 100 μL was taken, 800 μL of pre-cooled methanol was added, and it was placed in an ice bath for 30 min. 15,000 g, 4°C centrifugation for 10 min, collection of supernatant vacuum concentration (45°C, 2-4 h), with 100 μL of 20% methanol aqueous solution, 15000 g, 4°C centrifugation for 10 min, taking the supernatant for analysis.

[0083] ​The indole-3-lactic acid content in the fermentation supernatant of each of the above-mentioned strains was determined by high-performance liquid chromatography (HPLC). Qualitative analysis was performed using standards, and quantitative analysis was performed using the external standard method. 1 mg of standard was dissolved in 1 mL of 100% methanol, and standard solutions of different concentrations were prepared using 10% methanol aqueous solution. A standard curve was constructed and fitted under the mobile phase conditions to obtain the quantitative analysis results. The chromatographic conditions were as follows: Waters Atlantis T3 column (5 μm, 4.6 × 250 mm), flow rate 1.0 mL / min, excitation wavelength 280 nm, absorption wavelength 320 nm, injection volume 10 μL. The mobile phase consisted of PBS (phase A) at pH 4 and 100% chromatographic grade acetonitrile (phase B), and the column temperature was 35℃. Gradient elution program: 0–3 min, 5% B; 3–9 min, 30% B; 9–12 min, 100% B; 12–13.5 min, 100% B rinse; 13.5–15 min, 5% B. Experimental results are as follows: Figure 18 .

[0084] Representative subspecies of Bifidobacterium longum (CCFM1269) and subspecies of Bifidobacterium breve (CCFM1025) were used as positive controls. Figure 18 As shown, after 48 hours of fermentation, the ILA content of tryptophan metabolite in the supernatant of Bifidobacterium longum subsp. infantis CCFM1269 and Bifidobacterium breve CCFM1025 was 33.1 μg / mL and 34.04 μg / mL, respectively, while the ILA content of tryptophan metabolite in the supernatant of Bifidobacterium breve CCFM1078 after 48 hours of fermentation was 51.31 μg / mL, which was 1.55 times that of CCFM1269 and 1.5 times that of CCFM1025, respectively.

[0085] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Bifidobacterium breve (B. breve) CCFM1078 for use in the preparation of a medicament or food for preventing and / or treating atopic dermatitis. Bifidobacterium breve ) CCFM1078 for use in the preparation of a medicament or food for preventing and / or treating atopic dermatitis.

2. Use according to claim 1, characterized in that, In the aforementioned medicine, the Bifidobacterium breve ( Bifidobacterium breve The bacterial count of CCFM1078 is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

3. Use according to claim 2, characterized in that, The pharmaceutical product contains the Bifidobacterium breve (B. breve) Bifidobacterium breve ) CCFM1078, a pharmaceutical carrier and / or a pharmaceutical excipient.

4. Use according to claim 3, wherein the compound is ###0002### The pharmaceutical carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents commonly used in medicine.

5. The use according to claim 4, wherein the compound is ###0002### The pharmaceutical excipient includes excipients and / or additional agents.

6. Use according to claim 5, characterized in that, The pharmaceutical product includes viable cells, freeze-dried cells, fermentation products, or lysates of Bifidobacterium breve (B. breve) Bifidobacterium breve ) CCFM1078.

7. Use according to claim 6, characterized in that, The pharmaceutical product is a microbial preparation of Bifidobacterium breve (B. breve) Bifidobacterium breve ) CCFM1078.

8. Use according to claim 7, wherein the compound is ###0002### The dosage form of the pharmaceutical product includes a liquid preparation, a solid preparation, or a semi-solid preparation.

9. Use according to claim 8, wherein the compound is ###0002### The solid preparation includes tablets, granules, powders, or blocks.

10. Use according to claim 9, wherein The liquid preparation includes aqueous solutions, suspensions, emulsions.

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