Cutibacterium acnes, its uses, compositions and medicaments

Abstract

We provide the Cutibacterium acnes AD3 strain, which has the accession number GDMCC No. 62625. This strain has a high yield of propionic acid and can be used to treat inflammatory skin diseases and to manufacture skin-improving products.

Description

[Technical Field]

[0001] This application claims priority from a Chinese patent application filed with the State Intellectual Property Office of the People's Republic of China on August 4, 2022, bearing application number 202210935678.3 and entitled "Cutibacterium acnes, its use, composition and medicine," the entire contents of which are incorporated herein by reference.

[0002] The present invention belongs to the field of biomedical technology, and specifically relates to Cutibacterium acnes, its uses, compositions and medicaments. [Background technology]

[0003] Atopic dermatitis (AD) is a common chronic, recurrent, and inflammatory skin disease characterized by intense itching, eczematous dermatitis (characterized by erythema, infiltrates / papules, exudation with crusting, epidermal peeling, and lichenification), and a family history of dry skin and hypersensitivity. It is also associated with an increased risk of multiple comorbidities, including food allergies, asthma, allergic rhinitis, and mental health disorders, severely damaging patients' physical and mental health. The incidence of atopic dermatitis has gradually increased in recent years, affecting people of almost all ages and races. Its relapsing course and intractable skin itch have significant socioeconomic and psychological impacts on patients and their families, making it one of the major causes of the global burden of skin diseases. The cause of atopic dermatitis remains unknown, but research suggests it may be related to skin barrier dysfunction, immune microenvironment bias, skin microbial imbalance, genetics, and environmental factors. Currently, treatment for atopic dermatitis is primarily focused on conventional topical medications (topical corticosteroids, calcineurin inhibitors, antibiotics, topical antipruritics, emollients, etc.), oral medications (antihistamines, systemic corticosteroids, immunosuppressants, etc.), and biologics.

[0004] In most cases, topical corticosteroids (TCS) are the most commonly prescribed medications in clinical practice. However, due to risks associated with skin atrophy, pigmentation disorders, acneiform rash, and systemic absorption (e.g., effects on the hypothalamic-pituitary axis, Cushing's disease), long-term use of TCS is not recommended. Topical calcineurin inhibitors (TCIs) are generally effective and safe when used for short-term treatment; however, due to concerns about skin malignancies and an increased risk of lymphoma, regulatory agencies require that prescribing information for topical tacrolimus and pimecrolimus include a safety warning regarding long-term use. Repeated application of any topical medication over a long period of time or over a large area of the body can also lead to poor patient compliance. Oral immunosuppressants and glucocorticoids are effective in treating AD, but can be associated with serious toxicities and side effects, limiting their use to short-term and / or intermittent therapy. Systemic corticosteroid administration is associated with adverse effects such as diabetes, hypertension, and osteoporosis, and carries the risk of rebound after discontinuation of the medication. Biologic agents such as anti-tumor necrosis factor-α (infliximab and etanercept), anti-IgE (omalizumab), anti-IL-5 (mepolizumab), and anti-CD11a (efalizumab) have often been ineffective in clinical trials. Although the biologic agent anti-IL-4Rα (dupilumab) has demonstrated favorable therapeutic efficacy, approximately one-third of patients still experience poor responses. Small molecule targeted JAK inhibitors, such as upadacitinib and abrocitinib, have also demonstrated favorable therapeutic efficacy, but their action in blocking T-helper cell response signals can have the adverse effect of immunosuppression. Therefore, there is a significant unmet medical need for alternative treatments for AD.

[0005] Similar to the evidence that oral probiotics have a therapeutic effect on atopic dermatitis, there has been some progress in the study of external skin microbiota in atopic dermatitis. For example, animal studies have demonstrated that lysates of the nonpathogenic bacterium Vitreoscilla filiformis can inhibit skin inflammation in mice. Small-scale clinical trials have demonstrated that topical Roseomonas can alleviate symptoms in patients with atopic dermatitis. A phase I randomized, double-blind clinical trial has demonstrated that Staphylococcus aureus has a therapeutic effect on patients with atopic dermatitis. These studies suggest that topical administration of skin microbiota is an important therapeutic strategy for atopic dermatitis.

[0006] Compared with normal individuals, the skin microbiota of patients with atopic dermatitis has a reduced diversity of bacterial species, particularly in the areas of skin lesions, and an increased relative abundance of Staphylococcus aureus and Staphylococcus epidermidis. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide Cutibacterium acnes, uses thereof, compositions and medicines thereof, and the Cutibacterium acnes provided by the present invention has a high propionic acid production rate and is used in the manufacture of skin improvement products which are at least one of treating inflammatory skin diseases, promoting the balance and homeostasis of the normal skin flora, promoting skin barrier function, repairing skin damage or inhibiting skin itching.

[0008] The present invention provides Cutibacterium acnes, which has accession number GDMCC No: 62625 and is named Cutibacterium acnes AD3, and whose Latin name is Cutibacterium acnes AD3.

[0009] The above-mentioned Cutibacterium acnes and at least one of its cellular components, metabolites, derivatives of metabolites, and secreted products are all within the scope of the present invention. The cellular components include at least one of cells, culture media containing the cells, and various chemical components constituting the cells, the metabolites include at least one of intermediate metabolic products and final metabolic products, and the secreted products include at least one of nucleic acids, enzymes, antibodies, exosomes, and hormones.

[0010] The Cutibacterium acnes of the present invention is obtained by extracting from the surrounding area of facial skin lesions of atopic dermatitis patients, screening, culturing, and purifying the extract, and has high propionic acid productivity. In some embodiments of the present invention, the amount of propionic acid produced per 100,000 CFU of Cutibacterium acnes when cultured in an oxygen-free environment for 24 hours is 200 μg to 1,000 μg.

[0011] The Cutibacterium acnes described herein inhibits the expression of inflammatory factors via the metabolite propionic acid, thereby achieving the effect of treating inflammation, for example, inhibiting the expression of inflammatory factors in keratinocytes via the metabolite propionic acid, thereby achieving the effect of treating inflammatory skin diseases. In some embodiments, the present invention provides use of at least one of the Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions in the manufacture of an inhibitor of inflammatory factor expression, wherein the inflammatory factors include at least one of IL-33, IL-1, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-18, IL-25, IL-31, TSLP, IL-17, IL-22, IL-23, TNF-α, and IFN-γ. In some embodiments, the present invention provides use of at least one of Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions in the manufacture of a skin-improvement product for treating an inflammatory skin disease. In one embodiment, the inflammatory skin disease includes at least one of autoimmune skin diseases such as atopic dermatitis, eczema, rosacea, seborrheic dermatitis, contact dermatitis, psoriasis, acne, vitiligo, urticaria, alopecia areata, androgenetic alopecia, systemic lupus erythematosus, scleroderma, and dermatomyositis; bullous skin diseases such as pemphigus and bullous pemphigoid; infectious skin diseases such as fungal, bacterial, and viral infections; and skin tumors including benign skin tumors such as seborrheic keratosis and actinic keratosis, and malignant skin tumors such as basal cell carcinoma and squamous cell carcinoma.

[0012] The Cutibacterium acnes described in the present invention can treat diseases caused by Staphylococcus aureus by inhibiting the growth of Staphylococcus aureus, and can also achieve the effect of stabilizing the balance of the normal skin flora. In some embodiments of the present invention, the present invention provides use of at least one of the Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions in the manufacture of a Staphylococcus aureus inhibitor. In some embodiments of the present invention, the present invention provides use of at least one of the Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions in the manufacture of a skin-improving product that promotes the balance and homeostasis of the normal skin flora.

[0013] The Cutibacterium acnes described herein can promote the maintenance of barrier function by promoting the expression of barrier-associated molecules by keratinocytes. In some embodiments, the present invention provides use of at least one of the Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions in the manufacture of an agent for promoting the expression of skin tissue barrier-associated molecules, wherein the skin tissue barrier-associated molecules include at least one of filaggrin, loricrin, and involucrin. In some embodiments, the present invention provides use of at least one of the Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions in the manufacture of a skin-improvement product that promotes skin barrier function.

[0014] The Cutibacterium acnes described in the present invention can inhibit skin itching by inhibiting the activation of dorsal root ganglion cells, and the inhibition of itching is manifested by inhibiting acute and chronic itching induced by histamine, IL-4, etc., and inhibiting ion influx and action potentials in dorsal root ganglion cells. In some embodiments of the present invention, the present invention provides use of at least one of the Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions in a neuron inhibitor. In some embodiments of the present invention, the present invention provides use of at least one of the Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions in the manufacture of a skin improvement product that inhibits skin itching.

[0015] The Cutibacterium acnes described in the present invention can also repair skin damage. In some embodiments, the present invention provides use of at least one of the Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions in the manufacture of a skin improvement product for repairing skin damage.

[0016] The Cutibacterium acnes is used for manufacturing a skin improvement product which is at least one of treating inflammatory skin diseases, promoting the balance and homeostasis of normal skin flora, promoting skin barrier function, repairing skin damage, or inhibiting skin itching, and specifically, the skin improvement product is a pharmaceutical or skin care product.

[0017] The Cutibacterium acnes concentration, dilution ratio, duration of application, and frequency of application vary depending on the type of disease and the specific condition of the individual. The supernatant (metabolites) obtained by culturing the Cutibacterium acnes for different periods of time, its dilution ratio, topical administration concentration, duration of application, and frequency of application may also vary depending on the specific condition of the individual and the type of inflammatory skin disease. In addition to the aforementioned mechanisms of action—high propionic acid production, inhibition of Staphylococcus aureus growth, promotion of skin barrier function, and inhibition of itching—the Cutibacterium acnes described in the present invention also possesses other potential protective mechanisms, such as the production of specific antibacterial metabolites and modulation of host immunity.

[0018] The present invention provides a composition comprising at least one of Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions, and an excipient. In some embodiments of the present invention, the excipient comprises at least one of a protective agent, a moisturizer, an emollient, an abrasive, a salt, and a topical formulation. In one embodiment, the excipient comprises a thickened topical formulation of a protective agent, a moisturizer, an emollient, an abrasive, a salt, and / or a surfactant.

[0019] The present invention also provides a skin improvement product comprising the above composition and a carrier, wherein the skin improvement product is a pharmaceutical product or a skin care product, and the carrier is a pharmaceutically acceptable carrier or an acceptable carrier for skin care products.

[0020] Here, the pharmaceutically acceptable carrier may be a sugar such as lactose, glucose, or sucrose, a starch such as corn starch or potato starch, a cellulose or derivative thereof such as sodium carboxymethylcellulose, ethyl cellulose, or methyl cellulose, malt, gelatin, vegetable oil, peanut oil, corn oil, cocoa butter, or sesame oil, a polyol such as glycerol, sorbitol, or mannitol, a flavoring agent, a tableting agent, a stabilizer, an isotonic saline solution such as normal saline, or a phosphate buffer solution. The skin care product has moisturizing, repairing, barrier function recovery, anti-aging, whitening, antioxidant, and sun protection functions, and the skin care product is not particularly limited in terms of other ingredients other than the above-mentioned composition. The skin care product may include excipient ingredients having moisturizing, whitening, desensitizing, soothing, or other effects, as well as auxiliary ingredients such as skin texture adjusters, preservatives, and thickeners, and those skilled in the art can select the appropriate ingredients depending on the formulation of the skin care product. In some embodiments of the present invention, the pharmaceutical formulation is a lotion, cream, salve, ointment, spray, powder, medicated oil, thickener, or poultice. In some embodiments of the present invention, the skin care product formulation is an aqueous, oil, or gel.

[0021] The present invention further provides a method for isolating and culturing the above-mentioned Cutibacterium acnes, which comprises the following steps:

[0022] Step 1: Collect skin bacterial flora from around the skin lesions of patients with atopic dermatitis.

[0023] Step 2: Select Cutibacterium acnes from the skin bacterial flora described in step 1 and culture it under anaerobic conditions to obtain purified Cutibacterium acnes.

[0024] Step 3: The purified Cutibacterium acnes described in Step 2 is cultured in broth containing 2% to 10% glycerol to a McFarland turbidity of 0.5 to 1.0, and then cultured until mid-logarithmic growth phase to obtain the Cutibacterium acnes of the present invention.

[0025] In the present invention, first, skin bacterial flora is collected from around the skin lesions of a patient with atopic dermatitis. Specifically, the skin bacterial flora is collected from around the skin lesions on the cheek, armpit, or forearm of the patient with atopic dermatitis, immediately inoculated into a medium, streaked, and cultured at 37°C for 3 to 4 days in an anaerobic environment.

[0026] The present invention involves collecting skin microbiota, selecting Cutibacterium acnes from the skin microbiota, and culturing the bacteria under anaerobic conditions to obtain purified Cutibacterium acnes. Specifically, a single colony morphologically resembling Cutibacterium acnes is randomly selected from the skin microbiota, inoculated into a separate medium, placed in an anaerobic environment, and cultured at 37°C for 3-4 days. Subsequently, a single colony of Cutibacterium acnes is selected by detection, inoculated into a separate medium, placed in an anaerobic environment, and cultured at 37°C for 3-4 days until the third generation, thereby obtaining purified Cutibacterium acnes. In one embodiment, the detection method is matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, 16S rDNA sequencing, or whole genome sequencing.

[0027] In the present invention, purified Cutibacterium acnes is obtained, and then the resulting strain is cultured in a broth containing 2% to 10% glycerol until a McFarland turbidity of 0.5 to 1.0 is reached, followed by growth until the mid-logarithmic growth phase, thereby obtaining the Cutibacterium acnes strain described in the present invention. Specifically, the present invention involves taking a small amount of purified Cutibacterium acnes and culturing it in a broth containing 2% to 10% glycerol until a McFarland turbidity of 0.5 to 1.0 is reached, followed by culturing it in an anaerobic environment at 37°C for 20 to 25 hours until the strain reaches the mid-logarithmic growth phase, thereby obtaining the Cutibacterium acnes strain described in the present invention. The resulting supernatant is then centrifuged and frozen at -80°C to -100°C. In one embodiment, the broth is at least one of Brucella broth and brain heart broth.

[0028] The present invention provides Cutibacterium acnes, its uses, compositions, and pharmaceuticals. The present invention involves isolating and purifying skin microbiota from clinical skin microbiota samples, and identifying the Cutibacterium acnes strain AD3, which exhibits high propionic acid production, through mass spectrometry detection of targeted SCFA metabolism in the Cutibacterium acnes culture supernatant. This strain is derived from the skin microbiota of patients with atopic dermatitis. It has good safety and colonization properties for long-term use. When applied to the skin surface, it continuously secretes metabolic products such as propionic acid, effectively treating inflammatory skin diseases, maintaining the homeostasis of the skin's resident microbiota, and promoting long-term skin health. Skin-improvement products made using this strain are used to treat damaged skin in patients with inflammatory skin diseases. They have safe and effective therapeutic effects, promoting skin barrier function, reducing itching, and maintaining the homeostasis of the skin's resident microbiota, thereby promoting long-term skin health. The experimental results showed that the Cutibacterium acnes provided by the present invention has high propionic acid productivity, and in the supernatant of the strain described in the present invention containing 100,000 CFU after 24 hours of cultivation under anaerobic conditions, the propionic acid content was 291.695 μg / mL, which can significantly inhibit inflammatory factors and exert therapeutic effects on inflammatory skin diseases, promote the expression of barrier-related molecules, inhibit the growth of Staphylococcus aureus, inhibit itching, and also repair skin damage. [Brief explanation of the drawings]

[0029] [Figure 1] 1 is a graph showing the propionic acid content of the supernatant of Cutibacterium acnes strain. [Figure 2] This is a graph showing the effect of topical application of Cutibacterium acnes strain AD3 on mice with AD-like dermatitis. [Figure 3] 1 is a data graph of mouse scratching frequency. [Figure 4] 1 is a comparative chart of mouse serum IgE levels. [Figure 5] FIG. 1 shows the expression of various inflammatory factors in mouse ear tissue cells. [Figure 6]FIG. 1 shows the expression of mouse skin barrier-related molecules. [Figure 7] FIG. 1 is a comparison of transepidermal water loss and stratum corneum hydration in mouse ear skin. [Figure 8] This is a graph showing the effect of topical application of Cutibacterium acnes strain AD3 on mice with psoriasis-like dermatitis. [Figure 9] This is a graph showing the effect of topical application of Cutibacterium acnes strain AD3 on contact dermatitis in mice. [Figure 10] This is a graph showing the effect of topical application of a metabolite of Cutibacterium acnes strain AD3 to mice with AD-like dermatitis. [Figure 11] 1 is a data chart of scratching frequency in mice with AD-like dermatitis. [Figure 12] 1 is a data graph of serum IgE levels in mice with AD-like dermatitis. [Figure 13] 1 is a graph showing the expression of each cytokine in the ear tissue of mice with AD-like dermatitis. [Figure 14] FIG. 10 is a graph showing the gene expression of IL-33 in skin lesions of mice in each group. [Figure 15] 1 is a Western blot detection chart of human primary keratinocytes stimulated with different concentrations of propionic acid. [Figure 16] FIG. 1 shows differential gene expression, mRNA expression, and protein expression of IL-33 in keratinocytes after silencing HDAC2 with siRNA. [Figure 17] FIG. 1 shows differential gene expression, mRNA expression, and protein expression of IL-33 in keratinocytes after silencing HDAC3 with siRNA. [Figure 18] FIG. 16 shows differential gene expression of AhR, AhRR, and CYP1A1 in keratinocytes after propionic acid stimulation. [Figure 19] FIG. 10. Differential gene expression of AhR in keratinocytes after silencing HDAC2 and HDAC3. [Figure 20]FIG. 1 shows the degree of acetylation of AhR promoter sites in keratinocytes after propionic acid stimulation. [Figure 21] Schematic diagram of the intracellular location of AhR in keratinocytes after 6, 12, and 24 hours of propionic acid stimulation. [Figure 22] 1 shows the overall phenotype of AhR knockout mice (AhR KO) after topical application of propionic acid, HE staining of the lesional ear skin of the mice, and IL-33 immunohistochemical staining of the lesional ear skin of the mice. [Figure 23] FIG. 1 shows the effect of propionic acid on acute itching in mice induced by histamine, compound 48 / 80, and chloroquine. [Figure 24] 1 shows the AEW modeling and propionic acid treatment modes and a statistical chart of the number of scratchings in each group of mice during the modeling period. [Figure 25] Photographs of the appearance of skin lesions on the back of mice and HE staining. [Figure 26] 1 is a statistical chart of the epidermal thickness of mice in each group. [Figure 27] This is a distribution map of epidermal nerve fibers labeled with PGP9.5 and DAPI by immunofluorescence staining. [Figure 28] Calcium influx graphs of DRG responses to IL-4, histamine and chloroquine before and after propionic acid treatment. [Figure 29] This is a full-body photograph of the back of an acne mouse. [Figure 30] This is a HE stained image of a section of skin lesion on the back of an acne mouse. [Figure 31] Relative expression levels of each cytokine in skin lesions on the back of acne mice measured by RT-qPCR. [Figure 32] This is a full-body photograph of the back of a mouse with alopecia areata. [Figure 33] FIG. 1 shows HE-stained images of sections of lesioned skin on the back of a mouse with alopecia areata. [Figure 34] This shows the difference in hair growth scores between mice with alopecia areata in each group at different time points in step 1. [Figure 35] Step 2 shows the difference in hair growth scores between mice with alopecia areata in each group at different time points. [Figure 36] This is the ratio of the number of anagen / telogen hair follicles for each group in step 1. [Figure 37] This is the ratio of the number of anagen / telogen hair follicles for each group in step 2. [Figure 38] This is a full-body photograph of the back of a mouse with male pattern baldness. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention discloses Cutibacterium acnes, its use, compositions, and pharmaceuticals. Those skilled in the art can learn from the contents of this specification and realize appropriate improvements to process parameters. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and use of the present invention are described through preferred embodiments. It is clear that those skilled in the art can modify, adapt, and combine the methods and uses described herein to realize and use the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0031] The present invention will be described in more detail below in conjunction with examples.

[0032] Example 1 First, bacterial strains were obtained and cultured. Skin microbiota was swabbed using sterile cotton swabs moistened with saline from the cheeks, cubital fossa, and forearms of 12 healthy control subjects and from the cheeks, cubital fossa, and forearms of 11 patients with atopic dermatitis. The swabs were immediately inoculated onto Columbia blood agar plates (BIO-KONT) and streaked using a disposable sterile inoculation loop. The blood agar plates were immediately placed in an anaerobic bag (GENbag, BioMérieux) or an anaerobic tank (GasPak™, BD), sealed with an anaerobic packet (BioMérieux), and incubated at 37°C for 3–4 days.

[0033] A single colony morphologically resembling Cutibacterium acnes was randomly selected from the blood agar plate and inoculated onto a Columbia blood agar plate. The blood agar plate was immediately placed in an anaerobic bag or tank, added with an anaerobic packet, sealed, and incubated at 37°C for 3–4 days. A single colony was then selected and identified as a Cutibacterium acnes single colony using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (VITEK® MALDI-TOF-MS, BioMérieux). After identification as a single colony of Cutibacterium acnes, the resembling single colony was selected from the blood agar plate and inoculated onto a Columbia blood agar plate. The blood agar plate was immediately placed in an anaerobic bag or tank, added with an anaerobic packet, sealed, and incubated at 37°C for 3–4 days for up to three generations.

[0034] The strains were then purified. A small amount of purified Cutibacterium acnes strain was swabbed from a blood agar plate using a sterile cotton swab and measured for turbidity in Brucella Broth (BD) supplemented with 2% glycerol. The turbidity was measured to 0.5 McFarland turbidity using a turbidity meter (Sensititre, Thermo). For each subject, one Cutibacterium acnes strain was selected from each skin site. 4 mL of the turbid bacterial solution was transferred to a sterile, enzyme-free, 5 mL capped plastic centrifuge tube (Axygen), immediately placed in an anaerobic bag, an anaerobic packet was added, the tube was sealed, and incubated at 37°C for 24 minutes. After 24 hours, the strains were grown to mid-logarithmic growth phase and centrifuged at 2000 rpm for 10 minutes. The culture supernatant was collected, stored in a 2 mL capped sterile plastic centrifuge tube (Axygen), and frozen at -80°C.

[0035] Example 2 Culture supernatants of Cutibacterium acnes isolated from 34 strains of healthy human skin and 32 strains of Cutibacterium acnes isolated from the surrounding skin lesions of patients with atopic dermatitis were selected and used for target short-chain fatty acid (SCFA) metabolic mass spectrometry detection. The detection method was as follows.

[0036] Pure standard solutions of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid were weighed and prepared in diethyl ether into 10 mixed standard gradient concentrations of 0.02 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. All mother liquors and working standards were stored at -20°C. The sample was transferred to a 2 mL centrifuge tube, 50 μL of 15% phosphoric acid was added, and then 10 μL of a 75 μg / mL internal standard solution and 140 μL of diethyl ether were added and vortexed for 1 minute. The internal standard solution was an isocaproic acid solution, and the tube was centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was collected and subjected to on-machine detection (gas chromatograph: Thermo, Trace 1300, mass spectrometer: Thermo, ISQ7000).

[0037] The gas chromatograph conditions were: Agilent HP-INNOWAX chromatography column, 30 m × 0.25 mm ID × 0.25 μm capillary column, split injection, 1 μL injection volume, 10:1 split ratio, inlet temperature 250 °C, ion source temperature 300 °C, transfer line temperature 250 °C, program ramp starting temperature 90 °C, then ramp to 120 °C at 10 °C / min, then ramp to 150 °C at 5 °C / min, and finally ramp to 250 °C at 25 °C / min and hold for 2 minutes, helium gas, carrier gas flow rate 1.0 mL / min, and mass spectrometer conditions were: electron impact ionization (EI) source, SIM scan mode, electron energy 70 eV.

[0038] LC-MS detection was performed for each working standard solution, and a standard curve was constructed by plotting the linear range with the concentration of the working standard on the horizontal axis and the peak area ratio to the internal standard on the vertical axis. All samples were quantitatively analyzed according to the established sample preparation and instrumental analysis methods, and the contents (μg / mL) of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid in all samples were measured.

[0039] The propionic acid content in the culture supernatants of all 66 species of Cutibacterium acnes was analyzed, and the results are shown in Figure 1 and Table 1. Figure 1 shows the propionic acid content in the culture supernatants of Cutibacterium acnes, where NC represents a normal healthy individual and AD represents atopic dermatitis patients. Table 1 shows the propionic acid content in the culture supernatants of all 66 species of Cutibacterium acnes, where NC represents a strain extracted from a normal healthy individual, AD represents a strain extracted from atopic dermatitis patients, FA represents a strain isolated from the face, AM represents a strain isolated from the forearm, and AF represents a strain isolated from the cubital fossa.

[0040] [Table 1-1] [Table 1-2]

[0041] As shown in Figure 1 and Table 1, the supernatant of the Cutibacterium acnes strain with source number AD3-FA, which was collected with a cotton swab from the facial skin flora of a patient with atopic dermatitis, had the highest propionic acid content at 291.695 μg / mL. This indicates that the present invention has identified the Cutibacterium acnes strain AD3, which produces high levels of propionic acid, through screening.

[0042] Example 3 Experiments have demonstrated the inhibitory effect of topical Cutibacterium acnes strain AD3 on AD-like dermatitis, and the protocol for topical Cutibacterium acnes strain AD3 is as follows:

[0043] A mouse model of AD-like dermatitis was established by applying 2 nmol of calcipotriol (MC903) to the ears of mice, and simultaneously infusing 1 × 10 Cutibacterium acnes strain AD3 per day. 5 CFU / mL was applied topically for 14 days.

[0044] After topical application of the AD3 strain, AD-like inflammation in mice was significantly reduced. The results are shown in Figure 2. Figure 2 shows the effect of topical application of Cutibacterium acnes strain AD3 on mice with AD-like dermatitis. Figure 2A is a photograph of the entire mouse, Figure 2B is a schematic diagram of the overall ear thickness of the mouse, Figure 2C is a HE-stained image of a mouse ear section, and Figure 2D is a schematic diagram showing the epidermal thickness of the mouse ear. Figures 2A-D clearly show that AD-like dermatitis in mice treated with Cutibacterium acnes strain AD3 was significantly improved. The number of scratching episodes in the mice was measured, and the results are shown in Figure 3, which is a graph of the scratching frequency data. Serum IgE levels in the mice were measured, and the results are shown in Figure 4, which is a comparison chart of mouse serum IgE levels. Cytokine expression was also measured in mouse ear tissue, and the results are shown in Figure 5, which is a chart of the expression of various inflammatory factors in mouse ear tissue cells. Figures 3 to 5 further demonstrate the effect of topical application of Cutibacterium acnes strain AD3 on alleviating the symptoms of AD-like dermatitis. Furthermore, data on the overall ear thickness, ear epidermal thickness, scratching frequency, serum IgE levels, and expression of inflammatory factors in various cells in mouse ear tissue are shown in Table 2.

[0045] [Table 2]

[0046] Statistical differences between the MC903 group and the MC903+C. acnes group in Table 2: *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001

[0047] As shown in Figures 2-5 and Table 2, topical application of the AD3 strain significantly reduced AD-like inflammation in mice. Overall, the MC903 + C. acnes group showed reduced redness and swelling, reduced scaling, and significantly reduced ear thickness. HE staining revealed a decrease in epidermal thickness and a decrease in inflammatory cells infiltrating the dermis. Furthermore, topical application of the AD3 strain significantly inhibited scratching behavior in mice, inhibited the expression of inflammatory factors IL-33, IL-4, IL-5, IL-6, IL-13, and IL-25, and reduced total IgE levels in peripheral blood.

[0048] Topical application of the AD3 strain promoted the maintenance of skin barrier function, and the results are shown in Figure 6 and Table 3. Figure 6 is a diagram showing the expression of mouse skin barrier-related molecules.

[0049] [Table 3]

[0050] Statistical differences between the MC903 group and the MC903 + C. acnes group in Table 3: *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001

[0051] Figure 6 and Table 3 show that the expression of skin barrier-related molecules in mice with AD-like dermatitis was significantly reduced compared to healthy mice, and that topical application of the AD3 strain promoted an increase in the expression of skin barrier-related molecules.

[0052] The transepidermal water loss (TEWL) and stratum corneum hydration status of the skin at the mouse ear site were tested, and the results are shown in Figure 7 and Table 4. Figure 7 is a comparison chart of the transepidermal water loss (TEWL) and stratum corneum hydration status of the skin at the mouse ear site. The results of Figure 7 and Table 4 further support the ability of topical application of the AD3 strain to promote the maintenance of skin barrier function.

[0053] [Table 4]

[0054] Statistical differences between the MC903 group and the MC903 + C. acnes group in Table 4: *, P value < 0.05; **, P value < 0.01

[0055] In addition, in Figures 2 to 7, NC represents a normal healthy mouse, MC903 represents a mouse with AD-like dermatitis, and MC903+C. acnes represents a mouse with AD-like dermatitis to which the strain AD3 was applied topically.

[0056] In summary, topical application of strain AD3 promoted the expression of barrier-related molecules such as filaggrin, loricrin, and involucrin in mouse skin tissue, promoted stratum corneum hydration, and reduced transepidermal water loss, suggesting that topical application of strain AD3 is involved in regulating skin barrier function.

[0057] Example 4 Experiments have demonstrated that topical application of Cutibacterium acnes strain AD3 has an inhibitory effect on other inflammatory skin diseases, and the treatment protocol for topical application of Cutibacterium acnes strain AD3 is as follows:

[0058] Cutibacterium acnes strain AD3 was applied to the skin of mice with psoriasis-like dermatitis and mice with contact dermatitis at a dose of 1 × 10 per day. 5 The topical dose of 100 CFU / mL was applied daily. Here, psoriasis-like dermatitis was induced in mice by IMQ, and contact dermatitis was induced in mice by QXA.

[0059] The experimental results are shown in Figures 8 and 9 and Table 5. Figure 8 shows the effect of topical application of Cutibacterium acnes strain AD3 on psoriasis-like dermatitis in mice, and Figure 9 shows the effect of topical application of Cutibacterium acnes strain AD3 on contact dermatitis in mice. Figure 8A shows a general photograph of a mouse with psoriasis-like dermatitis, Figure 8B shows a data chart of the overall ear thickness of the mouse with psoriasis-like dermatitis, and Figure 8C shows an HE stained image of an ear section from a mouse with psoriasis-like dermatitis. Figure 9D shows a general photograph of a mouse with contact dermatitis, Figure 9E shows a data chart of the overall ear thickness of the mouse with contact dermatitis, and Figure 9F shows an HE stained image of an ear section from a mouse with contact dermatitis. In Figure 8, NC indicates a healthy mouse, IMQ indicates an IMQ-induced mouse, and IMQ+C. acnes indicates an IMQ-induced mouse to which the Cutibacterium acnes of the present invention was topically applied. In FIG. 9, NC denotes healthy mice, OXA denotes mice induced by OXA, and OXA+c.acnes denotes mice induced by OXA with topical application of Cutibacterium acnes according to the present invention.

[0060] [Table 5]

[0061] Statistical differences in Table 5: *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001

[0062] Figures 8, 9 and Table 5 show that inflammation in mice was significantly reduced after topical application of strain AD3, as manifested by reduced redness and swelling, reduced scaling, and a significant reduction in ear thickness in the group treated with strain AD3, as well as a reduction in the number of inflammatory cells infiltrating into the dermis.

[0063] Example 5 Experiments have demonstrated the inhibitory effect of the metabolites of Cutibacterium acnes strain AD3 on AD-like dermatitis. The preparation and topical application protocol of the metabolites of Cutibacterium acnes strain AD3 is as follows:

[0064] 1×10 5 CFU / mL of the AD3 strain was cultured in complete medium under an anaerobic environment at 37°C for 24 hours, and the culture supernatant was filtered through a 0.22 μm filter to obtain a sterile supernatant, which was a metabolic product of the AD3 strain. The sterile supernatant was diluted 5-fold and then applied topically to the ears of inflamed mice at a dose of 30 μl per ear per day.

[0065] The experimental results are shown in Figures 10 to 13 and Table 6. Figure 10 is a graph showing the effect of topical application of a metabolite of Cutibacterium acnes strain AD3 to mice with AD-like dermatitis. Figure 10A is a photograph of the entire mouse with AD-like dermatitis. Figure 10B is a graph showing the overall ear thickness of the mouse with AD-like dermatitis. Figure 10C is a HE-stained image of an ear section from the mouse with AD-like dermatitis. Figure 10D is a graph showing the thickness of the ear epidermis in the mouse with AD-like dermatitis. Figure 11 is a graph showing the scratching frequency of the mouse with AD-like dermatitis. Figure 12 is a graph showing the serum IgE levels of the mouse with AD-like dermatitis. Figure 13 is a graph showing the expression of various cytokines in the ear tissue of the mouse with AD-like dermatitis.

[0066] [Table 6]

[0067] Statistical differences between the MC903 group and the MC903 + metabolite group in Table 6: *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001

[0068] After topical application of the metabolites of the AD3 strain obtained above, AD-like inflammation in mice was significantly reduced. Overall, the group treated with the metabolites of the AD3 strain showed reduced redness and swelling, reduced scaling, and significantly reduced ear thickness, as well as a reduction in inflammatory cells infiltrating the dermis. Furthermore, topical application of the AD3 strain significantly inhibited scratching behavior in mice, inhibited the expression of inflammatory factors, and reduced total IgE levels in peripheral blood.

[0069] Example 6 The mechanism of action of topical Cutibacterium acnes strain AD3 was investigated, and the mechanism by which strain AD3 inhibits inflammation via its metabolite propionic acid was confirmed.

[0070] (1) As shown in Figure 14, Figure 14 shows the gene expression of IL-33 in the skin lesions of mice in each group. Figure 14A shows the differential gene expression of IL-33 in the skin lesions of mice in each group. Figure 14B shows the mRNA and protein expression of IL-33 in the skin lesions of mice in each group. Figure 14C shows the expression of IL-33 in the mouse epidermal cell line JB6 stimulated with propionic acid. In Figure 14, NC represents normal healthy mice, MC903 represents mice with AD-like dermatitis, MC903+Vehicle represents the vehicle control group of mice with AD-like dermatitis, and MC903+Prop represents mice with AD-like dermatitis treated with topical propionic acid. Figure 14 demonstrates that both in vivo and in vitro experimental results demonstrate that propionic acid inhibits the expression of the inflammation-initiating factor IL-33 in mouse and human primary keratinocytes.

[0071] (2) Short-chain fatty acids such as propionic acid are generally thought to be histone deacetylase (HDAC) inhibitors in the intestine. Further research has shown that propionic acid in the skin may also play a role in inhibiting HDAC. The research results are shown in Figure 15, which shows a Western blot analysis of human primary keratinocytes stimulated with different concentrations of propionic acid. Figure 15D shows the differential expression of the IL-33 gene in human primary keratinocytes stimulated with various concentrations of propionic acid. Figure 15E shows the mRNA and protein expression of IL-33 in human primary keratinocytes stimulated with various concentrations of propionic acid. Figure 15F shows the degree of acetylation in keratinocytes stimulated with propionic acid. The Western blot results in Figure 15 indicate that propionic acid promotes the degree of histone acetylation in keratinocytes.

[0072] Individual HDACs were sequentially silenced in keratinocytes to examine the transport mechanism between HDACs and IL-33. The results are shown in Figures 16 and 17. Figure 16 shows differential gene expression, mRNA expression, and protein expression of IL-33 in keratinocytes after silencing HDAC2 with siRNA. Figure 17 shows differential gene expression, mRNA expression, and protein expression of IL-33 in keratinocytes after silencing HDAC3 with siRNA. In Figures 16 and 17, Ctrl is the control group, SiHDAC2 represents HDAC2 silencing using siRNA, and SiHDAC3 represents HDAC3 silencing using siRNA.

[0073] Figures 16 and 17 show that by sequentially silencing each HDAC in keratinocytes, propionic acid inhibits IL-33 expression by inhibiting HDAC2 and HDAC3 in keratinocytes, but the degree of acetylation of the IL-33 promoter does not increase significantly, suggesting the possibility of the existence of an intermediate factor that transmits signals between HDAC and IL-33.

[0074] (3) Furthermore, by predicting the transcription factors that bind to the promoter site of the human IL-33 gene, we found that the aryl hydrocarbon receptor (AhR) showed significant changes after propionic acid stimulation of keratinocytes.

[0075] Specifically, as shown in Figures 18 to 22, Figure 18 is a diagram of the differential gene expression of AhR, AhRR, and CYP1A1 in keratinocytes after propionic acid stimulation, and Figure 19 is a diagram of the differential gene expression of AhR in keratinocytes after silencing HDAC2 and HDAC3. In Figure 19, J is a diagram of the differential gene expression of AhR in keratinocytes after silencing HDAC2, and K is a diagram of the differential gene expression of AhR in keratinocytes after silencing HDAC3. Figure 20 is a diagram of the acetylation level of the AhR promoter site in keratinocytes after propionic acid stimulation. Figure 21 is a schematic diagram of the intracellular location of AhR in keratinocytes after 6, 12, and 24 hours of propionic acid stimulation. Figure 22 shows the overall phenotype of AhR knockout mice (AhR KO) treated with topical propionic acid, HE staining of mouse ear skin lesions, and IL-33 immunohistochemical staining of mouse ear skin lesions. In Figures 18 to 21, Ctrl represents the control group, Prop represents the propionic acid group (i.e., mice whose keratinocytes were stimulated with propionic acid), and TSA represents the trichostatin A group (i.e., mice whose keratinocytes were stimulated with trichostatin A, a recognized HDAC inhibitor). In Figure 22, NC represents normal healthy mice, MC903 represents mice with AD-like dermatitis, and MC903+Prop represents mice with AD-like dermatitis treated with topical propionic acid.

[0076] As shown in Figures 18-22, RT-qPCR demonstrated that propionic acid stimulation of keratinocytes upregulated AhR expression and activated the AhR signaling pathway. Silencing HDAC2 or HDAC3, respectively, upregulated AhR expression and increased the acetylation level of the AhR promoter. Concurrently, confocal fluorescence imaging revealed that propionic acid promoted the nuclear translocation of AhR. These results suggest that propionic acid upregulated AhR expression by inhibiting HDAC2 / 3, and may also bind to AhR as a ligand, promoting its nuclear translocation and exerting its biological function. Furthermore, we found that silencing AhR in keratinocytes upregulated IL-33 expression. This upregulated IL-33 expression could not be inhibited by propionic acid, and propionic acid was unable to reverse AD-like inflammation in AhR knockout mice. These results indicate that propionate inhibits IL-33 by upregulating AhR expression and signaling.

[0077] Example 7 Propionic acid, a metabolite of Cutibacterium acnes, can inhibit skin itch by inhibiting the activation of dorsal root ganglion cells. It inhibits acute and chronic itch induced by histamine, IL-4, etc., and is achieved by inhibiting ion influx and action potentials in dorsal root ganglion cells. The specific implementation protocol is as follows:

[0078] (1) Effect of propionic acid on acute itch in mice: Mice were randomly assigned to a histamine treatment group and a vehicle control group (5–6 wild-type C57 mice per group). Histamine, Compound 48 / 80, or chloroquine (CQ) were injected intradermally into the cheek of the mice, and the number of scratches elicited within 30 minutes was counted. The treatment group received an intradermal injection of 10 μL of 4 mmol / L propionic acid, while the control group received 10 μL of PBS. 30 minutes later, mice in each group were intradermally administered various acute itch-inducing agents, including 100 μg histamine, 50 μg chloroquine, and 20 μg Compound 48 / 80. Immediately after administration, video recordings were taken to record the scratching behavior of the mice. The results are shown in Figure 23, which shows the effects of propionic acid on acute itching in mice induced by histamine, compound 48 / 80, and chloroquine. Figure 23A shows the effects of histamine, compound 48 / 80, and chloroquine, respectively. Figure 23B shows the effects of histamine, compound 48 / 80, and chloroquine, respectively. Figure 23C shows the effects of chloroquine on acute itching in mice. The data in Figure 23 are expressed as mean ± SEM, and unpaired two-tailed t-tests were used. *: p<0.05; **: p<0.01; ***: p<0.001.

[0079] As can be seen from Figure 23, the acute itching induced by histamine (59.06±4.29 vs. 33.51±4.11), chloroquine (47.80±3.57 vs. 34.62±3.83), and compound 48 / 80 (85.17±5.05 vs. 52.57±4.74) in the propionic acid treatment group was significantly lower than that in the vehicle control group, and the difference was statistically significant (p<0.01).

[0080] (2) Effect of propionic acid on chronic itch in mice: Furthermore, the effect of propionic acid on itch in an acetone-diethyl ether-water (AEW)-induced xerosis model was investigated. The results are shown in Figures 24 to 26. Figure 24 shows the AEW modeling and propionic acid treatment modes, as well as a statistical chart of the number of scratching episodes in each group of mice during the modeling period. Figure 25 shows the appearance and HE-stained photographs of skin lesions on the backs of mice. Figure 26 shows a statistical chart of epidermal thickness in each group of mice.

[0081] As can be seen in Figures 24-26, the AEW model successfully induced scratching behavior in mice, and propionic acid treatment significantly reduced the number of scratches. After modeling, desquamation and scratches were observed at the lesion site, and HE staining revealed acanthosis and hyperkeratosis. Propionic acid treatment significantly improved epidermal thickness (56.95±2.84μm vs 22.78±1.77μm).

[0082] Epidermal nerve fibers were labeled with immunofluorescent staining and observed at high magnification using a confocal microscope. The results showed that the number of nerve fibers growing from the dermal buds to the epidermis increased in the AEW group compared to the control group, but the distribution density of epidermal nerve fibers in the propionic acid-treated group was slightly reduced compared to the vehicle group, as shown in Figure 27, which is a distribution diagram of epidermal nerve fibers labeled with PGP9.5 and DAPI by immunofluorescent staining.

[0083] (3) Inhibition of dorsal root ganglion cell activation by propionic acid Calcium imaging experiments were used to record intracellular calcium transients. IL-4, capsaicin, histamine, menthol, and chloroquine were perfused for 30 seconds, respectively, and changes in calcium ion concentration were recorded. After ECS elution returned to baseline, pretreatment with 100 μmol / L propionic acid was performed. Then, stimulation with the same pruritus was resumed to observe changes in the DRG response rate and peak amplitude of calcium ion concentration. The results are shown in Figure 28, which shows calcium influx graphs of DRG responses to IL-4, histamine, and chloroquine before and after propionic acid treatment. Figure 28A shows calcium influx graphs of DRG responses to IL-4 before and after propionic acid treatment. Figure 28D shows calcium influx graphs of DRG responses to histamine before and after propionic acid treatment. Figure 28E shows calcium influx graphs of DRG responses to chloroquine before and after propionic acid treatment.

[0084] Figure 28 shows that the Th2 inflammatory factor IL-4 can induce calcium influx in some DRGs. After incubation with propionic acid, the rate of DRG response to IL-4 was significantly reduced, as was the amplitude of the calcium peak. However, the control group was incubated with solvent, and there was no significant change in the amplitude of the calcium peak induced by IL-4 before and after the two treatments. In acute itch, both histamine and chloroquine can induce strong calcium ion influx in DRGs. The calcium transient peaks activated by two consecutive administrations were similar. However, when the same DRGs were pre-incubated with propionic acid and then re-stimulated with histamine and chloroquine, the induced calcium influx was significantly inhibited.

[0085] In summary, the AD3 strain inhibits inflammatory factors via the metabolite propionic acid. Specifically, propionic acid exerts its anti-inflammatory effects by inhibiting the inflammation-initiating factor IL-33 in skin keratinocytes. The process by which propionic acid inhibits IL-33 is achieved by inhibiting HDAC2 / 3 and promoting the expression of the aryl hydrocarbon receptor (AhR), where propionic acid binds as a ligand and promotes the nuclear translocation of AhR.

[0086] In addition, strain AD3 inhibits the growth of Staphylococcus aureus and stabilizes the balance of the resident skin flora. When strain AD3 was co-cultured with S. aureus in vitro, the growth of S. aureus was inhibited, and when strain AD3 was applied topically to mouse skin, the number of S. aureus colonized on the skin surface was reduced. Strain AD3 promotes the expression of barrier-related molecules such as filaggrin by keratinocytes, promoting the maintenance of barrier function. Furthermore, propionic acid can inhibit skin itching in mice and inhibit the activation of in vitro-cultured neurons.

[0087] Example 8: Effect of topical application of Cutibacterium acnes strain AD3 on acne mouse model A sample of Cutibacterium acnes in the logarithmic growth phase was collected and the turbidity was measured using a turbidimeter to determine the turbidity of approximately 1 x 10 8 CFU / ml ~ 1 x 10 9 CFU / ml was produced and used in subsequent animal experiments.

[0088] 1. Preparation of artificial sebum mixture: A mixed artificial sebum was prepared with the ratio of squalene 15%, cholesterol 1.2%, liquid paraffin 10%, palmityl palmitate 15%, cholesterol oleate 2.4%, oleic acid 1.4%, stearic acid 2.5%, lauric acid 2.5%, palmitic acid 5%, and triolein 45%, and heated in a water bath at 70°C to promote dissolution. Tween 80 was added according to the ratio of mixed artificial sebum:Tween 80 = 1:3, and the mixture was heated in a water bath at 70°C to promote dissolution.

[0089] 2. Topical application of Cutibacterium acnes AD3 in a mouse model of acne: Balb / c mice (6-8 weeks old) were depilated on the back with a razor and depilatory cream and allowed to rest for 2 days. The total bacterial count in 50 μl was approximately 1 × 10 7 CFU of Cutibacterium acnes ATCC6919 was injected intradermally on the back, followed by application of 30 μl of artificial sebum to the back daily for a total of 7 days.8 50ul of live CFU of AD3 bacteria was applied to the back of the mice, and the positive control group received 50ul of sterile medium applied to the back every day.

[0090] 3. Topical application of strain AD3 reduces inflammation in Kibi mice: The results are shown in Figures 29, 30, and 31. Figure 29 is a photograph of the entire back of an acne mouse, where Vehicle is the positive control group and AD3 live is the group treated with live AD3 bacteria. Red (dashed) circles indicate inflammatory papules. Figure 30 shows HE staining of a section of skin lesions on the back of an acne mouse, where Vehicle is the positive control group and AD3 live is the group treated with live AD3 bacteria. Figure 31 shows the relative expression levels of each cytokine by RT-qPCR in the lesional skin tissues on the back of an acne mouse, where NC is the blank control, Acne is the positive control group, and Acne+AD3 is the group treated with live AD3 bacteria. Seven days after topical application of strain AD3, inflammation of acne on the backs of mice was significantly reduced, as manifested by a smaller volume of inflammatory papules and less erythema and scaling in the back skin in the AD3-treated group (Figure 29). HE staining revealed a decrease in the number of inflammatory cells infiltrating the dermis and thinner skin compared to the positive control group (Figure 30). Furthermore, topical application of strain AD3 significantly inhibited the expression of acne-related inflammatory factors IFN-γ and IL-1β, and inhibited the expression of acne-related inflammatory factor IL-6, without increasing the expression of acne-related inflammatory factor NLRP3 (Figure 31).

[0091] Example 9 Effect of topical application of Cutibacterium acnes strain AD3 on a mouse model of alopecia areata The turbidity of the logarithmic growth phase Cutibacterium acnes solution was measured using a turbidity meter to determine approximately 1 x 10 8 CFU / ml ~ 1 x 10 9 The resulting culture was then prepared to CFU / ml and used in subsequent animal experiments.

[0092] 1. Topical application of propionic acid in a mouse model of alopecia areata: The backs of 6-8 week-old Balb / c mice were depilated using a razor and depilatory cream. The mice's hair was confirmed to be in the telogen phase (pink skin) and measured an area of approximately 3cm x 4cm. On the 9th day after depilation, the normal control group received the same amount of saline, while the other groups received an intraperitoneal injection of cyclophosphamide 3mg / 20g. Treatment began on the 2nd day after depilation. The model group received daily application of 50µl of a 1:7 propylene glycol:isopropyl alcohol mixture. The propionic acid-treated group received 50µl of 1mM sodium propionate (dissolved in a 1:7 propylene glycol:isopropyl alcohol mixture) on the depilated area. The NC group received no treatment, and the positive control group received daily application of 200µl of 3% minoxidil. The mice were then treated for 18 consecutive days, with periodic photographs taken to monitor hair loss and growth.

[0093] 2. Topical application of Propionibacterium acnes AD3 to a mouse model of alopecia areata: The backs of 6-8 week-old C57BL / 6 mice were depilated with a razor and depilatory cream and then allowed to rest for 8 days. On the 9th day, the mice were intraperitoneally injected with 3 mg / 20 g of cyclophosphamide. After 5 days of observation, hair loss was observed on the head or back of the mice, indicating successful modeling of anagen effluvium in mice. Mice with successful modeling were selected for subsequent experiments. Except for the blank group, the intervention groups received 100 μl of 1 × 10 8 CFU AD3 live bacteria were applied to the backs of the mice every day, 100 ul of sterile medium was applied to the backs of the modeling group every day, and 200 ul of 3% minoxidil was applied to the positive control group. The intervention was carried out for 7 consecutive days, and photographs were taken regularly to observe the hair loss and growth status of the mice.

[0094] 3. Topical application of strain AD3 to the skin promotes hair regrowth in mice with alopecia areata: The results are shown in Figures 32 and 33. Figure 32 shows photographs of the entire back of mice with alopecia areata, where the photographs in the first column are photographs after treatment for each group in Step 1, and the photographs in the second column are photographs after treatment for each group in Step 2, where Blank: blank group, Vehicle: modeling group, Propionate: topical propionic acid group, PC: positive control, positive control group, AD3 live: topical AD3 bacteria group. Figure 33 shows HE staining of skin lesion sections on the back of mice with alopecia areata, where the first and second columns are staining results for each group after treatment in Step 1, and the photographs in the second column are staining results for each group after treatment in Step 2, where Blank: blank group, Vehicle: modeling group, Propionate: topical propionic acid group, PC: positive control, positive control group, AD3 live: topical AD3 bacteria group. Nine days after treatment, the hair on the bald areas of the backs of the modeling group was very short and grayish-white. In the blank group, the hair on the bald areas of the backs was longer than in the modeling group, and most of the hair was dense, with a few animals having sparse hair and a dark gray color. In the positive control group, the hair on the bald areas of the backs was dense, jet black, and shiny. In the propionic acid treatment group, the hair on the bald areas of the backs was long, dense, shiny, and dark gray (Figure 32). HE staining revealed a regular number of hair follicles and melanin formation in the skin of the blank group, while in the modeling group, the hair follicles were sparse, with no obvious melanin and in the catagen and telogen phases. Both the positive control group and the AD3 treatment group showed an increase in the number of hair follicles, active proliferation, hair papillae surrounded by hair bulbs, and obvious melanin formation (Figure 33).

[0095] The scoring criteria based on new hair growth at the hair loss site were: pink skin was scored as 0, gray skin as 1, black skin as 2, black skin with a little hair growth as 3, and no difference from other areas as 4. The hair growth of each group of mice was scored at different time points, and the results are shown in Figures 34 and 35. Figure 34 shows the difference in hair growth scores of each group of mice with alopecia areata at different time points in Step 1, and Figure 35 shows each group in Step 2, where Blank: blank group, Vehicle: modeling group, Propionate: propionic acid topical application group, PC: positive control group, and AD3 live: AD3 live topical application group. Figures 34 and 35 show that hair growth in the positive control group grew fastest over time, followed by the blank group, and that the hair growth rate of the propionic acid topical application group was significantly higher than that of the modeling group. In the AD3 live topical application experiment, the hair growth rate in the AD3 live topical application group was significantly higher than that of the blank group and the modeling group, but slightly lower than that of the positive control group.

[0096] The ratio of anagen / telogen hair follicles in each group was observed using HE, and the results are shown in Table 7, Table 8, Figure 36, and Figure 37. Table 7 shows the statistical results of the ratio of anagen / telogen hair follicles in each group in Step 1. Table 8 shows the statistical results of the ratio of anagen / telogen hair follicles in each group in Step 2. Figure 36 shows the ratio of anagen / telogen hair follicles in each group in Step 1. Figure 37 shows the ratio of anagen / telogen hair follicles in each group in Step 2. Here, Blank: blank group, Vehicle: modeling group, Propionate: propionic acid topical application group, AD3 live: AD3 live bacteria topical application group, Positive control: positive control group, A: anagen, anagen, T: telogen, telogen.

[0097] Table 7. Statistical results of the ratio of anagen / telogen hair follicles in each group in Step 1 [Table 7]

[0098] Table 8. Statistical results of the ratio of anagen / telogen hair follicles in each group in Step 2 [Table 8]

[0099] In Tables 7 and 8, A: anagen, anagen; T: telogen, telogen. a: P<0.05 compared with the model group.

[0100] Tables 7, 8, Figures 36 and 37 show that the ratio of anagen / telogen hair follicles in the modeling group was significantly lower than that in the blank group (P<0.05), while the ratio of anagen / telogen hair follicles in the positive control group, AD3 live bacteria topical application group and propionic acid group was significantly higher than that in the modeling group (P<0.01). The difference between the AD3 live bacteria topical application group, propionic acid group and modeling group is between the blank group and the positive control group.

[0101] 4. Topical Application of Cutibacterium acnes AD3 in a Mouse Model of Male Pattern Baldness: C57BL / 6 mice, 6–8 weeks old. Except for the blank group, the backs of the C57BL / 6 mice in the other groups were depilated with a razor and depilatory cream. Testosterone was then injected onto the backs of the mice at 10 mg kg -1 The mice were treated with 100ul of 1x10 ethanol at a dose of 1x10 for 21 consecutive days. After 21 days, the modeling was considered successful when the backs of the mice became smooth and pink and hairless. Successfully modeled mice were selected for subsequent experiments. Except for the blank group, the intervention group received 100ul of 1x10 ethanol at a dose of 1x10 8 CFU AD3 live bacteria were applied to the backs of the mice every day, 100ul of sterile medium was applied to the backs of the modeling group every day, and 200ul of 3% minoxidil paint was applied to the backs of the positive control group every day. The intervention was carried out for 10 consecutive days, and photographs were taken regularly to observe the hair loss and growth of the mice.

[0102] 5. Promotion of hair regrowth in mice with male pattern baldness by topical application of strain AD3 to the skin: The results are shown in Figure 38, which is a photograph of the entire back of a mouse with male pattern baldness, where Blank: blank group, Vehicle: modeling group, AD3 live: AD3 live bacteria topical application group, and PC: positive control, positive control group. As shown in Figure 38, 10 days after treatment, the hair at the hair loss site on the back of the modeling group was very short and grayish-white, the hair at the hair loss site on the back of the blank group was darker gray than that of the modeling group, the hair at the hair loss site on the back of the positive control group was dense, black, and shiny, and the hair at the hair loss site on the back of the AD3 live bacteria topical application group was long, dense, shiny, and dark gray.

[0103] The above are only preferred specific embodiments of the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent replacements or modifications made by those skilled in the art based on the technical solutions and inventive ideas of the present invention within the technical scope disclosed in the present invention will fall within the protection scope of the present invention. [Accession number]

[0104] Biological Deposit Description Biological material: Cutibacterium acnes AD3, category name: Cutibacterium acnes, accession number: GDMCC No: 62625. Deposited on July 15, 2022, to Guangdong Microbial Culture Collection Center, Address: Building 59, 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China, Institute of Microbiology, Guangdong Academy of Sciences.

Claims

1. Cutibacterium acnes AD3, characterized by having the accession number GDMCC No: 62625.

2. The Cutibacterium acnes according to claim 1, wherein the amount of propionic acid produced per 100,000 CFU of the Cutibacterium acnes when cultured in an anaerobic environment for 24 hours is 200 μg to 1,000 μg.

3. Use of at least one of Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions according to claim 1 or 2 in the manufacture of at least one of an inhibitor of inflammatory factor expression, an inhibitor of Staphylococcus aureus, an agent for promoting the expression of skin tissue barrier-related molecules, and an inhibitor of nerve cells.

4. The inflammatory factor includes at least one of IL-33, IL-1, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-18, IL-25, IL-31, TSLP, IL-17, IL-22, IL-23, TNF-α, and IFN-γ, The use according to claim 3, characterized in that the skin tissue barrier-associated molecule comprises at least one of filaggrin, loricrin, and involucrin.

5. Use of at least one of Cutibacterium acnes, its cellular components, its metabolites, derivatives of its metabolites, and its secretions according to claim 1 or 2 in the manufacture of a skin improvement product which is at least one of treating inflammatory skin diseases, promoting the balance and homeostasis of the normal skin flora, promoting skin barrier function, repairing skin damage, and inhibiting skin itching.

6. The use according to claim 5, characterized in that the skin improvement product is a medicine or a skin care product.

7. 6. The use according to claim 5, wherein the inflammatory skin disease comprises at least one of atopic dermatitis, eczema, rosacea, seborrheic dermatitis, contact dermatitis, psoriasis, acne, vitiligo, hives, alopecia areata, male pattern baldness, systemic lupus erythematosus, scleroderma, dermatomyositis, pemphigus, bullous pemphigoid, fungal infectious skin diseases, bacterial infectious skin diseases, viral infectious skin diseases, seborrheic keratosis, actinic keratosis, basal cell carcinoma, and squamous cell carcinoma.

8. A composition comprising at least one of the Cutibacterium acnes strains according to claim 1 or 2, their cellular components, their metabolic products, derivatives of their metabolic products, and their secretions, and an excipient.

9. 9. The composition of claim 8, wherein the excipient comprises at least one of a protective agent, a humectant, an emollient, an abrasive, a salt, and a topical preparation.

10. A skin improvement product comprising the composition of claim 8 and a carrier.

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