Parasaccharomyces hominis and its use in the preparation of a medicament for treating atopic dermatitis

Abstract

The application discloses a skin symbiotic bacterium Paracoccus aizawai, which is classified and named as Paracoccus aizawai (CGMCC NO. 33417 Paracoccus yeei ), and the preservation number is CGMCC NO. 33417, and the preservation time is January 15, 2025. The application further discloses application of the Paracoccus aizawai in treatment of atopic dermatitis. Local use of the Paracoccus aizawai DL-2 can significantly improve the atopic dermatitis-like skin lesions of a mouse induced by MC903, and the erythema and swelling are obviously reduced, the inflammatory cell infiltration is reduced, the mast cells are reduced, the inflammatory factor gene is down-regulated, the IgE in plasma is reduced, and the expression of barrier function related filaggrin is up-regulated. The skin symbiotic bacterium live bacterium preparation disclosed by the application can repair the skin barrier at the skin lesion, and has a wide application prospect in treatment of atopic dermatitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a skin symbiotic bacterium, Paracoccus eluti, and its application in the preparation of drugs for treating atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a common chronic inflammatory skin disease characterized by impaired skin barrier function, accompanied by intense itching and inflammation. The prevalence of AD has increased rapidly over the past 30 years, affecting up to 20% of children and 10% of adults in developed countries. Skin microbiota dysbiosis is considered one of the major environmental factors in the pathogenesis of AD. The skin microbiota plays a crucial role in the occurrence and development of AD. Recent studies have shown that skin microbiota dysbiosis is closely related to the occurrence and pathological process of atopic dermatitis. Skin microbiota dysbiosis may exacerbate skin lesions through mechanisms such as disrupting the skin barrier, promoting inflammatory responses, and enhancing allergic reactions. Recent studies have shown that the Phase I clinical trial results of transplantation of the skin commensal bacteria Staphylococcus hominis A9 (Sh A9) and Roseomonas mucosa for the treatment of atopic dermatitis are safe and effective, improving disease severity and effectively avoiding the side effects of systemic drugs acting on the host's immune system, representing a safe and low-toxicity new therapy.

[0003] Calcipotriol (MC903) is a synthetic vitamin D3 analog widely used to treat psoriasis and can inhibit the proliferation and differentiation of leukemia cells. In recent years, topical application of MC903 has induced atopic dermatitis syndrome in mice, and MC903 can also induce high expression of thymic stromal lymphopoietin (TSLP) through the vitamin D receptor on epithelial keratinocytes. TSLP is an epithelial cell-derived cytokine that plays a key role in initiating and promoting Th2 cell-mediated allergic inflammation. In the acute phase of Alzheimer's disease (AD), Th2 cytokines such as IL-4, IL-5, and IL-10 are overexpressed, stimulating high expression of immunoglobulin E (IgE) in mice. Simultaneously, in the chronic phase of AD, Th1 cells produce and highly express IFN-γ, IL-6, and TNF-α. TNF-α is a well-known pro-inflammatory cytokine, and its inhibition is considered a feasible strategy for preventing allergies. Summary of the Invention

[0004] Purpose of the invention: This invention provides *Paragonimus westermani* and its use in the preparation of medicaments for the treatment of atopic dermatitis.

[0005] The present invention further provides a method for preparing Paracoccus eluti and its live bacterial preparations, and the present invention further provides the application of the live bacterial preparations of Paracoccus eluti in the treatment of atopic dermatitis.

[0006] To address the aforementioned technical problems, this invention discloses a skin symbiotic bacterium, *Paracoccus yeei*, classified as *Paracoccus yeei*, strain number DL-2. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 15, 2025, with accession number CGMCC NO.33417. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. This strain was screened by the inventors in February 2023 from non-lesioned areas of atopic dermatitis patients.

[0007] The *Paracoccus eluti* DL-2 has the following properties:

[0008] Colony morphological characteristics:

[0009] After incubation at 32℃ for 16-24 hours on R2A medium, the colonies are large and slimy. Microscopic observation reveals that the bacteria are spherical, mostly diplococci, with some short rods. They are classified as belonging to the order Pseudomonas, family Paracoccidae, and genus *Paragonimus*. They are Gram-negative bacteria. Their growth temperature range is 5-40℃, with an optimum temperature of 28-37℃, and their growth pH range is 6-8, with an optimum pH of 6.5-7.5. They can grow normally in LB, R2A, TSB, and BAB media.

[0010] 16S rDNA sequence analysis

[0011] The 16S rDNA sequence is 1350 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1. A phylogenetic tree based on the full 16S rDNA sequence was constructed by comparing the 16S rDNA sequence with related species in the GeneBank database. The results showed that strain DL-2 is 99.48% homologous to Paracoccus yeei G1212. Therefore, the strain of this invention is identified as Paracoccus yeei.

[0012] The present invention further proposes a live bacterial preparation of the skin symbiotic *Paragonimus westermani*, wherein the live bacterial preparation comprises the aforementioned *Paragonimus westermani* DL-2.

[0013] The viable count of *Paragonimus westermani* in the live bacterial preparation is 1 × 10⁻⁶. 4 -9×10 11 Preferably, the viable count is 1×10⁻⁶. 6 -1×10 9 CFU / mL.

[0014] This invention further proposes a method for preparing the above-mentioned live *Paragonimus westermani* preparation, wherein the *Paragonimus westermani* is inoculated into an activation medium and activated at 25-37°C for 12-24 hours; the activated *Paragonimus westermani* is then transferred to a fermentation medium and cultured at 30-37°C for 24-36 hours; then, *Paragonimus westermani* cells are obtained by centrifugation at 4000-9000 rpm for 5-10 minutes; and a drug solvent is added to obtain the final product. The drug solvent is any one of sucrose, glycerol, and propylene glycol. The activation medium is either R2A medium or TSB medium; generally, activation is first performed on a plate using R2A medium, followed by activation in a shake flask using TSB medium.

[0015] The R2A culture medium consisted of the following components: yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15.0 g / L, and pH 7.2.

[0016] The TSB medium consists of 17.0 g tryptone, 5.0 g sodium chloride, 3.0 g soybean papain hydrolysate, 2.5 g dipotassium hydrogen phosphate, and 2.5 g glucose (monohydrate / anhydrous) per 1000 mL, with a pH of 7.3 ± 0.2 and a temperature of 25 °C.

[0017] Preferably, the fermentation medium is formulated as follows: carbon source 2-20 g / L, nitrogen source 1-30 g / L, inorganic salt 0.01-50 g / L, pH 5.0-9.0, and the solvent is water.

[0018] The carbon source is any combination of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol, starch, and molasses, preferably any combination of sucrose, glucose, lactose, citric acid, and starch; the nitrogen source is any combination of yeast extract, beef extract, peptone, yeast extract, corn steep liquor, soybean meal, cottonseed meal, urea, (NH4)2SO4, NH4Cl, (NH4)2HPO4, and NH4NO3, preferably any combination of peptone, yeast extract, corn steep liquor, (NH4)2SO4, NH4Cl, (NH4)2HPO4, and NH4NO3; the inorganic salt is any combination of sodium chloride, sulfate, phosphate, dihydrogen phosphate, dihydrogen phosphate, and hydrochloride.

[0019] In a preferred embodiment, the fermentation medium consists of 17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and a pH of 7.3.

[0020] The present invention further provides the use of dermatophyte *Paragonimus westermani* or a live bacterial preparation containing it in the preparation of a medicament or preparation for the treatment of atopic dermatitis.

[0021] This application utilizes MC903 to induce atopic dermatitis and investigates the efficacy of low- and high-dose *Paragonimus westermani* inducing MC903-induced atopic dermatitis.

[0022] Specifically, the live bacteria preparation of *Paragonimus westermani* DL-2 can significantly improve MC903-induced ear swelling, scaling, and increased mast cells in the ears of BALB / c mice; significantly improve MC903-induced ear thickening and epidermal thickening in BALB / c atopic dermatitis mice, and alleviate MC903-induced mast cell infiltration; inhibit the increase in transcriptional levels of one or more of the inflammatory factors IL-1β, TNF-α, IL-4, IL-5, IL-13, IL-6, TSLP, and IL-31 in the skin lesions of MC903-induced BALB / c mice; inhibit the increase in plasma IgE in MC903-induced BALB / c mice, and simultaneously inhibit the decrease in filaggrin content in the skin lesions of MC903-induced BALB / c mice.

[0023] In practical applications, a drug composition containing live bacteria Paracoccus eluti is applied topically to the subject to treat atopic dermatitis.

[0024] To promote the application of the invention, this invention specifically includes 10 3 10 4 10 8 CFU (Cellular Fusarium oxysporum) DL-2 was injected intravenously into 8-week-old C57BL / J mice, and the mice were observed until day 10 when they were sacrificed. The results showed that injecting 10... 3 10 4 10 8 Ten days after intravenous injection of CFU (paracoccus eluti) DL-2 into 8-week-old C57BL / J mice, there was no significant difference in body weight compared to the control group. 3 10 4 10 8 Ten days after intravenous injection of CFU of *Paragonimus westermani* DL-2 via the tail vein into 8-week-old C57BL / J mice, no infection was observed in the liver, spleen, or kidneys. These results demonstrate the safety of *Paragonimus westermani* DL-2.

[0025] Beneficial effects: Compared with the prior art, this application has the following advantages:

[0026] (1) The present application of Paracoccus eluti DL-2 can significantly improve the skin lesions of atopic dermatitis induced by MC903 in BALB / c mice, including ear erythema, swelling and scaling;

[0027] (2) The *Paragonimus westermani* DL-2 of this application is safe and non-toxic;

[0028] (3) It is of great significance to apply live skin symbiotic bacteria preparations to the treatment of skin lesions and local inflammation of immune skin diseases and to innovate and develop a new therapy with high efficiency and low toxicity. Attached Figure Description

[0029] Figure 1 Biological characteristics of Paracoccus eluti DL-2; A: Colony morphology, B: Gram staining, C: Scanning electron micrograph, D: Phylogenetic tree analysis of 16S rRNA sequence;

[0030] Figure 2 The therapeutic phenotype of AD-like dermatitis induced by MC903 in BALB / c mice by Paracoccus eluti DL-2; A: Simplified experimental design diagram; B: Typical representative phenotype diagram;

[0031] Figure 3 Pathological analysis of skin lesions in BALB / c mice induced by MC903-induced AD-like dermatitis by Paracoccus eluti DL-2;

[0032] Figure 4 The effect of Paracoccus eluti DL-2 on ear thickness induced by MC903 in BALB / c mice; A: Changes in ear thickness during the mouse experiment; B: Epidermal thickness of the mice.

[0033] Figure 5 The effect of Paracoccus eluti DL-2 on mast cell infiltration at MC903-induced skin lesions in BALB / c mice;

[0034] Figure 6 Statistical analysis of mast cell infiltration at skin lesions in BALB / c mice induced by MC903 in Paracoccus eluti DL-2;

[0035] Figure 7 The effect of Paracoccus eluti DL-2 on the transcriptional levels of inflammatory factors in MC903-induced skin lesions in BALB / c mice;

[0036] Figure 8 The effect of Paracoccus eluti DL-2 on plasma IgE levels in MC903-induced BALB / c mice;

[0037] Figure 9 The effect of Paracoccus eluti DL-2 on filaggrin content in skin lesions induced by MC903 in BALB / c mice (blue: DAPI, red: filaggrin), A: canonical plot, B: statistical plot;

[0038] Figure 10Effects of tail vein injection of Paracoccus eluti DL-2 on the physiological state of mice; A: Schematic diagram of experimental design; B: Changes in body weight during the experiment; C: Pathological images of the liver, spleen and kidneys of mice. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0040] Example 1: Screening and identification of Paracoccus eluti DL-2.

[0041] Paracoccus eluti DL-2 is a dermal commensal bacterium screened by the inventors from non-lesioned areas of subjects with atopic dermatitis. The screening process is as follows:

[0042] Wipe the human skin 50 times with a sterile cotton swab, immerse in sterile water, and shake to mix. Perform serial dilutions of the sample solution, taking 10... -4 10 -5 10 -6 Three concentration gradients of the dilution were evenly spread on solid culture medium (medium composition: yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15.0 g / L, pH 7.2 ± 0.2). The plates were incubated at 32℃ for 48 h. During this period, the plates were checked daily to observe colony characteristics. Single colonies were isolated by streak plating on solid culture plates. This process was repeated three times to obtain single bacterial strains.

[0043] The strains obtained in the previous step were inoculated into liquid culture medium (composition: tryptone 17.0 g / L, soybean peptone 3.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, pH 7.3 ± 0.2) and cultured on a shaker at 37°C. The isolated bacterial cells were resuspended in 10% sucrose solution and used to treat MC903-induced AD-like dermatitis. The single bacterium with the best effect was screened and named *Paragonimus westermani* DL-2. *Paragonimus westermani* DL-2 was identified as having the following properties:

[0044] (1) Colony morphological characteristics

[0045] After incubation at 32℃ for 16-24 hours on R2A medium, the colonies are large and slimy. Microscopic observation reveals that the bacteria are spherical, mostly diplococci, with some short rods. They are classified as belonging to the order Pseudomonas, family Paracoccidae, and genus *Paragonimus*. They are Gram-negative bacteria. Their growth temperature range is 5-40℃, with an optimum temperature of 28-37℃, and their growth pH range is 6-8, with an optimum pH of 6.5-7.5. They can grow normally in LB, R2A, TSB, and BAB media.

[0046] (2) 16S rDNA sequence analysis

[0047] The 16S rDNA sequence is 1350 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1. A phylogenetic tree based on the full 16S rDNA sequence was constructed by comparing the 16S rDNA sequence with related species in the GeneBank database. The results showed that strain DL-2 was 99.48% homologous to Paracoccus yeei G1212. Therefore, the strain of this invention was identified as Paracoccus yeei, strain number DL-2, and deposited at the China General Microbiological Culture Collection Center on January 15, 2025, with accession number CGMCC NO.33417.

[0048] Example 2: Culture of Paracoccus eluti DL-2.

[0049] Paracoccus eluti DL-2, preserved on a slant at 4℃, was activated on R2A medium and inoculated onto seed culture medium (i.e., activation medium TSB) at 32℃ for 16 hours. The seed fermentation broth was then inoculated into the fermentation medium at a volume ratio of 10%, and cultured at 32℃ for 24 hours at 200 rpm to obtain the fermentation broth. The viable cell count in the fermentation broth reached 5 × 10⁻⁶. 9 CFU / mL or higher.

[0050] The activation medium was R2A medium, and its components were: yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15.0 g / L, and pH 7.2.

[0051] The fermentation medium consisted of 17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and a pH of 7.3.

[0052] Example 3: Live bacterial preparation of Paracoccus eluti DL-2.

[0053] The fermentation broth obtained in Example 2 was centrifuged (9000 rpm, 5 min), and the bacterial culture was resuspended in 10 wt% sucrose to obtain a live bacterial preparation with a live bacterial count of 1 × 10⁻⁶. 4 -1×10 11 CFU / mL.

[0054] Example 4: Effect of Paracoccus eluti DL-2 on AD-like skin lesions induced by MC903 in BALB / c mice.

[0055] Breeding environment: SPF grade; Mice: BALB / c female mice, 8 weeks old; Modeling method: (1) After stabilizing the purchased 8-week-old BALB / c female mice of uniform size for one week, apply 1 nmol MC903 evenly to the ear of each mouse every day. The experiment set up 4 treatment methods, with 6 biological replicates for each treatment method and 24 mice. The drug administration treatment was uniformly as follows: local administration on the day of modeling, administration in the morning and modeling in the afternoon. Ear thickness was recorded on days 0, 3, 6 and 9. The treatment methods are as follows:

[0056] Treatment 1 (NC), without treatment, is the healthy control group;

[0057] Treatment 2 (Vehicle): Starting from the MC903 modeling, it was given 20 μL of 10% sucrose topically, which was the matrix group;

[0058] Process 3(10) 7 Starting from MC903 modeling, administer 20μL of topical application containing 10 [units of something] daily. 7 One CFU of Paracoccus eluti DL-2 constitutes the low-dose live bacteria treatment group.

[0059] Process 4(10) 9 Starting from MC903 modeling, administer 20μL of topical application containing 10 [units of something] daily. 9 One CFU of Paracoccus eluti DL-2 was used in the high-dose live bacteria treatment group.

[0060] Other routine management practices remained consistent across all four treatments. At the end of the experiment, tissue samples were collected from the sacrificed mice, and the skin lesions and pathological findings were analyzed using HE staining. Figure 2 As shown, compared with the healthy group, MC903 induced typical AD skin lesions, manifested as increased ear thickness, erythema, and scaling in mice. Compared with AD model mice, both the high-dose and low-dose live bacteria treatment groups of *Paragonimus westermani* DL-2 showed improvement in erythema and scaling, with the low-dose group showing a more significant allergic reaction. Pathological analysis is as follows: Figure 3As shown, MC903 induced swelling of the epidermis and dermis of mouse ears, with severe inflammatory cell infiltration. Compared with the matrix-treated group, both high- and low-dose DL-2 treatment groups improved ear swelling and inflammatory cell infiltration in mice, and there was no significant difference between the high- and low-dose treatment groups. Therefore, Paracoccus eluti DL-2 has a significant ameliorative effect on MC903-induced AD-like skin lesions in mice, and may become a safe and low-toxicity new therapy.

[0061] Example 5: Effects of Paracoccus eluti DL-2 on ear thickness and epidermal thickening induced by MC903 in BALB / c mice.

[0062] Ear thickness was recorded on days 0, 3, 6, and 9 of the experiment. Ear thickness of the mice in Example 4 was measured using vernier calipers. Three measurements were taken for each mouse, and the average value was calculated. The statistical results are as follows: Figure 4 As shown in Figure A, compared with the healthy group, the ear thickness of mice in the matrix group significantly increased from day 6. Compared with the matrix group, *Paragonimus westermani* DL-2 could alleviate the MC903-induced increase in ear thickness in mice, with statistically significant differences between the high and low dose treatment groups of *Paragonimus westermani* DL-2. Based on the pathological images in Example 4, the epidermal thickness was statistically analyzed using ImageJ, and the results are as follows... Figure 4 As shown in Figure B, compared with the healthy group, the epidermis of mice significantly thickened after continuous application of MC903 for 9 days. Compared with the matrix group, all treatment groups significantly improved MC903-induced epidermal thickening in mice, with statistically significant differences.

[0063] Example 6: Effect of Paracoccus eluti DL-2 on mast cell infiltration at skin lesions induced by MC903 in BALB / c mice.

[0064] The skin lesions from the sacrificed mice in Example 4 were embedded in paraffin and sectioned. Mast cells were stained with toluidine blue as follows: (1) The tissue was fixed in neutral formaldehyde solution or formaldehyde ethanol solution. (2) The tissue sections were dewaxed. (3) The sections were stained with toluidine blue solution (0.5g toluidine blue diluted to 100ml with distilled water) for 30min. (4) The sections were rinsed slightly with water. (5) The sections were differentiated with glacial acetic acid solution (0.5ml glacial acetic acid diluted to 100ml with distilled water) until the nuclei and granules were clear (controlled under a microscope). (6) The sections were rinsed slightly with water and dried with cold air. (7) The sections were sealed with clear neutral resin.

[0065] Based on the statistical analysis results, as follows Figure 5 As shown, mast cell granules appear reddish-purple with blue nuclei. MC903 treatment significantly induced mast cell infiltration in the mouse dermis. Compared with the matrix-treated group, both mometasone furoate and extracellular polysaccharide (EPS) treatment groups alleviated MC903-induced mast cell infiltration in mice. The statistical results were obtained by averaging the mast cell counts under each high-power microscope after full-scan scanning. Figure 6As shown, all Paracoccus eluti DL-2 treatment groups showed significant differences, with no significant differences between the low-dose and high-dose Paracoccus eluti DL-2 treatment groups.

[0066] Example 7: Effect of Paracoccus eluti DL-2 on the transcriptional level of inflammatory factors in MC903-induced skin lesions in BALB / c mice.

[0067] After grinding the skin lesion tissue from the sacrificed mice in Example 4, total RNA was extracted from the skin lesion tissue using a TRIZOL kit. The total RNA was then reverse transcribed into cDNA using a reverse transcription kit. The expression level of the target mRNA was detected using a SYBR Green kit on an ABI Picomm 7900 real-time quantitative PCR instrument. β-actin was used as an internal control, and the relative expression level of the target gene mRNA was calculated based on 2 - ΔΔCt.

[0068] The results are as follows Figure 7 As shown, MC903 significantly increased the transcriptional levels of inflammatory factors IL-1β, TNF-α, IL-4, IL-5, IL-13, IL-6, TSLP, and IL-31 in the ears of BALB / c mice. Compared with the matrix treatment group, Paracoccus eluti DL-2 significantly inhibited the increase in inflammatory factor levels induced by MC903. According to statistical analysis, the inhibitory effect of Paracoccus eluti DL-2 on the increase in transcriptional levels of inflammatory factors IL-1β, TNF-α, IL-4, IL-5, IL-13, IL-6, TSLP, and IL-31 induced by MC903 in skin lesions was significantly different.

[0069] Example 8: Effect of Paracoccus eluti DL-2 on plasma IgE levels in MC903-induced BALB / c mice.

[0070] Blood was collected from the mice in Example 4 before sacrifice in anticoagulant tubes, centrifuged at 1000 rpm for 10 min, and the supernatant was frozen and stored at -80°C. After equilibration at room temperature for 30 min, the plasma samples were tested according to the manufacturer's instructions for the IgE enzyme-linked immunosorbent assay kit (Huamei Biotechnology, CSB-E079083). The results are as follows... Figure 8 As shown, compared with the matrix-treated group, both the high- and low-dose Paracoccus eluti DL-2 groups significantly inhibited the MC903-induced increase in IgE in mouse plasma. Although there was no significant difference between the low-dose and high-dose Paracoccus eluti DL-2 treatment groups, the average IgE level was lower in the high-dose Paracoccus eluti DL-2 treatment group.

[0071] Example 9: Effect of Paracoccus eluti DL-2 on filaggrin content in skin lesions of MC903-induced BALB / c mice.

[0072] Immunofluorescence staining was performed on skin lesions from the sacrificed mice in Example 4, as follows:

[0073] 1) Remove the collected skin lesion tissue from the back of the mouse from the -80℃ freezer, embed it using OCT, and cut the tissue into 5mm thin slices using a cryostat.

[0074] 2) Place the slide in a humidified chamber and allow it to warm for 10 minutes.

[0075] 3) Place the tissue sections in PBS and place them on a horizontal shaker at 80 rpm for 5 minutes each time, for a total of 3 times.

[0076] 4) After smearing the tissue, wipe away the PBS around the tissue with a tissue and draw circles around the tissue with an immunohistochemistry pen.

[0077] 5) Add rapid immunostaining blocking solution and incubate at room temperature for 10 minutes.

[0078] 6) Wash twice with PBS, 5 minutes each time.

[0079] 7) Drop FLG Rabbit pAb primary antibody (1:1000 dilution, Abclonal, catalog number A20011) diluted with immunostaining primary antibody dilution solution onto the tissue surface and incubate overnight at 4°C in a humidified chamber.

[0080] 8) Wash 3 times with PBS at 80 rpm for 5 minutes each time.

[0081] 9) Drop IgG-Alexa Fluor 594 secondary antibody (1:200 dilution, Absin, catalog number abs20021) diluted with secondary antibody dilution buffer onto the tissue surface and incubate at room temperature in the dark for 60 min.

[0082] 10) Wash 3 times with PBS at 80 rpm for 5 min each time.

[0083] 11) Add two drops of DAPI-containing mounting medium to the tissue, incubate at room temperature in the dark for 5 minutes, then mount the slide and observe under a fluorescence confocal microscope.

[0084] MC903 can induce atopic dermatitis-like skin barrier dysfunction in BALB / c mice. Filagrin, a structural protein of the epidermis, plays a crucial role in the skin barrier. Results are as follows... Figure 9 As shown, MC903 can induce a significant decrease in filaggrin content in the ears of BALB / c mice. Compared with the matrix treatment group, both the low-dose and high-dose Paracoccus eluti DL-2 treatment groups can inhibit the decrease in filaggrin content induced by MC903. The high-dose treatment group and the low-dose extracellular polysaccharide treatment group have very significant effects.

[0085] Example 10: Toxicity analysis of Paracoccus eluti DL-2.

[0086] Housing environment: SPF grade; Mice: BALB / c female mice, 8 weeks old; The experiment set up 4 treatments, with 5 biological replicates for each treatment, 20 mice in total. Drug administration was uniformly performed via tail vein injection on day 1, and body weight was recorded on days 0, 3, 6, and 10. The treatment methods are as follows:

[0087] Treatment 1 (Saline): 100 μL of normal saline was injected into the tail vein, serving as the healthy control group;

[0088] Process 2(10) 3 ), 100μL containing 10 3 One CFU of Paracoccus eluti DL-2;

[0089] Process 3(10) 4 ), 100μL containing 10 4 One CFU of Paracoccus eluti DL-2;

[0090] Process 4(10) 8 ), 100μL injected via tail vein. 8 One CFU of Paracoccus eluti DL-2.

[0091] Other routine management practices remained consistent across all four treatments. At the end of the experiment, tissue samples were collected from the sacrificed mice, and liver, spleen, and kidneys were analyzed using hematoxylin and eosin (HE) staining. Figure 10 As shown in Figure B, compared with the saline treatment group, the tail vein injection of *Paragonimus westermani* DL-2 treatment group had no effect on mouse body weight. Liver, spleen, and kidney pathology of mice in each group were as follows (HE). Figure 10 As shown in Figure C, no infections were observed in any of the groups treated with tail vein injection of Paracoccus eluti DL-2. The results indicate that Paracoccus eluti DL-2 is safe and non-toxic.

[0092] In summary, this invention provides a method for preparing and applying Paracoccus yeei DL-2, a dermal commensal bacterium that alleviates atopic dermatitis. Paracoccus yeei DL-2 can inhibit MC903-induced ear margin thickening, inflammatory factor accumulation, and skin barrier damage in BALB / c mice. Furthermore, Paracoccus yeei DL-2 is safe and non-toxic, thus showing broad application prospects in the treatment of atopic dermatitis.

Claims

1. A dermal commensal bacterium, Paracoccus eludiensis, classified as Paracoccus eludiensis (… Paracoccus yeei The strain number is DL-2, which has been deposited at the China General Microbiological Culture Collection Center on January 15, 2025, with the accession number CGMCC NO.33417.

2. A live bacterial preparation of *Paragonimus westermani*, a dermal commensal bacterium, characterized in that, The live bacteria preparation includes the skin-symbiotic Paracoccus eluti as described in claim 1.

3. The live bacterial preparation of *Paragonimus westermani*, a dermal commensal bacterium, according to claim 2, is characterized in that... The viable count of *Paragonimus westermani* in the live bacterial preparation is 1 × 10⁻⁶. 4 -9×10 11 CFU / mL.

4. The method for preparing the live bacterial preparation of *Paragonimus westermani*, a dermal commensal bacterium, as described in claim 2 or 3, is characterized in that... The *Paracoccus eluti* was inoculated into an activation medium and activated at 25-37°C for 12-24 h. The activated *Paracoccus eluti* was then transferred to a fermentation medium at a volume ratio of 10-15% and cultured at 30-37°C for 24-36 h. *Paracoccus eluti* cells were obtained by solid-liquid separation and added to a drug delivery solvent to obtain a live bacterial preparation.

5. The preparation method according to claim 4, characterized in that, The fermentation medium is composed of: 2-20 g / L carbon source, 1-30 g / L nitrogen source, 0.01-50 g / L inorganic salt, and pH 5.0-9.

0. Preferably, the fermentation medium is composed of: 17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and pH 7.

3.

6. The use of the skin symbiotic *Paragonimus westermani* as described in claim 1 or the live bacterial preparation as described in claim 2 in the preparation of a medicament or preparation for the treatment of atopic dermatitis.

7. The application according to claim 6, characterized in that, The live bacteria preparation improved MC903-induced ear swelling, scaling, and increased mast cells in BALB / c mice.

8. The application according to claim 6, characterized in that, The live bacteria preparation improved the ear thickening and epidermal thickening induced by MC903 in BALB / c atopic dermatitis mice and alleviated MC903-induced mast cell infiltration in mice.

9. The application according to claim 6, characterized in that, The live bacteria preparation inhibits the increase in transcriptional levels of one or more of the inflammatory factors IL-1β, TNF-α, IL-4, IL-5, IL-13, IL-6, TSLP and IL-31 in MC903-induced skin lesions in BALB / c mice.

10. The application according to claim 6, characterized in that, The live bacteria preparation inhibited the increase of IgE in the plasma of BALB / c mice induced by MC903 and inhibited the decrease of filaggrin content in the skin lesions of BALB / c mice induced by MC903.

Images