CN116103205B

CN116103205B - Rosemonas mucilaginosa, microbial inoculum and extracellular polysaccharide as well as preparation method and application thereof

Info

Publication number: CN116103205B
Application number: CN202310227440.XA
Authority: CN
Inventors: 陆前进; 詹伊婧; 梁金宇; 刘倩梅; 张博; 高长醒; 辛月
Original assignee: Institute of Dermatology and Skin Disease Hospital of CAMS
Current assignee: Institute of Dermatology and Skin Disease Hospital of CAMS
Priority date: 2023-03-10
Filing date: 2023-03-10
Publication date: 2023-06-27
Anticipated expiration: 2043-03-10
Also published as: CN116103205A

Abstract

The invention discloses a myxorhodomonas, further discloses a microbial inoculum containing the myxorhodomonas and extracellular polysaccharide thereof, and simultaneously discloses application of the myxorhodomonas and extracellular polysaccharide thereof in preventing and/or treating inflammation-related and/or T/B cell overactivation diseases and treating cutaneous lupus erythematosus. The local application of the myxorosea DL-1 and secretion thereof can obviously improve the skin damage of skin lupus erythematosus model mice induced by the combination of UVB and Pristane, reduce the lymphocyte infiltration of dermis layer at the skin damage, reduce immune complex IgG deposition in basal mulch tape area and the like. The skin symbiotic bacteria live bacteria preparation and the extracellular polysaccharide thereof can relieve inflammation at skin lesions, have wide application prospect in treating cutaneous lupus erythematosus, and provide a new idea for clinical and transformation researches of other autoimmune diseases.

Description

Rosemonas mucilaginosa, microbial inoculum and extracellular polysaccharide as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a rhodomonas mucilaginosa, a microbial inoculum containing the rhodomonas mucilaginosa, extracellular polysaccharide, a preparation method and application of the extracellular polysaccharide, and particularly application of the rhodomonas mucilaginosa in treating cutaneous lupus erythematosus.

Background

Autoimmune diseases are diseases in which the body is immune-reacted to autoantigens to cause damage to self tissues, and mainly comprise rheumatoid arthritis, scleroderma, systemic lupus erythematosus, hyperthyroidism, ulcerative colitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura and the like, which affect about 5-8% of the world population and cause great pain to patients. T, B lymphocyte immunomodulators are likely to play an important role in the treatment of autoimmune diseases, for example, inhibiting T cell proliferation, activating and regulating T cell subset balance, and inhibiting B cell proliferation, differentiation and antibody secretion may be potential for conducting immune modulation therapy of autoimmune diseases. Because of the important role of abnormal activation of B cells in various autoimmune diseases, therapeutic drugs for autoimmune diseases targeting B cells have been a research hotspot. The classical B cell scavenger CD20 mab has achieved significant efficacy in rheumatoid arthritis, anti-neutrophil cytoplasmic antibody-associated vasculitis and even in sjogren syndrome in addition to the significant efficacy in classical B cell aberrant activation disease systemic lupus erythematosus (systemic lupus erythematosus, SLE).

The CD28 pathway is the most important co-stimulatory signal in the alloantigen immune response, and it induces T cell activation, enhances the recognition ability of the immune system, results in immune tolerance, and reduces the immune response in the body.

Lupus erythematosus is a classical autoimmune disease, which can cause multiple organ involvement, with the skin involvement patient accounting for 70% -85%. Cutaneous lupus erythematosus (Cutaneous lupus erythematosus, CLE) affects mainly the skin, generally without or with minor involvement of internal organs. CLE can be classified into acute cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, and chronic cutaneous lupus erythematosus. Most of the patients with systemic lupus erythematosus, except CLE, show skin manifestations up to 90% and 25-28% of patients have skin as the first symptom. Clinically, some CLE subtypes may progress to SLE, and the occurrence of conditions including kidney and brain involvement leads to renal failure and neurological disease. Successful treatment of CLE may therefore significantly reduce the risk of systemic involvement.

The etiology and pathogenesis of CLE are complex, which is not completely clear until now, and more researches indicate that genetic and environmental factors can cause immune cells such as T cells, B cells, neutrophils, antigen presenting cells and NK cells to infiltrate in the skin lesions of the CLE, and the pro-inflammatory cytokines HMGB-1, interferon, IL-6, IL-17 and IL-12 at the skin lesions of the CLE, and the chemokines CXCL9, CXCL10, CXCL11, CXCL12 and the like are obviously up-regulated. At present, the treatment drugs comprise antimalarial drugs or external glucocorticoids, calcineurin inhibitors and the like, but partial patients have poor curative effects, and development of safe and low-toxicity new therapies is urgently needed.

The surface of the skin has a large amount of symbiotic bacteria, plays an important role in maintaining the stable state of the skin of a host, playing a role in the barrier function of the skin, resisting invasion of pathogenic microorganisms, participating in host immune response, maintaining normal structure of epidermis, regulating damage repair and regeneration of the skin and the like. Recent studies have shown skin commensal bacteriaStaphylococcus hominisA9（Sh A9 Sum of (d)R. mucosaThe phase I clinical test result of the transplanting treatment of atopic dermatitis shows that the method is safe and effective, can improve the severity of diseases, effectively avoids side effects generated by systemic drugs acting on the host immune system, and is a safe and low-toxicity novel therapy.

Disclosure of Invention

The invention aims to: in order to relieve the skin damage problem of cutaneous lupus erythematosus, the invention provides a rhodomonas mucilaginosa.

The invention further provides a preparation method of the active bacterial preparation of the myxoroseomonas and extracellular polysaccharide thereof, and further provides application of the active bacterial preparation of the myxoroseomonas and the extracellular polysaccharide in relieving cutaneous lupus erythematosus.

In order to solve the technical problems, the invention discloses a rhodomonas mucilaginosa which is classified and named as rhodomonas mucilaginosa @Roseomonas mucosa) The strain number is DL-1, which is preserved in China Committee for culture Collection of microorganismsThe general microbiological center (CGMCC) has a preservation time of 2022, 10 months and 26 days, a preservation number of CGMCC 25967, and a preservation address of Beijing Chaoyang area North Chenxi Lu No. 1, 3, national academy of sciences microbiological institute, and a postal code 100101. The strain is a skin symbiotic strain screened from the skin of a healthy person by the inventor in 2021 at 11.

The rhodomonas mucilaginosa DL-1 has the following properties:

colony morphology characterization

After 16-24 h culture in R2A medium at 32 ℃, the colony was observed to be a single colony by a microscope, and the morphology is shown in FIG. 1A. The strain is classified into rhodospirillum, acetobacter, globose, which belongs to gram-negative bacteria, produces red pigment, has a mucilage shape, is arranged singly, in pairs or in short chains, and has flagellum capable of moving. The bacterial cells can grow in a large quantity when being cultivated in the culture medium for 12 hours at 32 ℃, the growth temperature is 5-40 ℃, the optimal temperature is 30-35 ℃, the growth pH is 5.0-9.5, and the optimal pH is 6.5-7.5. Can grow normally in LB, R2A, TSB, BAB and other culture mediums.

16S rDNA sequence analysis

16S rDNA sequence length 1464 bp. The similarity of the strain DL-1 with the rhodomonas mucilaginosa reaches 99.86% by comparing the 16S rDNA sequence with related species in the GeneBank database. Therefore, the strain of the invention is regarded as the rhodomonas mucilaginosaRoseomonas mucosa) The strain number is DL-1 and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation time of 2022, 10 months and 26 days and the preservation number of CGMCC 25967.

The invention further provides a living bacterial preparation of skin symbiotic bacteria rhodopseudomonas mucilaginosa, which comprises the rhodopseudomonas mucilaginosa DL-1 (abbreviated asR. mucosa DL-1）。

Wherein the number of the active bacteria of the rhodomonas mucilaginosa in the active bacteria preparation is 1 multiplied by 10 ⁴ -9×10 ¹¹ Preferably, the viable count is 1X 10 ⁶ -1×10 ⁹ CFU/mL。

The invention further provides a preparation method of the active microbial preparation of the myxoroseomonas, the myxoroseomonas is inoculated in an R2A culture medium, activated at 25-37 ℃ for 12-24 h, the activated myxoroseomonas is transferred in a fermentation culture medium, cultured at 30-37 ℃ for 24-36 h, and then an administration solvent is added, wherein the administration solvent is any one of glycerol and propylene glycol.

Wherein, the R2A culture medium comprises the following components: yeast extract powder 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.

Preferably, the fermentation medium is formulated as follows: 10-100 parts of carbon source g/L, 1-30 parts of nitrogen source g/L, 0.01-50 parts of inorganic salt g/L and pH5.0-9.0, wherein the solvent is water.

Wherein the carbon source is any one or more of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol, starch and molasses, preferably any one or more of sucrose, glucose, lactose, citric acid and starch; the nitrogen source is yeast extract powder, beef extract, peptone, yeast extract, corn steep liquor, bean cake powder, cotton seed cake powder, urea, (NH) ₄ ) ₂ SO ₄ 、NH ₄ Cl、(NH ₄ ) ₂ HPO ₄ And NH ₄ NO ₃ Any one or a combination of more of peptone, yeast extract, corn steep liquor, (NH) ₄ ) ₂ SO4、NH ₄ Cl、(NH ₄ ) ₂ HPO ₄ And NH ₄ NO ₃ Any one or a combination of a plurality of the above; the inorganic salt is any one or the combination of a plurality of sodium chloride, sulfate, phosphate, dihydrogen phosphate, hydrogen phosphate dibasic and hydrochloride.

In a preferred embodiment, the fermentation medium composition is tryptone 17.0g/L, soytone 3.0g/L, sodium chloride 5.0g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, pH 7.3.

The invention further provides a rhodomonas mucilaginosa extracellular polysaccharide which is obtained by performing sterilization, deproteinization, absolute ethanol precipitation and DEAE centrifugal exchange resin separation and purification on the fermentation liquor of the rhodomonas mucilaginosa.

Specifically, sterilizing by centrifugation at 3000-5000rpm for 10min, adding 1/2 volume of sevage reagent to remove protein 3 times, adding 1 times volume of absolute ethanol, standing overnight at 4deg.C to obtain crude extracellular polysaccharide precipitate, re-dissolving with deionized water, purifying with DEAE-52, and lyophilizing to obtain extracellular polysaccharideR. mucosaDL-1 EPS，EPS）。

Wherein the weight average molecular weight of the mucus rosacea extracellular polysaccharide is 3000-3500Da, and the monosaccharide composition comprises arabinose, rhamnose, galactose, glucose, xylose, mannose, ribose, galacturonic acid, glucuronic acid, mannuronic acid and guluronic acid.

The monosaccharide composition of the rhodomonas mucilaginosa extracellular polysaccharide is as follows: arabinose: rhamnose: galactose: glucose: xylose: mannose: ribose: galacturonic acid: glucuronic acid: mannuronic acid: guluronic acid=0.50-0.6:1.3-1.5:1.3-2:72-78:4-7:9-11:0.9-1.1:0.5-2.4:0.6-1.4:0.6-1.4:0.9-1.7.

In a specific embodiment, the extracellular polysaccharide has a weight average molecular weight of 3000Da and the monosaccharide composition is arabinose: rhamnose: galactose: glucose: xylose: mannose: ribose: galacturonic acid: glucuronic acid: mannuronic acid: guluronic acid=0.51:1.47:1.8: 73.98:4.67:9.96:0.94:2.33:1.35:1.32:1.67.

The invention further provides application of the rhodomonas mucilaginosa extracellular polysaccharide in preparing medicines for preventing and/or treating inflammation-related and/or T/B cell overactivation diseases.

In one aspect, the present application finds that the rhodomonas mucilaginosa exopolysaccharide inhibits the in vitro induced differentiation of B cells into plasmablasts and antibody secreting cells. Specifically, 25 μg/mL-75 μg/mL of the L-1 exopolysaccharide from Rosemonas viscosa can inhibit 30 ng/mL IL-4 and 100 ng/mL R848 induced in vitro B cells from differentiating into plasmablasts.

Meanwhile, T cells induced by the extracellular polysaccharide anti-CD28 of the rhodomonas mucilaginosa in vitro are activated into Tfh cells. Specifically, 5 μg/mL-500 μg/mL of the L-1 extracellular polysaccharide of the rhodomonas mucilaginosa can inhibit T cells induced by 5 μg/mL of anti-CD3 and 2 μg/mL of anti-CD28 in vitro from activating to Tfh cells.

The invention further provides application of the mucus rosacea extracellular polysaccharide in preparation of medicines or preparations for treating skin lupus erythematosus skin lesions.

When the preparation is applied, the living bacterial preparation or extracellular polysaccharide is locally applied to skin lesions of skin type lupus erythematosus mice induced by UVB and Pristane. The live bacteria preparation can be used together with medicines in diagnosis and treatment consensus of cutaneous lupus erythematosus, and can also be used independently to regulate and control immune microenvironment at skin lesions, thereby reducing the deposition of immune complex IgG, relieving skin lesions and reducing the dosage of clinical glucocorticoid.

Preferably, topical applicationR. mucosa DL-1 live bacteria preparationR. mucosa DL-1 viable count of 1×10 ⁷ -1×10 ⁹ CFU/mL。

Preferably, topical applicationR. mucosaThe DL-1 live bacteria preparation is administered after UVB irradiation, and the topical dosage is 1×10 ⁷ -1×10 ⁹ CFU/cm ² 1 time a day.

Topical applicationR. mucosaDL-1 extracellular polysaccharide is used after UVB irradiation, wherein the topical dosage is 0.1 mg/cm ² -0.3 mg/cm ² 1 time a day.

The skin type lupus erythematosus skin lesion comprises one or more of acute skin type lupus erythematosus, subacute skin type lupus erythematosus and chronic skin type lupus erythematosus and subtypes thereof.

The research of the application shows that the living bacterial preparation or the extracellular polysaccharide of the rosacea can regulate and control the immune microenvironment at the skin lesion, and reduce the deposition of immune complex IgG.

The beneficial effects are that: the invention screens out skin symbiotic bacteria with environmental skin type lupus skin lesions from healthy peopleR. mucosa DL-1, and byR. mucosa DL-1 is bacterial strain fermentation culture and separation to obtainR. mucosa DL-1 live bacteria preparation and its extracellular polysaccharide,R. mucosa DL-1 colonizes the skin lesionEpidermis, regulate and control the immune microenvironment of the skin lesions, thereby reducing the deposition of immune complex IgG, relieving the skin lesions and reducing the dosage of clinical glucocorticoid. The invention providesR. mucosa The DL-1 live bacteria preparation and extracellular polysaccharide are safe and effective for treating skin lupus erythematosus skin lesions, do not produce side effects on skin or systems, and have good application prospects. The living bacteria preparation of the skin symbiotic bacteria and the extracellular polysaccharide of the metabolite thereof are applied to relieving skin damage and local inflammation of the immune skin disease, and have great significance to application and development of skin microbiome in the immune skin disease.

Drawings

FIG. 1 is a diagram of the biological characteristics of roseomonas mucilaginosa, A: colony morphology, B: gram staining, C: scanning electron micrograph, D: phylogenetic tree analysis of the 16S rRNA sequence;

FIG. 2 shows the elution profile of the extracellular polysaccharide EPS of R.viscosus DL-1;

FIG. 3 is a standard sample ion chromatogram;

FIG. 4 is an ion chromatogram of an extracellular polysaccharide EPS sample;

FIG. 5 is a graph of the scattering spectrum of an EPS sample of exopolysaccharides;

FIG. 6 is the effect of roseomonas mucilaginosa DL-1 extracellular polysaccharide EPS on B cell induced differentiation in vitro;

FIG. 7 is the effect of roseomonas mucilaginosa DL-1 extracellular polysaccharide EPS on T cell activation;

FIG. 8 shows symbiotic bacteria for skinR. mucosaViable bacteria preparation of DL-1 on Pristane and UVB combined induced C57BL/6J mouse CLE skin damage and pathological influence;

FIG. 9 shows symbiotic bacteria for skinR. mucosa Effect of viable bacterial formulations of DL-1 on immune complex deposition at CLE lesions in pristate and UVB-induced C57BL/6J mice;

FIG. 10 shows symbiotic bacteria for skinR. mucosa Effect of viable bacteria preparation of DL-1 on Pristane and UVB combined induced C57BL/6J mouse CLE skin lesion local immunocytes;

FIG. 11 shows symbiotic bacteria for skinR. mucosa DL-1 extracellular polysaccharide EPS has pathological influence on Pristane and UVB combined induced C57BL/6J mouse CLE skin damage;

FIG. 12 shows symbiotic bacteria for skinR. mucosa Effect of DL-1 exopolysaccharide EPS on immune complex deposition at CLE lesions in pristate and UVB-induced C57BL/6J mice;

FIG. 13 shows skin symbiotic bacteriaR. mucosa Effect of DL-1 exopolysaccharide EPS on the transcriptional levels of inflammatory factors IL-6, IL-1 beta and IL-17A at CLE lesions in pristate and UVB-induced C57BL/6J mice.

Description of the embodiments

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

Example 1 screening and identification of roseomonas mucilaginosa DL-1.

The rhodomonas mucilaginosa DL-1 is skin symbiotic bacteria obtained by the inventor through screening from healthy human skin, and the screening process is as follows:

wiping the aseptic cotton swab on the human epidermis for 50 times, immersing in aseptic water, vibrating, and mixing. Subjecting the sample solution to gradient dilution to obtain 10 ^-4 、10 ^-5 、10 ^-6 The three concentration gradient dilutions were uniformly coated on solid media (media composition: yeast extract powder 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). Culturing in a constant temperature incubator at 32deg.C for 48 h times, checking the plate every day during the culturing period, observing colony characteristics, separating single colony on a solid culture hard plate by streaking to obtain single colony, repeating for three times to obtain single strain.

The strains obtained in the previous step are respectively inoculated into a liquid culture medium (medium components: tryptone 17.0g/L, soybean peptone 3.0g/L, sodium chloride 5.0g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L and pH value 7.3+/-0.2) for culture, shake fermentation culture is carried out on a shaking table at 37 ℃, the separated thalli are resuspended by physiological saline and then are used for treating lupus erythematosus skin lesions induced by combining UVB and Pristane, and single bacteria with the best effect are obtained through screening, and the single bacteria are named as the rosacea mucilaginosa DL-1.

The rhodomonas mucilaginosa DL-1 was identified to have the following properties:

(1) Colony morphology characterization

After 16-24 h culture in R2A medium at 32 ℃, the colony was observed to be a single colony by a microscope, and the morphology is shown as A in FIG. 1. Gram staining is carried out on the strain, the result is shown as B in figure 1, the strain belongs to gram-negative bacteria, and the strain is scanned by an electron microscope, the form is shown as C in figure 1, a large amount of biological membranes are secreted, and the strain has flagellum capable of moving. Cells of 12h can grow in large quantity in the culture medium at 32 ℃, produce red pigment, have a mucilage shape and are arranged singly, in pairs or in short chains. The growth temperature is 5-40 ℃, the optimal temperature is 30-35 ℃, the growth pH is 5.0-9.5, and the optimal pH is 6.5-7.5. Can grow normally in LB, R2A, TSB, BAB and other culture mediums.

(2) 16S rDNA sequence analysis

16S rDNA sequence length 1464 bp. The 16S rDNA sequences were compared with related species in the GeneBank database, and 16S rDNA full sequence based phylogenetic tree was constructed as shown in FIG. 1D. The results show that: the similarity of the strain DL-1 and the rosacea mucilaginosa reaches 99.86 percent. Therefore, the strain of the invention is regarded as the rhodomonas mucilaginosaRoseomonas mucosa) The strain is classified into rhodospirillum, acetobacter, the strain number is DL-1, and the rhodospirillum is preserved in China general microbiological culture Collection center (CGMCC), the preservation time is 2022, 10 months and 26 days, and the preservation number is CGMCC 25967.

Example 2 cultivation of roseomonas mucilaginosa DL-1.

The rhodomonas mucilaginosa DL-1 preserved at the inclined plane of 4 ℃ is activated and inoculated on a seed culture medium (namely an activation culture medium), wherein the activation temperature is 32 ℃, and the activation time is 16 h. Inoculating 10% of seed fermentation broth into fermentation medium at rotation speed of 200 rpm at 32deg.C for 24 hr to obtain fermentation broth with viable bacteria number of 5×10 ⁹ CFU/mL or more.

The activating culture medium is an R2A culture medium, and the culture medium comprises the following components: yeast extract powder 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.

The components of the fermentation medium are tryptone 17.0g/L, soyase peptone 3.0g/L, sodium chloride 5.0g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L and pH value 7.3.

Example 3 live bacterial preparation of roseomonas mucilaginosa DL-1.

Centrifuging the fermentation broth obtained in example 2 (9000 rpm, 5 min), and suspending the bacterial body weight to 60% glycerol (volume fraction) to obtain viable bacteria preparation with viable bacteria number of 1×10 ⁴ -1×10 ¹¹ CFU/mL。

Example 4 preparation of a roseomonas mucilaginosa DL-1 extracellular polysaccharide.

The fermentation broth in example 2 was centrifuged (3000-4000 rpm, 10 min) for sterilization, 1 volume of the fermentation broth was added to 2 volumes of sevag reagent (chloroform: n-butanol=4:1, volume ratio) and repeated 2-3 times to remove proteins, then 1 volume of absolute ethanol was added, and the mixture was centrifuged overnight at 4 ℃ for 3000-4000rpm, 10min to obtain crude extracellular polysaccharide precipitate, which was dried and redissolved with deionized water. And (3) measuring about 5-6 g extracellular polysaccharide crude products by a sulfuric acid-phenol method, separating and purifying by using DEAE centrifugal exchange resin, collecting 0.2M NaCl elution components for freeze-drying, and respectively measuring extracellular polysaccharide EPS monosaccharide composition and molecular weight.

The extracellular polysaccharide EPS monosaccharide assay method is as follows: thermo ICS5000 ion chromatography system (ICS 5000, thermo Fisher Scientific, USA) uses electrochemical detectors to analyze and detect mixed label and sample monosaccharide components. And respectively and accurately weighing the standard substances required by the project, adding water to prepare a standard solution mother solution single standard of 10mg/mL, then mixing a proper amount of standard solution single standard to prepare a standard substance mixed standard with the highest index concentration of 60 mug/mL, 50 mug/mL or 40 mug/mL, and preparing a series of standard substances required by the machine according to the following concentration gradient.

TABLE 1 monosaccharide mix gradient concentration information

* Standards are mainly from sigma company

A Dionex ™ CarboPac ™ PA20 (150 x 3.0mm,10 μm) liquid chromatography column was used; the sample loading was 5. Mu.l. Mobile phase A (H) ₂ O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH,0.2M NaAc), flow rate 0.5ml/min; the column temperature is 30 ℃; elution gradient: 0min A/B/C (95:5:0, V/V), 26min A/B/C (85:5:10, V/V), 42min A/B/C (85:5:10, V/V), 42.1min A/B/C (60:0:40, V/V), 52.1min A/B/C (60:40:0, V/V), 52.1min A/B/C (95:5:0, V/V), 60min A/B/C (95:5:0, V/V).

The results of the standard measurement are shown in FIG. 3, and 13 standards are all unimodal. The sample chromatogram is shown in fig. 4, and is calculated according to the standard curve of the retention time of the standard substance and the concentration of the sample, and the extracellular polysaccharide EPS monosaccharide composition is determined to be arabinose: rhamnose: galactose: glucose: xylose: mannose: ribose: galacturonic acid: glucuronic acid: mannuronic acid: guluronic acid=0.51:1.47:1.8: 73.98:4.67:9.96:0.94:2.33:1.35:1.32:1.67.

The extracellular polysaccharide EPS molecular weight measurement method is as follows: the chromatographic system used was a gel chromatography-differential-multi-angle laser light scattering system, the liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOS II (Wyatt technology, CA, USA). The gel exclusion chromatography column (Ohpak SB-805 HQ (300X 8 mm), ohpak SB-804 HQ (300X 8 mm) and Ohpak SB-803 HQ (300X 8 mm) were used in series, the column temperature was 45 ℃, the sample injection amount was 100. Mu.L, and mobile phase A (0.02% NaN) ₃ ，0.1M NaNO ₃ ) Flow rate 0.4mL/min, elution gradient: isocratic, 100min. As shown in FIG. 5, the retention time of the sample of the extracellular polysaccharide EPS was 63.5min, the sample was calculated according to the Mark-Howink equation, the weight average molecular weight of the extracellular polysaccharide EPS was 3000Da, and the sample was detected as a single peak, and the sample was determinedIs an extracellular polysaccharide EPS pure product.

Example 5 influence of the roseomonas mucilaginosa DL-1 extracellular polysaccharide EPS on the in vitro induced B cell differentiation of IL-4 and R848.

B cell in vitro induced differentiation: fresh spleens of 3C 57BL/J mice of 8 weeks old were ground and filtered, B cells were sorted according to B cell negative selection kit (Biolegend, 480088), and a 24-well plate was plated with a medium containing 500. Mu.L 1640 (containing diabody, 50. Mu.M. Beta. -mercaptoethanol, 30 ng/mL IL-4 and 100 ng/mL R848) at 10 wells ⁵ The B cells after sorting are synchronously added with 5-75 mug/mL of the rhodomonas mucilaginosa DL-1 extracellular polysaccharide EPS for treatment, 3 biological repeats are treated each, the extracellular polysaccharide is not added in a blank group, and the flow detection is carried out after the cell culture box is cultured for 48 and h.

The flow type results are analyzed by using Flowjo, and the results are shown in FIG. 6, compared with a control group, 5-75 mug/mL of the treatment of the DL-1 extracellular polysaccharide EPS of the rhodomonas mucilaginosa can significantly lower the proportion of plasma cells and antibody secretion cells, wherein 75 mug/mL of the treatment of the DL-1 extracellular polysaccharide EPS of the rhodomonas mucilaginosa has the most significant proportion of the plasma cells, and 5 mug/mL of the treatment of the DL-1 extracellular polysaccharide EPS of the rhodomonas mucilaginosa has the most significant proportion of the antibody secretion cells. Therefore, the mucus roseomonas DL-1 extracellular polysaccharide EPS can inhibit the in-vitro induction and differentiation of B cells into plasma cells and antibody secreting cells, and has potential application prospects in autoimmune diseases of B cell overactivation and differentiation.

Example 6 influence of the roseomonas mucilaginosa DL-1 extracellular polysaccharide EPS on anti-CD3 and anti-CD28 induced T cell activation in vitro.

T cell in vitro activation: 300. Mu.L of 1 XPBS containing 5. Mu.g/mL of anti-CD3 was plated in 24-well plates at 4℃for 6-12h, 3C 57BL/J mice of 8 weeks age were freshly spleen ground and filtered and T cells were sorted according to T cell negative selection kit (Biolegend, 480033), 1 XPBS was removed from each well, and 500. Mu.L of 1640 medium (containing diab, 50. Mu.M beta-mercaptoethanol and 2. Mu.g/mL of anti-CD 28) was plated in 24-well plates, 10. Mu.L of each well ⁵ The T cells after sorting are synchronously added with 5-500 mug/mL of the rhodopseudomonas mucilaginosa DL-1 extracellular polysaccharide EPS for treatment, and 3 biological repeats are treated each timeThe blank group was subjected to flow assay after 58-h cell culture in a cell incubator without exopolysaccharide.

The flow chart results are analyzed by using Flowjo, and as shown in FIG. 7, compared with a control group, 5-500 mug/mL of the treatment of the polysaccharide EPS of the rhodomonas mucilaginosa DL-1 has no influence on cell proliferation and apoptosis in the T cell activation process, and the proportion of Treg cells is not obviously influenced, but the proportion of Tfh cells can be obviously reduced, and the influence of 50 mug/mL of the treatment of the polysaccharide EPS of the rhodomonas mucilaginosa DL-1 on the proportion of Tfh cells in the T cell activation is most obvious, so that the T cell activation to Tfh cells can be inhibited. Therefore, the mucus roseomonas DL-1 extracellular polysaccharide EPS can inhibit the activation of T cells into Tfh cells, and has potential application prospect in autoimmune diseases of T cell overactivation and differentiation.

Example 7A viable bacterial agent of roseomonas mucilaginosa DL-1 can alleviate cutaneous lupus erythematosus skin lesions.

The preparation method of the viable bacteria preparation used in the invention is the same as that of example 3.

The skin lupus erythematosus molding method refers to the specific molding method of the issued patent (a skin lupus erythematosus mouse model, a construction method and application thereof, ZL 202110542671.0) as follows;

feeding environment: SPF grade, mice: c57BL/6 female mice, 8 weeks old; sterile Pristane (Sigma-Aldrich, P2870); the molding method comprises the following steps: (1) The purchased C57BL/6 female mice with uniform size and 8 weeks old are stably bred for 2cm multiplied by 2cm of back shaving after one week; (2) Each insulin needle is used for intradermal injection of Pristane, 20-30 needles are counted, the total amount is 250 mu L, and the insulin needle is immunized for one week; (3) Mice without hair growth were selected for UVB induction for 7 days at a UVB dose of 200 mJ/cm ² The strength is 900 mu W/cm ² The method comprises the steps of carrying out a first treatment on the surface of the (4) Mice without hair growth were selected for UVB maintenance for 14 days at a UVB dose of 100 mJ/cm ² The strength is 900 mu W/cm ² . The experiment was set up with 5 treatments, 6 replicates each, 48 mice. The UVB irradiation intensity meter used for the molding is SIGMA SS-07B (280 nm-320 nm), and is calibrated by SOLAR irradiation meter in 2022 and 11 months. The experiment sets 4 treatment modes, and the administration treatment is unified as UVB irradiationStarting on the same day, local administration is carried out once a day, and the treatment mode is as follows:

treatment 1 (HC): the healthy control group is obtained after no treatment;

treatment 2 (CLE): pristane+UVB combined modeling is carried out, namely a CLE model group;

treatment 3 (cle+halometasone): pristane+UVB combination moulding, which was given a topical application of 50 mg/4 cm from UVB irradiation ² Halominosone is a positive control group;

treatment 4 (cle+dl-1): pristane+UVB combined modelling, topical application of a formulation of active rhodopseudomonas mucilaginosa DL-1 (4×10) from UVB irradiation ⁹ CFU/mL, 250 μL/ 4cm ² ) Namely, a live bacteria treatment group.

Other daily management of the 4 treatments remained consistent. After the end of the test, the sacrificial mice were sampled and analyzed for skin lesions and pathological HE staining. As shown in FIG. 8, pristane intradermal injection for 1 week in combination with UVB irradiation induced mild CLE lesions, manifested as a dark red scar on the back of mice, a liquefied degeneration of basal cells seen by HE staining, a small amount of lymphocyte infiltration, and a typical lesion of the hair follicle horn plug, as compared to the healthy panel. Compared with CLE model mice, the active bacteria preparation of the myxorosea DL-1 improves pathological characteristics of bright red or dark red scar-like erythema, hair follicle angular embolism, hyperkeratosis and the like, and has a curative effect equivalent to that of positive control halometasone. This demonstrates that the viable preparation of roseomonas mucilaginosa DL-1 can alleviate skin lesions in CLE mice.

Example 8 effect of a roseomonas mucilaginosa DL-1 inoculant on immune complex deposition in CLE mice.

The immunofluorescent staining of the back tissue of the mice in the different treatment groups of example 7 was performed by the following method for immunofluorescent IgG staining of the skin tissue of the mice:

1) The back skin lesion tissue of the mice, which had taken example 5, was removed from the-80 ℃ refrigerator, embedded with OCT, and the tissue was cut into 5 mm slices using a frozen microtome;

2) Placing the slide in a wet box for rewarming for 10min;

3) Placing the tissue slices into PBS and placing the PBS into a horizontal shaking table at 80rpm for 5min each time for 3 times;

4) After the slice is thrown, PBS around the tissue is wiped off by using paper towel, and the immunohistochemical pen circles around the tissue;

5) Dripping rapid immunostaining sealing liquid, and incubating for 10min at room temperature;

6) PBS is washed for 2 times, each time for 5min;

7) Dripping a direct-labeling secondary antibody FITC-Mouse IgG (H+L) (diluted 1:200, abclon, cat. No. AS 001) on the surface of the tissue, and incubating in a wet box at 37 ℃ in a dark place for 60min;

8) PBS is washed 3 times at 80rpm for 5min each time;

9) Two DAPI-containing caplets were added dropwise to the tissue, incubated at room temperature in the dark for 5min and then observed under a fluorescence confocal microscope.

The results are shown in figure 9, where Pristane and UVB combined induction resulted in deposition of IgG immune complexes in basal membrane bands at the back skin lesions of mice compared to healthy panelists. Both the rhodomonas mucilaginosa DL-1 live preparation and the positive control halominosone group reduced deposition of IgG immune complexes on the basal membrane band at the back skin lesions of mice compared to CLE model mice.

Example 9 effect of roseomonas mucilaginosa DL-1 on local immune cells on skin lesions in CLE mice.

Skin flow experiments were performed with 3 mice back tissues per treatment group in example 7, wherein the skin digestion procedure was as follows:

(1) After washing each mouse skin tissue three times with 1mL HBSS, the droplets were squeezed out as much as possible, transferred to a 6-well plate, and 1mL of pre-chilled skin digestion buffer (containing 100 μg/mL DNase I (ROCHE, 10104159001) and 1 mg/mL Collagenase P (ROCHE, 11213857001), prepared ready-to-use with high sugar DMEM (Hyclone, SH 30022), pre-chilled at 4 ℃);

(2) Cutting into pieces on ice quickly with curved scissors as small as possible;

(3) According to 3.5 mL/cm ² Adding skin digestion buffer solution according to skin injury area, incubating at 37deg.C for 90min, and mixing for multiple times during digestion;

(4) Filtered through a 40 μm cell filter into a 50 mL centrifuge tube, washed with 12 mL high sugar DMEM containing 10% FBS and collected, centrifuged at 400g at 4℃for 5min, the supernatant discarded, resuspended in 400. Mu.L PBS and counted.

The flow dyeing method is shown in table 2:

TABLE 2

(5) 200. Mu.L of cells were plated in 24-well plates, 300. Mu.L of medium was added, and 0.5. Mu.L of ionomycin Golgi apparatus inhibitor stimulation (BD, 550583) was added, and incubated at 37℃for 5-6h;

(6) Centrifuging at 4deg.C for 5min at 400g, discarding supernatant, resuspending 1mL of 1 XPBS cells, adding 1 mu L Fixable Viability Stain dead dye, and incubating at room temperature in the absence of light for 15min;

(7) Centrifuging at 4deg.C for 5min at 400g, discarding supernatant, adding 0.5 μl Fc block vortex, and incubating at 4deg.C for 10min;

(8) Adding a streaming antibody: after adding CD45 (9. Mu.L) CD4 (9. Mu.L) ICOS (18. Mu.L) CD19 (9. Mu.L) to 1780. Mu.L 1 XPBS and mixing, 100. Mu.L of each sample was added, vortexed, and incubated at 4℃for 25 min;

(9) Adding 1mL of PBS, centrifuging at 4deg.C for 5min, discarding supernatant, treating with a nuclear membrane disruption kit (eBioscience, 00-5523-00), adding 1mL of Fixation/Permeablization working solution (1X) into each tube, and incubating at 4deg.C for 50-60min in the absence of light;

(10) 400g is centrifuged for 5min, the supernatant is discarded, and 1mL of 1X Permeabilization buffer,4 ℃ is added for 10min of incubation;

(11) Centrifuging 500g for 5min, discarding supernatant, adding IL-4 (18 μL), IL-17A (18 μL), IFN-gamma (18 μL) and Bcl-6 (27 μL), adding 1760 μL Perm/Wash Buffer (1X), mixing, adding 100 μL of each sample, vortexing, and incubating at 4deg.C for 40-50 min;

(12) 500g centrifugation for 5min, removing supernatant, adding 1mL PBS, filtering with 70 μm cell strainer, transferring to flow tube, centrifuging at 4deg.C for 5min, removing supernatant, and loading with 200 μLPBS vortex.

Analysis of flow results using Flowjo results as shown in figure 10, pristane and UVB combined induction resulted in a significant increase in B cells, tfh cells, th1, th2 and Th17 cells at the back lesions in mice compared to healthy panelists. Compared with a CLE model mouse, the treatment of the myxoroseona DL-1 live bacterial preparation and the positive control halominosone can inhibit the increase trend of local B cells, tfh cells, th1, th2 and Th17 cells of the back skin damage of the CLE mouse, and has the effect of relieving inflammatory cell infiltration at the CLE skin damage.

Example 10 Rosemonas mucilaginosa DL-1 extracellular polysaccharide EPS can alleviate cutaneous lupus erythematosus skin lesions.

The preparation method of the roseomonas mucilaginosa DL-1 extracellular polysaccharide EPS used in the invention is the same as that of example 4.

The method for molding cutaneous lupus erythematosus was the same as in example 7. But the UVB radiation intensity meter used in this molding was a UV-B ultraviolet radiation meter (probe 279) from north master optoelectronics, inc.

The test sets 4 treatment modes, the administration treatment is unified as starting on the day of UVB irradiation, local administration is carried out once a day, and the treatment modes are as follows:

treatment 1 (HC): the healthy control group is obtained after no treatment;

treatment 4 (cle+eps): pristane+UVB combination modeling, administration of topical Rosemonas viscosa DL-1 exopolysaccharide EPS (40 mg/mL, 25 μL/4 cm) from UVB irradiation ² ) I.e., exopolysaccharide treatment groups.

Other routine daily management of the 4 treatments remained consistent. After the end of the test, the sacrificial mice were sampled and analyzed for skin lesions and pathological HE staining. As shown in FIG. 11, pristane intradermal injection for 5 weeks in combination with UVB irradiation induced severe CLE lesions, manifested as bright red scars on the backs of mice, HE staining with visible liquefied degeneration of basal cells, massive lymphocyte infiltration, and hair follicle horn plugs, among other typical lesions, compared to healthy panelists. Compared with CLE model mice, the mucus rosacea DL-1 extracellular polysaccharide EPS obviously improves pathological features such as bright red scar-like erythema, hair follicle angular embolism, hyperkeratosis and the like, has obvious curative effect compared with positive control halometasone, and the mucus rosacea DL-1 viable bacteria preparation can relieve skin damage of the CLE mice.

Example 11 influence of roseomonas mucilaginosa DL-1 extracellular polysaccharide EPS on immune complex deposition in CLE mice.

Using the method of example 10 for staining the back skin lesions of mice, igG immunocomplex deposition was similar to example 8, and the results are shown in FIG. 12, in which Pristane and UVB combined induction resulted in the deposition of IgG immunocomplexes in the basal membrane band at the back skin lesions of mice, compared to the healthy group. Compared with the matrix group treatment group, the deposition of the IgG immune complex of the basal mulch tape at the back skin lesion of the mice is not seen in the mucus roseomonas DL-1 extracellular polysaccharide EPS and the positive control halominosone treatment group.

Example 12 influence of the extracellular polysaccharide EPS of D.viscosus DL-1 on the transcription levels of local inflammatory factors IL-6, IL-1. Beta. And IL-17A in CLE mice.

The mouse skin lesion tissue in example 10 was sufficiently ground by a tissue grinder, and then total cellular RNA was extracted using TRIZOL kit. The total RNA was reverse transcribed into cDNA using a reverse transcription kit, and target mRNA expression levels were detected on an ABI Prism 7900 real-time quantitative PCR instrument using a SYBR Green kit. The relative expression level of mRNA of the target gene was determined according to 2 using beta-actin as a reference ^－ΔΔCt And (5) calculating.

As shown in FIG. 13, pristane and UVB combined induction can cause significant increase of the transcription level of IL-6, IL-1 beta and IL-17A of local main inflammatory factors of back skin lesions of mice, and the transcription increase of IL-6, IL-1 beta and IL-17A of the local main inflammatory factors of skin lesions caused by Pristane and UVB combined induction can be inhibited by adopting the treatment of the extracellular polysaccharide EPS of the DL-1 of the rhodomonas mucilaginosa.

The invention provides a skin symbiotic bacteria rhodopseudomonas mucilaginosaRoseomonas mucosa) Viable bacteria preparation and extracellular polypore thereofA preparation method of sugar and application thereof in treating cutaneous lupus erythematosus. Topical application to UVB and Pristane induced skin lesions in CLE model miceR. mucosaThe living bacteria and extracellular polysaccharide thereof can obviously improve the skin damage of mice with CLE model. The invention is simple, safe and effective, and provides a new idea for clinical and transformation researches of other autoimmune diseases. The method and means for implementing the technical solution are numerous, the above description is only a preferred embodiment of the present invention, it should be noted that, for a person skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. A kind of rhodomonas mucilaginosa is named as rhodomonas mucilaginosa @Roseomonas mucosa) The strain number is DL-1, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 2022, 10 months and 26 days, and the preservation number is CGMCC No. 25967.

2. A viable bacterial preparation of roseomonas mucilaginosa, characterized in that it comprises roseomonas mucilaginosa according to claim 1.

3. The viable rhodomonas mucilaginosa preparation according to claim 2, wherein the viable number of viable rhodomonas mucilaginosa in the viable bacterial preparation is 1x 10 ⁴ -9×10 ¹¹ CFU/mL。

4. A method for preparing a viable bacterial preparation of the myxorhodomonas according to claim 2 or 3, wherein the myxorhodomonas according to claim 1 is inoculated in an R2A culture medium, activated for 12-24 h at 25-37 ℃, the activated myxorhodomonas is transferred in a fermentation culture medium, cultured for 24-36 h at 30-37 ℃, and centrifuged for 5-10 min at 4000-9000 rpm to obtain myxorhodomonas cells, and a dosing solvent is added to obtain the viable bacterial preparation.

5. A method for producing a polysaccharide from a fermentation broth comprising the bacterium of claim 1, wherein the fermentation broth is purified by sterilization, deproteinization, absolute ethanol precipitation and DEAE centrifugal exchange resin.

6. The roseomonas mucilaginosa extracellular polysaccharide according to claim 5, wherein the weight average molecular weight of the roseomonas mucilaginosa extracellular polysaccharide is 3000-3500Da.

7. The mucorosacea extracellular polysaccharide according to claim 6, wherein the mucorosacea extracellular polysaccharide consists of arabinose, rhamnose, galactose, glucose, xylose, mannose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, and guluronic acid.

8. Use of a viable bacterial formulation according to claim 2 or a rhodomonas mucilaginosa extracellular polysaccharide according to any one of claims 5 to 7 in the manufacture of a medicament or formulation for the treatment of cutaneous lupus erythematosus lesions.

9. The use according to claim 8, wherein the cutaneous lupus erythematosus lesions comprise one or more of acute cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus and chronic cutaneous lupus erythematosus and subtypes thereof.

10. The use according to claim 8, wherein the live bacterial preparation or the extracellular polysaccharide of rhodomonas mucilaginosa modulates the immune microenvironment at the skin lesion, reducing immune complex IgG deposition.

11. The use according to claim 8, wherein the rhodomonas mucilaginosa exopolysaccharide inhibits the induced differentiation of B cells into plasmablasts and antibody secreting cells in vitro.

12. The use according to claim 8, wherein the rhodomonas mucilaginosa exopolysaccharide inhibits anti-CD28 in vitro induced T cell activation into Tfh cells.