Staphylococcus epidermidis H62-3 metabolite gel preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a staphylococcus epidermidis H62-3 metabolite gel preparation as well as a preparation method and application thereof. The staphylococcus epidermidis H62-3 metabolite gel preparation improves skin erythema and scale symptoms of a psoriasis mouse model, remarkably reduces the skin lesion thickness, can reduce the mRNA expression level of inflammatory factors interleukin-17A, interleukin-17F and interleukin-23 at the skin lesion of the psoriasis mouse model, and can be used for treating psoriasis. The compound has a prospect of being developed into drugs for treating inflammatory dermatosis. The staphylococcus epidermidis H62-3 metabolite gel preparation disclosed by the invention is used for improving a psoriasis mouse model, and a new thought for safely and effectively treating inflammatory skin diseases by external use of viable bacteria metabolite is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a gel preparation of Staphylococcus epidermidis H62-3 metabolites and a preparation method and application thereof. Background Art

[0002] Inflammatory skin diseases are a group of diseases caused by the destruction of skin tissue due to immune system disorders, including eczema, atopic dermatitis, acne, psoriasis, chronic urticaria, vitiligo, pemphigus, pemphigoid, lupus erythematosus, etc. These diseases may have been classified into different categories in the past due to their different clinical manifestations. Now people realize that these diseases have a common feature - inflammation, including inflammation mediated by T cells and inflammation mediated by antigen-antibody. By summarizing the above diseases, the following common characteristics can be drawn: (1) It is a chronic, recurrent, inflammatory, systemic disease induced by the combined effects of genetics and the environment, mediated by immunity; (2) There are many influencing factors, complex etiology, and diverse clinical manifestations; (3) It is limited or widely distributed and non-infectious; (4) It is difficult to treat and often suffers from it for life; (5) The course of the disease is long.

[0003] The skin is the largest organ in the human body. As one of the four major bacterial reservoirs, the skin surface is colonized by hundreds of millions of microorganisms, called the skin microbiome, which can regulate the host innate and adaptive immunity and help the host resist the invasion of external pathogens. With the advancement of next-generation sequencing technologies, including shotgun metagenomic sequencing and amplicon studies based on phylogenetic marker genes (such as bacterial 16S or fungal internal transcribed spacer sequencing), human microbiome research has undergone a revolutionary change to reveal the possible interactions between the human microbiome and microorganisms. The skin microbiome plays an important role in promoting defense and immune responses, inhibiting the colonization and infection of opportunistic or pathogenic microorganisms, and promoting tissue repair and barrier function. All these functions of the skin microbiome are achieved through their interactions and through mutualistic or symbiotic interactions with mammalian host cells. Dysbiosis of the skin microbiome is associated with altered immune responses and promotes the development of skin diseases such as atopic dermatitis, psoriasis, acne, and even skin cancer.

[0004] Studies have found that psoriasis patients have severe skin microbiome imbalance, and psoriasis-related flora show higher diversity and heterogeneity than healthy skin bacterial communities. Specific microbial characteristics are associated with psoriasis lesional skin, psoriasis non-lesional skin and healthy skin. Staphylococcus aureus and Proteus are more abundant in non-lesional and lesional skin of psoriasis. In contrast, the incidence of Staphylococcus epidermidis and Propionibacterium acnes in psoriasis lesions is lower than that in healthy skin. Based on the close correlation between skin microbiota and inflammatory skin diseases such as psoriasis, the present invention studies the relationship between skin microbiota and its metabolites and inflammatory skin diseases such as psoriasis, screens out metabolites with disease treatment potential, and prepares them into gels, ointments or creams with the addition of specific metabolites to antagonize the growth of other harmful bacteria, or interact with the immune system to enhance its ability to repair skin lesions and maintain skin health. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a gel preparation of Staphylococcus epidermidis H62-3 metabolites and a preparation method and application thereof.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions: In one aspect, the present invention provides a gel preparation containing metabolites of Staphylococcus epidermidis, characterized in that the gel preparation contains Staphylococcus epidermidis ( Staphylococcus epidermidis ) H62-3 strain metabolites, the Staphylococcus epidermidis H62-3 strain has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, the deposit date is February 21, 2023, and the deposit number is CGMCC No. 26640.

[0007] In some embodiments of the present invention, the gel preparation comprises the following ingredients, measured by mass percentage: 10-40% metabolites of Staphylococcus epidermidis H62-3, 0.2-1.5% carbomer 940, 0.2-1.5% triethanolamine, 0.05-0.5% low molecular weight sodium hyaluronate, 5-20% glycerol, 0.5-2.5% azone, and the balance is pure water.

[0008] In a preferred embodiment, the gel preparation comprises the following ingredients, measured in percentage by mass: 20% of Staphylococcus epidermidis H62-3 metabolites, 0.79% of carbomer 940, 0.87% of triethanolamine, 0.12% of low molecular weight sodium hyaluronate, 12% of glycerol, 1% of azone, and the balance is pure water.

[0009] In another aspect, the present invention provides a method for preparing a gel preparation, the method comprising the following steps: (1) Fully mixing low molecular weight sodium hyaluronate and glycerol to obtain a compound moisturizer; (2) The metabolites of Staphylococcus epidermidis H62-3 are fully mixed with an appropriate amount of a compound moisturizer and pure water, azone is added, and the mixture is mixed again to obtain a drug solution; (3) Add Carbomer 940 to the remaining compound moisturizer, then add the remaining pure water, stir thoroughly to allow Carbomer 940 to swell and disperse, and then stir evenly; (4) Add the solution obtained in step (3) to the drug solution obtained in step (2) and stir evenly; then add triethanolamine and continue stirring until it becomes a gel, thereby obtaining the gel preparation.

[0010] In some embodiments of the present invention, the preparation method of the metabolite of Staphylococcus epidermidis H62-3 is as follows: a single colony of the activated Staphylococcus epidermidis strain H62-3 is picked and inoculated into a TSB liquid culture medium, placed in a 32-38°C shaker, and shaken and cultured for 14-18 hours to serve as a seed solution; the seed solution is inoculated into a TSB liquid culture medium at an inoculation rate of 3%-5% by volume, and shaken and cultured in a 32-38°C shaker for 14-18 hours to obtain a fermentation solution; the fermentation solution is centrifuged and the bacterial precipitate is discarded; the supernatant of the fermentation solution is concentrated by rotary evaporation to a volume of 5 times, which is the metabolite of Staphylococcus epidermidis H62-3.

[0011] In some embodiments of the present invention, the swelling and dispersion time of Carbomer 940 in step (3) is 30 minutes.

[0012] In another aspect, the present invention provides use of the gel preparation in preparing drugs for inflammatory skin diseases.

[0013] In some embodiments of the present invention, the inflammatory skin disease is eczema, atopic dermatitis, acne, psoriasis, chronic urticaria, vitiligo, pemphigus, pemphigoid or lupus erythematosus, etc.

[0014] In some embodiments of the present invention, the psoriasis is at least one of psoriasis vulgaris, erythrodermic psoriasis, articular psoriasis or pustular psoriasis.

[0015] In some embodiments of the present invention, the gel preparation is used to improve the symptoms of skin erythema and scaling in a psoriasis mouse model; the gel preparation is used to reduce the thickness of skin lesions and reduce inflammatory factors in the skin lesions of a psoriasis mouse model.

[0016] In some embodiments of the present invention, the inflammatory factors are the mRNA expression level of interleukin-17A, the mRNA expression level of interleukin-17F, and the mRNA expression level of interleukin-23.

[0017] The beneficial effects of the present invention are: 1. Gas chromatography (GC) and liquid chromatography (LC) tandem mass spectrometry (MS) analysis revealed that the metabolites of Staphylococcus epidermidis H62-3 are rich in variety, containing more than 7,000 metabolites, and have certain differences in metabolite composition from the model strain of Staphylococcus epidermidis CGMCC 1.4260.

[0018] 2. Experiments have shown that the gel preparation of Staphylococcus epidermidis H62-3 metabolites can improve skin erythema, scaling and other symptoms in psoriasis mouse models, significantly reduce the thickness of skin lesions, and reduce the mRNA expression levels of inflammatory factors such as interleukin-17A, interleukin-17F, and interleukin-23 in the skin lesions of psoriasis mouse models. It has the prospect of being developed into a drug for the treatment of inflammatory skin diseases.

[0019] 3. Experiments have shown that the gel preparation of Staphylococcus epidermidis H62-3 metabolites can improve the psoriasis mouse model, providing a new idea for the safe and effective treatment of inflammatory skin diseases with topical live bacterial metabolites.

[0020] 4. Experiments have shown that Staphylococcus epidermidis H62-3 grows rapidly in TSB liquid culture medium, enters the logarithmic phase in about 2 hours, and enters the stable phase in about 12 hours, which is convenient for large-scale production and application in the later stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 This is the colony morphology of Staphylococcus epidermidis H62-3; Figure 2 This is the Gram stain of Staphylococcus epidermidis H62-3; Figure 3 is the growth curve of Staphylococcus epidermidis H62-3; Figure 4 The sequence alignment results and ANI analysis of Staphylococcus epidermidis H62-3; Figure 5 It is composed of the metabolites of Staphylococcus epidermidis H62-3; Figure 6 To study the effect of a gel preparation of Staphylococcus epidermidis H62-3 metabolites on imiquimod-induced psoriasis in C57BL / 6 mice. Figure 7 To study the effect of the gel preparation of Staphylococcus epidermidis H62-3 metabolites on the PASI score of psoriasis lesions induced by imiquimod in C57BL / 6 mice; Figure 8To study the effect of the metabolite gel preparation of Staphylococcus epidermidis H62-3 on the psoriasis-like skin pathology induced by imiquimod in C57BL / 6 mice; Fig. 9 To study the effect of the metabolite gel preparation of Staphylococcus epidermidis H62-3 on the thickness of the stratum spinosum of psoriasis lesions induced by imiquimod in C57BL / 6 mice. Fig.10 The effect of the gel preparation of Staphylococcus epidermidis H62-3 metabolites on the spleen index of C57BL / 6 mice induced by imiquimod; Fig.11 To study the effect of the metabolite gel preparation of Staphylococcus epidermidis H62-3 on the transcriptional levels of inflammatory factors in psoriasis lesions of C57BL / 6 mice induced by imiquimod; Fig.12 To investigate the effect of a gel preparation of Staphylococcus epidermidis H62-3 metabolites on the Ki67 protein level in imiquimod-induced psoriasis lesions in C57BL / 6 mice. DETAILED DESCRIPTION

[0022] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0023] The present invention provides a Staphylococcus epidermidis metabolite gel preparation for use in a drug for treating inflammatory skin diseases. The present invention is further illustrated below by way of examples, but the present invention is not limited to the scope of the examples. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The experimental methods in the following examples that do not specify specific conditions are operated according to conventional methods and conditions, or according to the product instructions.

[0024] The bacterial culture medium used in the following examples is as follows: Tryptone soy broth liquid culture medium (TSB): tryptone 17.0 g / L, soy peptone 3.0 g / L, sodium chloride 5.0 g / L, potassium dihydrogen phosphate 2.5 g / L, glucose 2.5 g / L, autoclave at 121°C for 15 min and set aside.

[0025] Tryptone soy broth solid medium (TSA): tryptone 17.0 g / L, soy peptone 3.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, agar powder 15 g / L, autoclave at 121°C for 15 min, pour into plates and set aside.

[0026] Example 1: Isolation, screening and purification of Staphylococcus epidermidis H62-3, and microbiological identification (1) Sample collection ① Volunteers of different ages (18-90 years old) with healthy facial skin were recruited. They were required to have no acne, pustules, inflammation, erythema, etc., and had not taken oral antibiotics in the past 2 months or used antibiotics internally or externally in the past 2 weeks. The face had not been cleaned or washed within 12 hours of sampling.

[0027] ② The sampler wears a mask and disposable sterile gloves. Soak a sterile sampling cotton swab with sterile saline. The cotton swab is 2 cm long, and the sampling area is 4 cm long. Wipe back and forth 50 times for about 30 seconds, covering an area of ​​about 8 cm. 2 After wiping, place the cotton swab in a glycerin storage tube, seal it tightly with the lid, and record the number.

[0028] (2) Cultivation, isolation, purification and preservation of skin flora ① Take 0.05 mL of glycerol preservative solution and add 0.15 mL of sterile water to dilute it. Spread the dilution on a TSA solid plate and place it in a 37°C constant temperature incubator for inversion culture for 24 hours.

[0029] ② According to the colony characteristics of Staphylococcus epidermidis, single colonies were picked and separated by four-zone lines, and placed in an inverted incubator at 37°C for 24 hours.

[0030] ③ Perform 16S rRNA sequencing and confirm that the single colony is Staphylococcus epidermidis and store it in glycerol at -80℃.

[0031] (3) Screening of functional bacteria The whole genome RNA of bacteria from different strains of Staphylococcus epidermidis was extracted and reverse transcribed into cDNA. qRT-PCR experiments were performed to screen out the Staphylococcus epidermidis strain H62-3 that highly expressed genes closely related to moisturizing efficacy.

[0032] (4) Colony characteristics ① The colony morphology of the strain on TSA plate is as follows Figure 1 As shown, the colonies are white and small, forming round protrusions with a smooth and opaque surface and no hemolysis zone.

[0033] ② Gram stain positive, such as Figure 2As shown in the figure, the bacteria are spherical, some are arranged in grape-like shapes, some are arranged singly, without flagella, and do not produce spores.

[0034] (5) Cultivation characteristics ① The optimal growth temperature of this strain is 32-38℃, and the optimal pH is 5.0-8.0.

[0035] ② Pick a single colony of the activated strain and inoculate it into TSB liquid culture medium, place it in a 32-38℃ shaker, and shake and culture it for 14-18h as seed liquid; inoculate the seed liquid into TSB liquid culture medium with an inoculum volume fraction of 3%-5%, shake and culture it in a 37℃ shaker for 24h, measure the OD value of the culture solution at 600nm every 1h, and draw the growth curve of the strain with OD600 as the vertical axis and time as the horizontal axis, as shown in the figure. Figure 3 As shown, it can be found that Staphylococcus epidermidis H62-3 grows rapidly in TSB liquid culture medium, enters the logarithmic phase in about 2 hours, and enters the stable phase in about 12 hours.

[0036] (6) Genetic characteristics The genomic DNA of strain H62-3 was extracted, and the sample was sequenced using the Illumina Hiseq 2000 sequencing platform to obtain second-generation data, and PacBio sequencing was used to obtain third-generation data. After quality control, Denovo assembly was performed, and gene prediction was performed after splicing and correction into a complete genome. The predicted gene protein was aligned to various databases such as the NR library to obtain the species information of the strain. As shown in Table 1, it can be determined that strain H62-3 is Staphylococcus epidermidis.

[0037] Table 1: Sequence alignment results of Staphylococcus epidermidis H62-3

[0038] Based on the above-mentioned characteristics of colony, culture, genetics, etc., strain H62-3 was determined to be Staphylococcus epidermidis. Since the ANI of Staphylococcus epidermidis H62-3 and the model strain of Staphylococcus epidermidis CGMCC 1.4260 is 97.1%, Figure 4 As shown, it can be determined that the two are not the same strain. Single nucleotide diversity (SNP) and insertion and deletion (InDel) analysis found that many bases of Staphylococcus epidermidis H62-3 mutated, increased or deleted, resulting in changes in the protein and affecting its biological function. The strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) on February 21, 2023, and its deposit number is CGMCC No. 26640.

[0039] Example 2: Composition Analysis of Metabolites of Staphylococcus epidermidis H62-3 The fermentation liquid of Staphylococcus epidermidis H62-3 was centrifuged and the bacterial precipitate was discarded; the supernatant of the fermentation liquid was concentrated by 5 times of volume by rotary evaporation, which was the metabolite of Staphylococcus epidermidis H62-3. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used for detection.

[0040] Liquid chromatography-mass spectrometry (LC-MS) detection includes: sample pretreatment, metabolite extraction, LC-MS full scan detection, data preprocessing and statistical analysis. Based on non-targeted metabolomics of ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry, combined with the metabolomics data processing software Progenesis QI v2.3, the raw data are qualitatively and relatively quantitatively analyzed, and the raw data are standardized and preprocessed. Data analysis includes multivariate statistical analysis (PCA, PLS-DA, OPLS-DA) and univariate statistical analysis (T test, fold difference), screening of metabolites, and enrichment of metabolic pathways. The results of liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are more than 7,800 metabolites of Staphylococcus epidermidis H62-3, including lipids and lipid-like molecules, organic heterocyclic compounds, organic acids and their derivatives, benzene compounds, organic oxygen compounds, phenylpropanoids and polyketide compounds, nucleosides, nucleotides and their analogs, organic nitrogen compounds, etc. Figure 5 shown.

[0041] Gas chromatography-mass spectrometry (GC-MS) detection includes: sample pretreatment, metabolite extraction, metabolite derivatization, GC-MS detection, data preprocessing and statistical analysis, etc. Non-targeted metabolomics based on quadrupole mass spectrometry, combined with the metabolomics data qualitative software MS-DIAL, performs qualitative and relative quantitative analysis on the raw data, and performs standardized preprocessing on the raw data. Data analysis includes multivariate statistical analysis (PCA, PLS-DA, OPLS-DA) and univariate statistical analysis (T test, multiple difference), screening metabolites, and enrichment of metabolic pathways. The results of gas chromatography-mass spectrometry (GC-MS) analysis showed that there are more than 600 metabolites of Staphylococcus epidermidis H62-3, including lipids and lipid-like molecules, organic heterocyclic compounds, organic acids and their derivatives, benzene compounds, organic oxygen compounds, phenylpropanoids and polyketide compounds, nucleosides, nucleotides and their analogs, organic nitrogen compounds, etc. Figure 6 shown.

[0042] Example 3: Experiment on the improvement of imiquimod-induced psoriasis skin lesions in C57BL / 6 mice by the metabolite gel preparation of Staphylococcus epidermidis H62-3 Feeding environment: SPF grade; mice: C57BL / 6 female mice, 7 weeks old; modeling method: After the purchased 7-week-old C57BL / 6 female mice of uniform size were stably fed for one week, 2cm×2cm hair removal was performed on the back and they were randomly divided into 4 groups, 5 mice in each group. 62.5mg imiquimod was evenly applied to the back of each mouse every morning for 6 consecutive days. Four treatment methods were set up in the experiment, and 5 biological replicates were set for each treatment method. The drug administration was unified on the day of modeling, local administration, modeling was done every morning, and the drug was administered 12h later. The treatment method is as follows: ① Treatment 1 (control), normal control mice, no treatment; ② Treatment 2 (imiquimod): 62.5 mg of imiquimod was evenly applied to the back of each mouse every morning, and 12 hours later, a blank matrix of the topical gel preparation was given for 6 consecutive days, which was the psoriasis model group; ③ Treatment 3 (calcipotriol): 62.5 mg of imiquimod was evenly applied to the back of each mouse every morning, and 12 h later, 12 mg / 4 cm2 of calcipotriol ointment was applied topically. 2 , for 6 consecutive days, which was the positive control group; ④ Treatment 4 (H62-3 metabolite): 62.5 mg of imiquimod was evenly applied to the back of each mouse every morning, and 12 h later, a topical gel preparation of Staphylococcus epidermidis H62-3 metabolite 12 mg / 4 cm 2 , for 6 consecutive days, which was the experimental group.

[0043] The mice in each group were photographed every other day and the PASI score of psoriasis lesions was calculated. The mice were killed on the 7th day, and the back lesions were taken for HE staining analysis of the lesions and pathology. Figure 7 As shown in the figure, compared with normal control mice, imiquimod induced typical psoriasis lesions, which were manifested as thickening of the skin on the back of mice, erythema and scaling. Compared with psoriasis model mice, the topical Staphylococcus epidermidis H62-3 metabolite gel group improved erythema, scaling and skin thickening, and the efficacy was comparable to that of the calcipotriol positive control group. However, the mice in the calcipotriol positive control group generally lost weight and had obvious side effects. Therefore, the topical Staphylococcus epidermidis H62-3 metabolite gel group may become a new safe and low-toxic therapy.

[0044] Example 4: Experiment on the improvement of PASI score of psoriasis lesions induced by imiquimod in C57BL / 6 mice by the metabolite gel preparation of Staphylococcus epidermidis H62-3 Mice were killed according to Example 3, and the erythema, scaling and thickening of the back skin lesions were scored. The severity of the back skin lesions, including skin erythema, scaling and infiltration thickness, was evaluated according to the Psoriasis Area and Severity Index (PASI) scoring system. Each parameter was independently scored on a scale of 0 to 4, where 0 = no symptoms, 1 = mild symptoms, 2 = moderate symptoms, 3 = obvious symptoms, and 4 = very obvious symptoms, as shown in Table 2. The cumulative score represents the statistical results of the severity of the disease. Compared with normal control mice, after continuous application of imiquimod for 6 days, obvious erythema, scaling and thickening of the back lesions appeared in the mice. The calcipotriol positive control group and the Staphylococcus epidermidis H62-3 metabolite gel preparation group significantly alleviated the back erythema, scaling and skin thickening induced by imiquimod, as shown in Table 2. Figure 8 shown.

[0045] Table 2: Scoring details of mouse psoriasis-like dermatitis severity (PASI)

[0046] Example 5: Experiment on the improvement of imiquimod-induced psoriasis skin lesions in C57BL / 6 mice by the metabolite gel preparation of Staphylococcus epidermidis H62-3 The back skin lesions of the mice killed in Example 3 were stained with HE. The typical pathological results are as follows: Fig. 9 Compared with normal control mice, after continuous application of imiquimod for 6 days, the mice in the modeling group showed phenotypes such as epidermal thickening, inflammatory cell infiltration, hyperkeratosis, incomplete keratosis, reduced granular layer, and elongated epidermal protrusions. The calcipotriol positive control group and the Staphylococcus epidermidis H62-3 metabolite gel preparation group can effectively alleviate the phenotypes of epidermal thickening, inflammatory cell infiltration, hyperkeratosis, incomplete keratosis, reduced granular layer, and elongated epidermal protrusions induced by imiquimod. Fig. 9 shown.

[0047] Example 6: Experiment on the improvement of stratum spinosum thickness of psoriasis lesions induced by imiquimod in C57BL / 6 mice by the metabolite gel preparation of Staphylococcus epidermidis H62-3 The back skin lesions of the mice killed in Example 3 were taken for HE staining, observed under a microscope, scanned and photographed, and histopathological analysis was performed, and the acanthosis and dermal inflammatory cell infiltration indicators were quantified. The quantification method of acanthosis is to select the visual field of the scanned image under the microscope after HE staining, fix the magnification (×20), and use AdobePhotoshop 2023 software to select all epidermal areas in the visual field in the visual field to obtain the pixel area of ​​the epidermal area, and the degree of acanthosis = epidermal pixels / total pixels in the visual field. The quantification method of dermal inflammatory cell infiltration is to select the visual field of the scanned image under the microscope after HE staining, fix the magnification (×20), and use Adobe Photoshop 2023 software in the visual field to randomly select 5 non-overlapping square areas (the side length is fixed to 1 unit) in the dermis area, and use the counting tool to calculate the number of inflammatory cells in each area, then the dermal inflammatory cell infiltration = average unit area inflammatory cell count. Compared with normal control mice, after continuous application of imiquimod for 6 days, the model group mice had acanthosis and dermal inflammatory cell infiltration. The calcipotriol positive control group and the Staphylococcus epidermidis H62-3 metabolite gel preparation group could effectively alleviate the acanthosis and dermal inflammatory cell infiltration induced by imiquimod in mice. Fig.10 shown.

[0048] Example 7: Experiment on the expression of inflammatory factors transcription level in psoriasis lesions of C57BL / 6 mice induced by imiquimod using a gel preparation of Staphylococcus epidermidis H62-3 metabolites The back lesion tissue of the mice killed in Example 3 was taken, and the total RNA was extracted by Trizol method, which was reverse transcribed into cDNA, and then qPCR was performed to detect the expression of three inflammatory factor genes. Compared with normal control mice, continuous application of imiquimod for 6 days can induce psoriatic dermatitis in mice, involving the release of cytokines and cell infiltration related to psoriasis in the lesion tissue. Compared with normal control mice, the contents of psoriasis-related cytokines IL-17A, IL-17F, and IL-23 in the lesion tissue of mice in the imiquimod modeling group were significantly increased. The calcipotriol positive control group and the Staphylococcus epidermidis H62-3 metabolite gel preparation group can reduce the transcription levels of the above cytokines to varying degrees, such as Fig.11 As shown. In the calcipotriol positive control group, the inhibition rates of IL-17A, IL-17F, and IL-23 after local administration can reach 68.3%, 72.2%, and 63.0%, respectively. In the Staphylococcus epidermidis H62-3 metabolite gel preparation group, the inhibition rates of IL-17A, IL-17F, and IL-23 after local administration can reach 68.5%, 60.8%, and 63.7%, respectively.

[0049] Example 8: Experiment on the effect of the gel preparation of Staphylococcus epidermidis H62-3 metabolites on the expression of Ki67 protein in imiquimod-induced psoriasis lesions in C57BL / 6 mice The back skin lesions of the mice killed in Example 3 were fixed, paraffin-embedded, sliced, and Ki67 immunohistochemically stained. They were observed under a microscope and scanned and photographed. Compared with the normal control mice, the expression level of Ki67 in the skin lesions of the mice in the imiquimod modeling group was significantly increased, and the calcipotriol positive control group and the Staphylococcus epidermidis H62-3 live bacterial preparation group could effectively reduce the expression level of Ki67, such as Fig.12 shown.

[0050] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A gel preparation containing metabolites of Staphylococcus epidermidis, characterized in that: The gel preparation contains Staphylococcus epidermidis ( Staphylococcus epidermidis ) H62-3 strain metabolites, the Staphylococcus epidermidis H62-3 strain has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, the deposit date is February 21, 2023, and the deposit number is CGMCC No. 26640.

2. The gel preparation according to claim 1, characterized in that The gel preparation comprises the following ingredients in percentage by mass: 10-40% of Staphylococcus epidermidis H62-3 metabolites, 0.2-1.5% of carbomer 940, 0.2-1.5% of triethanolamine, 0.05-0.5% of low molecular weight sodium hyaluronate, 5-20% of glycerol, 0.5-2.5% of azone, and the balance is pure water.

3. A method for preparing a gel preparation, wherein the gel preparation is the gel preparation according to any one of claims 1 or 2, characterized in that: The preparation method comprises the following steps: (1) Fully mixing low molecular weight sodium hyaluronate and glycerol to obtain a compound moisturizer; (2) The metabolites of Staphylococcus epidermidis H62-3 are fully mixed with an appropriate amount of a compound moisturizer and pure water, azone is added, and the mixture is mixed again to obtain a drug solution; (3) Add Carbomer 940 to the remaining compound moisturizer, then add the remaining pure water, stir thoroughly to allow Carbomer 940 to swell and disperse, and then stir evenly; (4) Add the solution obtained in step (3) to the drug solution obtained in step (2) and stir evenly; then add triethanolamine and continue stirring until it becomes a gel, thereby obtaining the gel preparation.

4. The preparation method according to claim 3, characterized in that: The preparation method of the Staphylococcus epidermidis H62-3 metabolite comprises the following steps: picking a single colony of the activated Staphylococcus epidermidis strain H62-3 and inoculating it in a TSB liquid culture medium, placing it in a shaking incubator at 32-38° C., and shaking and culturing it for 14-18 hours to obtain a seed liquid; inoculating the seed liquid into the TSB liquid culture medium at an inoculation amount of 3%-5% by volume, and shaking and culturing it in a shaking incubator at 32-38° C. for 14-18 hours to obtain a fermentation liquid; centrifuging the fermentation liquid and discarding the bacterial precipitate; and concentrating the supernatant of the fermentation liquid by 5 times of volume by rotary evaporation to obtain the Staphylococcus epidermidis H62-3 metabolite.

5. The preparation method according to claim 3, characterized in that: The swelling and dispersion time of Carbomer 940 in step (3) is 30 minutes.

6. Use of the gel preparation according to any one of claims 1 or 2 in the preparation of a medicament for inflammatory skin diseases.

7. The use according to claim 6, characterized in that The inflammatory skin disease is eczema, atopic dermatitis, acne, psoriasis, chronic urticaria, vitiligo, pemphigus, pemphigoid or lupus erythematosus.

8. The use according to claim 7, characterized in that The psoriasis is at least one of psoriasis vulgaris, erythrodermic psoriasis, articular psoriasis or pustular psoriasis.

9. The use according to claim 7, characterized in that The gel preparation is used to improve the symptoms of skin erythema and scaling in a psoriasis mouse model; the gel preparation is used to reduce the thickness of skin lesions and reduce inflammatory factors at the skin lesions of the psoriasis mouse model.

10. The use according to claim 9, characterized in that The inflammatory factors are the mRNA expression level of interleukin-17A, the mRNA expression level of interleukin-17F, and the mRNA expression level of interleukin-23.

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