Phytobacterium plantarum capable of relieving allergic rhinitis and application thereof

Abstract

The invention relates to a plant lactobacillus capable of relieving allergic rhinitis and application of the plant lactobacillus, and belongs to the technical field of biological medicines. The invention provides a plant lactobacillus XA-8187 strain and a preparation method thereof. The plant lactobacillus XA-8187 can relieve symptoms of allergic rhinitis of mice, reduce the content of histamine in serum of the mice with the allergic rhinitis, reduce the content of pro-sensitive cytokines in the serum of the mice with the allergic rhinitis, reduce the level of pro-inflammatory cytokines in the serum of the mice with the allergic rhinitis and repair damaged nasal mucosa of the mice with the allergic rhinitis. The lactobacillus plantarum XA-8187 has the advantages that the lactobacillus plantarum XA-8187 can be used for preventing and / or treating allergic rhinitis, the anti-DNP-IgE induced histamine release can be reduced, the anti-DNP-IgE induced RBL-2H3 cell degranulation degree can be reduced, and the LPS induced release of peripheral blood mononuclear cells on sensitizing cytokines can be reduced, so that the lactobacillus plantarum XA-8187 has a huge application prospect in the preparation of products for preventing and / or treating allergic rhinitis.

Description

Technical Field

[0001] The present invention relates to a Lactiplantibacillus plantarum strain capable of alleviating allergic rhinitis and its application, belonging to the field of biomedical technology. Background Art

[0002] Allergic rhinitis, also known as allergic rhinitis (AR), refers to a non-infectious inflammatory disease of the nasal mucosa mainly mediated by immunoglobulin E (IgE) after a susceptible individual contacts an allergen, involving immune active cells and cytokines of the body, with paroxysmal sneezing, clear watery nasal discharge, nasal itching and nasal congestion as the main symptoms, and belongs to one of the chronic allergic diseases. When allergic rhinitis attacks, the nasal mucosa is pale and edematous. If there is also an allergic constitution, it will cause urticaria. In severe cases, it may also evolve into sinusitis, asthma or ear infections.

[0003] At present, allergic rhinitis can only control symptoms through drug treatment, and there is no complete cure method. For example, antihistamine drugs such as cetirizine hydrochloride and loratadine can be prescribed when the patient is ill. In addition to antihistamine drugs, hormonal drugs such as corticosteroids can also reduce the attack frequency of allergic rhinitis or alleviate its allergic reaction, and decongestants can relieve the congestion and edema of the nasal mucosa and improve the nasal congestion symptoms of allergic rhinitis. However, traditional antihistamine drugs will cause side effects such as drowsiness, mental fatigue, headache and constipation in patients, hormonal drugs are also prone to cause side effects such as endocrine disorders, decreased immunity and osteoporosis, and decongestants will lead to the occurrence of high blood pressure, angina attacks, arrhythmia, migraine and ischemic stroke. Therefore, there is an urgent need to find a treatment drug for allergic rhinitis with lower side effects, higher effectiveness and less prone to immune tolerance. Summary of the Invention

[0004] To solve the above problems, the present invention provides a Lactiplantibacillus plantarum strain XA-8187, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 32482 and the deposit date of November 4, 2024.

[0005] The Lactiplantibacillus plantarum strain XA-8187 is derived from fresh fecal samples of healthy people in the Shenzhen area. After sequencing analysis, its 16S rDNA sequence is shown in SEQ ID NO. 1. The sequenced sequence is compared with the nucleic acid sequence in the Blastn program of NCBI, and the result shows that the strain is Lactiplantibacillus plantarum, named Lactiplantibacillus plantarum XA-8187.

[0006] The present invention also provides the use of the above-mentioned Lactiplantibacillus plantarum in the preparation of a medicament for preventing and / or treating allergic diseases, and the allergic diseases include allergic rhinitis.

[0007] In one embodiment of the present invention, the allergic disease is allergic rhinitis; the prevention and / or treatment of allergic rhinitis includes relieving the symptoms of allergic rhinitis, inhibiting the immune response caused by allergic rhinitis, inhibiting the inflammatory response caused by allergic rhinitis, and / or repairing the nasal mucosa damage caused by allergic rhinitis.

[0008] In one embodiment of the present invention, relieving the symptoms of allergic rhinitis includes reducing the frequency of nose scratching, sneezing and / or runny nose in patients with allergic rhinitis.

[0009] In one embodiment of the present invention, in the medicament, the viable count of the above-mentioned Lactiplantibacillus plantarum XA-8187 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] In one embodiment of the present invention, the medicament contains the above-mentioned Lactiplantibacillus plantarum XA-8187 and pharmaceutical excipients.

[0011] In one embodiment of the present invention, the pharmaceutical excipients include excipients and / or additives.

[0012] In one embodiment of the present invention, the excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, absorbents, diluents, flocculants, deflocculants, filter aids, and / or release retardants.

[0013] In one embodiment of the present invention, the additives include microcrystalline cellulose, hydroxypropyl methylcellulose, and / or refined lecithin.

[0014] In one embodiment of the present invention, the dosage form of the medicament is powder, granule, capsule, tablet, pill or oral liquid.

[0015] The present invention also provides the use of the above-mentioned Lactiplantibacillus plantarum in the preparation of an anti-inflammatory medicament.

[0016] In one embodiment of the present invention, the anti-inflammation includes inhibiting the inflammatory response by inhibiting the secretion of pro-inflammatory cytokines by immune cells.

[0017] In one embodiment of the present invention, in the medicament, the viable count of the above-mentioned Lactiplantibacillus plantarum XA-8187 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0018] In one embodiment of the present invention, the drug contains the above-mentioned Lactiplantibacillus plantarum XA-8187 and pharmaceutical excipients.

[0019] In one embodiment of the present invention, the pharmaceutical excipients include excipients and / or additives.

[0020] In one embodiment of the present invention, the excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, absorbents, diluents, flocculants, deflocculants, filter aids and / or release retardants.

[0021] In one embodiment of the present invention, the additives include microcrystalline cellulose, hypromellose and / or refined lecithin.

[0022] In one embodiment of the present invention, the dosage form of the drug is powder, granule, capsule, tablet, pill or oral liquid.

[0023] The present invention also provides a product containing the above-mentioned Lactiplantibacillus plantarum XA-8187.

[0024] In one embodiment of the present invention, in the product, the viable count of the above-mentioned Lactiplantibacillus plantarum XA-8187 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0025] In one embodiment of the present invention, the product includes food or drugs; the food is medical food, health food or functional beverage.

[0026] In one embodiment of the present invention, the drug has any one of the following functions:

[0027] (a) Treating allergic diseases;

[0028] And / or, (b) anti-inflammatory;

[0029] The allergic diseases include allergic rhinitis.

[0030] In one embodiment of the present invention, the drug contains the above-mentioned Lactiplantibacillus plantarum XA-8187 and pharmaceutical excipients.

[0031] In one embodiment of the present invention, the pharmaceutical excipients include excipients and / or additives.

[0032] In one embodiment of the present invention, the excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, absorbents, diluents, flocculants, deflocculants, filter aids and / or release retardants.

[0033] In one embodiment of the present invention, the adjuvant comprises microcrystalline cellulose, hydroxypropyl methylcellulose and / or refined lecithin.

[0034] In one embodiment of the present invention, the dosage form of the drug is powder, granule, capsule, tablet, pill or oral liquid.

[0035] In one embodiment of the present invention, the food contains the above-mentioned Lactiplantibacillus plantarum XA-8187 and food additives.

[0036] In one embodiment of the present invention, the food additives comprise antioxidants, bleaching agents, colorants, color retention agents, enzyme preparations, flavor enhancers, preservatives and / or sweeteners.

[0037] In one embodiment of the present invention, the food is a probiotic beverage or a fermented dairy product.

[0038] The technical solution of the present invention has the following advantages:

[0039] The present invention provides a strain of Lactiplantibacillus plantarum XA-8187, and the preservation number of this Lactiplantibacillus plantarum XA-8187 is CGMCC No. 32482.

[0040] Research shows that Lactiplantibacillus plantarum XA-8187 can significantly relieve the symptoms of allergic rhinitis in mice (i.e., reduce the number of nose scratching, sneezing and runny nose in mice with allergic rhinitis), can significantly reduce the histamine content in the serum of mice with allergic rhinitis, can significantly reduce the content of pro-allergic cytokines (IL-2, IL-4, IL-5 and IL-13) in the serum of mice with allergic rhinitis, can significantly reduce the levels of pro-inflammatory cytokines (IL-6 and THF-α) in the serum of mice with allergic rhinitis, can significantly repair the damaged nasal mucosa of mice with allergic rhinitis, can significantly reduce the histamine concentration released by anti-DNP-IgE-induced RBL-2H3 cells, can significantly reduce the degree of degranulation of anti-DNP-IgE-induced RBL-2H3 cells, and can significantly reduce the release of pro-allergic cytokines (IL-2, IL-5 and IL-13) by LPS-induced peripheral blood mononuclear cells (PBMCs). It can be seen that Lactiplantibacillus plantarum XA-8187 has great application prospects in the preparation of products (such as foods or drugs, etc.) for preventing and / or treating allergic rhinitis.

[0041] At the same time, research shows that Lactiplantibacillus plantarum XA-8187 can significantly reduce the release of pro-inflammatory cytokines (IL-6 and THF-α) by peripheral blood mononuclear cells (PBMCs). It can be seen that Lactiplantibacillus plantarum XA-8187 has great application prospects in the preparation of anti-inflammatory drugs.

[0042] In addition, research shows that Lactiplantibacillus plantarum XA-8187 does not have clear virulence factors and drug resistance genes. The growth of Lactiplantibacillus plantarum XA-8187 can be controlled by various antibiotics. Moreover, Lactiplantibacillus plantarum XA-8187 did not show hemolytic traces in the hemolytic test. It can be seen that Lactiplantibacillus plantarum XA-8187 has high safety and has great application prospects in the preparation of products for preventing and / or treating allergic rhinitis (such as foods or drugs, etc.) and the preparation of anti-inflammatory drugs.

[0043] Biological material preservation

[0044] A strain of Lactiplantibacillus plantarum XA-8187, taxonomically named Lactiplantibacillus plantarum, was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 04, 2024. The deposit number is CGMCC No. 32482, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the drawings

[0045] Figure 1 : Plate growth status (a) and staining (b) of Lactiplantibacillus plantarum XA-8187.

[0046] Figure 2 : Utilization of main nutrients by Lactiplantibacillus plantarum XA-8187.

[0047] Figure 3 : Whole genome map of Chr1 of Lactiplantibacillus plantarum XA-8187.

[0048] Figure 4 : Phylogenetic analysis of Lactiplantibacillus plantarum XA-8187 and reference genomes.

[0049] Figure 5 : Effect of Lactobacillus paracasei GOLDGUT-Lpc969 on the secretion of IL-2 by LPS-induced human PBMC.

[0050] Figure 6 : Effect of Lactobacillus paracasei GOLDGUT-Lpc969 on the secretion of IL-5 by LPS-induced human PBMC.

[0051] Figure 7 : Effect of Lactobacillus paracasei GOLDGUT-Lpc969 on the secretion of IL-13 by LPS-induced human PBMC.

[0052] Figure 8:Effect of Lactobacillus paracasei GOLDGUT-Lpc969 on the secretion of IL-6 by LPS-induced human PBMCs.

[0053] Figure 9 :Effect of Lactobacillus paracasei GOLDGUT-Lpc969 on the secretion of THF-α by LPS-induced human PBMCs.

[0054] Figure 10 :Effect of Lactiplantibacillus plantarum XA-8187 on the DAI score of OVA-induced allergic rhinitis mice.

[0055] Figure 11 :Effect of Lactiplantibacillus plantarum XA-8187 on the nasal mucosa pathology of OVA-induced allergic rhinitis mice.

[0056] Figure 12 :Effect of Lactiplantibacillus plantarum XA-8187 on the serum IL-2 concentration of OVA-induced allergic rhinitis mice.

[0057] Figure 13 :Effect of Lactiplantibacillus plantarum XA-8187 on the serum IL-4 concentration of OVA-induced allergic rhinitis mice.

[0058] Figure 14 :Effect of Lactiplantibacillus plantarum XA-8187 on the serum IL-5 concentration of OVA-induced allergic rhinitis mice.

[0059] Figure 15 :Effect of Lactiplantibacillus plantarum XA-8187 on the serum IL-13 concentration of OVA-induced allergic rhinitis mice.

[0060] Figure 16 :Effect of Lactiplantibacillus plantarum XA-8187 on the serum IL-6 concentration of OVA-induced allergic rhinitis mice.

[0061] Figure 17 :Effect of Lactiplantibacillus plantarum XA-8187 on the serum TNF-α concentration of OVA-induced allergic rhinitis mice.

[0062] Figure 18 :Effect of Lactiplantibacillus plantarum XA-8187 on the production of histamine by anti-DNP-IgE-induced RBL-2H3 cells.

[0063] Figure 19 :Effect of Lactiplantibacillus plantarum XA-8187 on the degranulation release of anti-DNP-IgE-induced RBL-2H3 cells.

[0064] Figure 20 :Hemolysis test results of Lactiplantibacillus plantarum XA-8187.

[0065] Figures 5 to 19 Among them, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Specific implementation manners

[0066] The following embodiments are provided to better further understand the present invention, and are not limited to the described optimal implementation manner. They do not constitute a limitation to the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0067] For those not specifying specific experimental steps or conditions in the following embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0068] The culture media involved in the following embodiments are as follows:

[0069] MRS solid medium: peptone 10 g / L, beef extract powder 5 g / L, yeast extract powder 4 g / L, glucose 20 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 g / L, agar 15 g / L, cysteine hydrochloride 0.5 g / L, pH 6.8.

[0070] MRS liquid medium: peptone 10 g / L, beef extract powder 5 g / L, yeast extract powder 4 g / L, glucose 20 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 g / L, cysteine hydrochloride 0.5 g / L, pH 6.8.

[0071] Experimental example 1: Obtaining, screening and identification of Lactiplantibacillus plantarum

[0072] The specific steps are as follows:

[0073] Using fresh feces from healthy people in the Shenzhen area as a sample, take 0.5 g of the fresh feces sample and add it to 5 mL of MRS liquid medium, and culture at 37 °C for 24 h for enrichment to obtain an enriched sample; aspirate 0.5 mL of the enriched sample and add it to 4.5 mL of sterile physiological saline to obtain 10 -1 dilution, and then aspirate 0.5 mL of 10 -1 dilution into 4.5 mL of physiological saline to obtain 10 -2 dilution. According to this operation, 10 -3, 10 -4 , 10 -5 , 10 -6 Diluent; Pipette 100 μL of the gradient diluent and spread it on the MRS solid medium, 10 -4 , 10 -5 , 10 -6 One plate for each gradient, incubate at 37 °C for 48 h to obtain colonies; Select the colonies with typical characteristics of Lactobacillus paracasei on the MRS solid medium according to the colony shape, size, edge, transparency, etc., pick the colonies with an inoculation loop and streak them on the MRS solid medium, incubate at 37 °C for 48 h to obtain purified single colonies; Pick the purified single colonies and inoculate them into 5 mL of MRS liquid medium respectively, incubate at 37 °C for 24 h to obtain bacterial solutions; After numbering each strain corresponding to each bacterial solution, perform Gram staining, strain identification, physiological and biochemical experiments, and genomic identification and analysis according to the steps recorded in the textbook "Microbiology" (edited by Shen Ping and Chen Xiangdong), select the strains with typical characteristics of Lactobacillus plantarum to obtain strain XA-8187.

[0074] Among them, the process of Gram staining is as follows:

[0075] Pick a single colony of XA-8187 for bacterial smear, heat-fix it after drying; Drop crystal violet for staining for 10 s, wash with water and shake dry, drop iodine solution for staining for 10 s, wash with water and shake dry, drop decolorizing solution for decolorization for 10 s, wash with water and shake dry, drop safranin solution for counterstaining for 10 s, wash with water; After the specimen slide dries naturally, drop 1 drop of cedarwood oil at the smeared bacteria area for oil immersion observation, and the observation result is: XA-8187 is purple by Gram staining and is a rod-shaped Gram-positive bacterium (see the plate growth condition and staining result of XA-8187 in Figure 1 ).

[0076] The process of strain identification is as follows:

[0077] Take the XA-8187 bacterial cells, extract the genome of XA-8187 using a bacterial genomic DNA extraction kit, and use the primer pair 27F / 1492R with sequences shown in SEQ ID NO.2 and SEQ ID NO.3 respectively (27F: AGAGTTTGATCMTGGCTCA, 1492R: TGTACGGYTACCTTGTTACGACTT; in 27F and 1492R, M and Y are degenerate bases, M = A or C, Y = C or T). Using the extracted genome of XA-8187 as a template for amplification, obtain the 16S rRNA of XA-8187 (the 16S rDNA sequence of XA-8187 is shown in SEQ ID NO.1); perform nucleic acid sequence alignment of the 16S rDNA of XA-8187 using the Blastn program of NCBI. The results show that this strain is Lactiplantibacillus plantarum, named Lactiplantibacillus plantarum XA-8187. Deposit Lactiplantibacillus plantarum XA-8187 in the General Microbiology Center of the China Microbial Culture Collection Center, with the deposit number CGMCC No. 32482 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0078] The physiological and biochemical experiment process is as follows:

[0079] Use a sterile cotton swab to pick a single colony of XA-8187 and inoculate it into sterile saline. Use a calibrated 2DensiCHEKTM Plus to prepare a homogeneous bacterial suspension equivalent to a McFarland turbidity of 3.0, and load it onto API 20A for physiological and biochemical identification within 30 minutes of preparation. The results show that XA-8187 can utilize substances such as N-acetyl-D-glucosamine, L-alanine glycine, D-xylose, β-methyl-D-glucoside, D-ribose, D-arabinose, etc. as carbon sources (see Figure 2 ) for the main nutrient utilization of XA-8187.

[0080] The genomic identification and analysis process is as follows:

[0081] Take the XA-8187 bacterial cells, extract the DNA of the bacterial cells by the SDS method, detect the quality of the extracted DNA by agarose gel electrophoresis, and use 2.0 Perform DNA quantification; the genome was sequenced using the Nanopore PromethION platform and the Illumina NovaSeq platform, and the sequencing strategy was a 10Kb library with a sequencing depth of ≥100×. The sequencing results showed that strain XA-8187 contained a circular genome with a size of 2.97 Mbp, and three circular plasmids with sizes of 12.88 kbp, 49.33 kbp, and 78.09 kbp respectively (see the whole genome map of XA-8187 in Figure 3 ). Download the reference genome of Lactiplantibacillus plantarum for phylogenetic analysis based on the core genome to determine the species information of strain XA-8187, and analyze the similarity between strain XA-8187 and the reference genome. Among them, comparative genomic analysis and core genome construction were performed using the orthorfinder software, and phylogenetic analysis was performed using the best calculation model evaluated by the iqtree software (see the phylogenetic tree results of XA-8187 in Figure 4 ).

[0082] The preparation of the XA-8187 bacterial suspension is as follows:

[0083] Streak Lactiplantibacillus plantarum XA-8187 on MRS solid medium and culture at 37 °C for 48 h to obtain single colonies; pick single colonies and inoculate them into MRS liquid medium, culture at 37 °C for 18 h for activation to obtain an activated solution; inoculate the activated solution into MRS liquid medium at an inoculation amount of 4% (v / v) and culture at 37 °C for 18 h to obtain a bacterial solution; centrifuge the bacterial solution at 3000 g for 10 min to obtain Lactiplantibacillus plantarum cells; wash the Lactiplantibacillus plantarum cells with 0.9% (m / v, g / 100 mL) physiological saline and resuspend them in 0.9% (m / v, g / 100 mL) physiological saline until the bacterial concentration is 1×10 10 CFU / mL to obtain the XA-8187 bacterial suspension.

[0084] Example 2: Effects of Lactiplantibacillus plantarum XA-8187 on LPS-induced human PBMCs

[0085] 1. Experimental procedure

[0086] Peripheral blood mononuclear cells (PBMCs) are a cell population isolated from peripheral blood, mainly including lymphocytes and monocytes, which play an important role in the immune system and are involved in various physiological and pathological processes such as the body's response to infection, tumors, and inflammation. Due to their good representativeness (able to reflect the body's immune response to various stimuli) and good responsiveness (able to show a significant response to inflammatory stimuli such as lipopolysaccharide LPS and release various cytokines and inflammatory mediators). Therefore, PBMCs have become an important cell line for studying in vitro anti-inflammatory models.

[0087] After taking out human PBMC cells (purchased from Shanghai Anneng) from the liquid nitrogen tank, thaw them by shaking in a 37°C water bath for 2 minutes to obtain thawed human PBMC cells; first wash the human PBMC cells with pre-warmed PBS buffer at 37°C, and then add 8 mL of cell culture medium (RPMI-1640 medium containing 10% fetal bovine serum inactivated at 56°C for 30 minutes, % refers to volume ratio, RPMI-1640 medium and fetal bovine serum are purchased from Gibco), and culture at 5% (v / v) CO 2 at 37°C for 48 h until the logarithmic growth phase; adjust the density of human PBMC cells in the logarithmic growth phase to 1×10 6 cells / mL with cell culture medium, then inoculate into a 96-well plate, 125 μL per well, that is, 8×10 5 cells / well.

[0088] Set a control well (CON) in the 96-well plate, without adding any stimulant (only add the same amount of RPMI 1640 medium as the bacterial suspension added to the strain treatment well in the control well). Add lipopolysaccharide (LPS, purchased from Sigma-Aldrich) with a final concentration of 1 μg / mL to other wells, and stimulate at 5% (v / v) CO 2 at 37°C for 2 h; after the stimulation, wash three times with PBS buffer, replace with fresh RPMI-1640 medium (purchased from Gibco), and set up model wells (RPMI1640) without adding drugs and strain interactions (add the same amount of RPMI 1640 medium as the bacterial suspension added to the strain treatment well in the model well), positive control wells treated with the control drug sodium butyrate (SB) (add the same amount of drug solution as the bacterial suspension added to the strain treatment well in the positive control well, the concentration of the drug solution is 5 mg / mL, and the solvent is physiological saline), and several groups of strain treatment wells with different strain interactions (including Lactiplantibacillus plantarum XA-8187 and other Lactiplantibacillus plantarum screened from the same sample as Lactiplantibacillus plantarum XA-8187), and set the MOI of the strain treatment well to 1:1 (add bacterial suspensions of different strains). After the addition, incubate the 96-well plate at 5% (v / v) CO 2 at 37°C for 22 hours. After the incubation, centrifuge to collect the cell culture supernatant, and use a multiplex cytokine kit (LEGENDplex TM HU Th1 / Th2 Panel (8-plex) w / VbP V02, purchased from Biolegend, catalog number 741030) to detect the cytokine levels in the cell culture supernatant, and the experimental results are shown in Figures 5 to 9 .

[0089] 2. Experimental Results

[0090] IL-2, IL-5, and IL-13 in the interleukin (IL) family have been shown to be positively correlated with the severity of allergic rhinitis as pro-sensitizing factors. As Figures 5 to 7 shown, the levels of the four cytokines IL-2, IL-5, and IL-13 secreted by PBMC cells after LPS stimulation all showed an upward trend (increased by about 20%, 100%, and >100% respectively). After the intervention treatment with the strain, the levels of cytokines in some experimental groups showed an obvious downward trend, among which Lactiplantibacillus plantarum XA-8187 was the most obvious. It tended to be the control group and showed a decrease compared with the model group (decreased by about 66%, 80%, and 90% respectively). It was proved that Lactiplantibacillus plantarum XA-8187 could relieve LPS-induced PBMC inflammatory factors by regulating the cytokine levels in the supernatant, which had a positive effect on the treatment of allergic rhinitis.

[0091] IL-6 and TNF-α have been shown to be positively correlated with inflammation in the body as pro-inflammatory factors. As Figures 8 to 9 shown, the secretion of IL-6 by PBMC cells after LPS stimulation increased significantly (increased by about 1000%), and TNF-α also showed an upward trend (increased by about 1200%). After the intervention treatment with the strain, the levels of IL-6 and THF-α in some experimental groups decreased. Among them, Lactiplantibacillus plantarum XA-8187 was the most obvious, with a decrease of about 51% and 90% respectively, indicating that Lactiplantibacillus plantarum XA-8187 could inhibit the inflammatory phenotype of LPS-stimulated PBMC, which had a positive effect on the treatment of allergic rhinitis and inflammation.

[0092] Example 3: Effect of Lactiplantibacillus plantarum XA-8187 on a murine model of ovalbumin (OVA)-induced allergic rhinitis

[0093] 1. Experimental procedure

[0094] Thirty-six 6- to 8-week-old SPF-grade BABL / C female mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., weighing 18 ± 2 g) were randomly divided into 4 groups, with 9 mice in each group. The 3 groups were: control group (CON), model control group (AR), positive control group (AZE), and probiotic intervention group (XA-8187) intragastrically administered with XA-8187 bacterial suspension.

[0095] Mice in the model group, positive control group, and probiotic intervention group were intraperitoneally injected with 200 μL of OVA suspension (OVA concentration was 1 mg / mL, and the solvent was normal saline) on days 0, 7, and 14 of the experiment to sensitize the mice. Mice in the probiotic intervention group started intragastric administration of 200 μL of a bacterial concentration of 1×10 10XA-8187 bacterial suspension at CFU / mL (with 0.9% normal saline as the solvent). In the positive control group, mice were intragastrically administered with 15 mg / kg of AZE (azelastine hydrochloride) according to their body weights every day. In the control group and the model control group, the same amount of 0.9% normal saline was intragastrically administered. Starting from the 21st day of the experiment, in the model group, the positive control group, and the probiotic intervention group, 500 μg of OVA was instilled into the nasal cavity every day to create a mouse model of allergic rhinitis. Starting from the 28th day of the experiment, the allergic rhinitis symptoms (number of nose scratching, number of sneezing, and number of nasal discharge) of all groups of mice within ten minutes after nasal instillation were recorded. The 29th day was the end point of the experiment. After all mice were euthanized, inflammatory evaluation was carried out according to the evaluation criteria. Among them, the main indicators for evaluating allergic rhinitis were DAI score, histopathological section evaluation, and cytokine content in mouse serum.

[0096] The DAI score includes the number of nose scratching, the number of sneezing, and the number of nasal discharge. For specific details, please refer to the literature "Zhu Y, Yu J, et al. Experimental observation of the effect of immunotherapy on CD4+T cells and Th1 / Th2 cytokines in mice with allergic rhinitis, Scientific Reports, 2023, 13:5273". When the experiment reached the end point, the DAI score results of all groups of mice are shown in Figure 10 。

[0097] At the end point of the experiment, mice were euthanized and nasal mucosa was taken, fixed with 4% (m / v, g / 100 mL) paraformaldehyde; after fixation, paraffin section preparation, H&E staining, and PAS staining experiments were carried out to evaluate the pathological results of the nasal mucosa. The evaluation results are shown in Figure 11 。

[0098] After mice were euthanized at the end point of the experiment, blood of all groups of mice was collected and serum was separated. The LEGENDplex MUT h1 / Th2 panel (8-plex) cytokine detection kit (product number 741053, purchased from Shenzhen Dakewei) was used to detect the cytokine content in the serum. The statistical results are shown in Figures 12 to 17 。

[0099] 2. Experimental Results

[0100] The DAI score is one of the important indicators for evaluating the rhinitis symptoms of mice. This score is composed of the number of nose scratching, sneezing, and nasal discharge. The higher the score, the more severe the allergic rhinitis symptoms of the mice. As Figure 10As shown, compared with the control group (about 0.2 points), the DAI score of the model group mice increased significantly (increased to about 2 points). After treatment with strain XA-8187, the DAI score of the mice decreased significantly compared with the model group (decreased to about 1 point). The above results indicate that XA-8187 can alleviate the increase in DAI score caused by OVA, and XA-8187 can significantly reduce the behaviors of itching and scratching the nose, sneezing, and running nose in mice caused by OVA.

[0101] In the pathological sections of the nasal mucosa of allergic rhinitis mice, compared with normal mice, there will be phenomena such as cilia shedding, vascular congestion, vascular hyperplasia, nasal mucosa thickening, and inflammatory cell infiltration. As Figure 11 shown, the mice in the control group were normal, the surface of the nasal mucosa was intact and neat, and there was no phenomenon of mucosal swelling. In the model group, the mucosal layer was significantly thickened, and the arrangement of the mucosal surface was not neat. In the mice treated with strain XA-8187, the nasal mucosa was significantly restored, the epithelial structure was intact and neatly arranged, and the mucosal thickness was restored to a level similar to that of the control group.

[0102] IL-2, IL-4, IL-5, and IL-13 in the interleukin (IL) family have been proven to be positively correlated with the severity of allergic rhinitis as pro-sensitizing factors. As Figures 12 to 15 shown, after OVA stimulation, the levels of the four cytokines IL-2, IL-4, IL-5, and IL-13 in the serum of mice all showed an upward trend (increased by about 20%, 200%, 100%, and 220% respectively). After treatment with XA-8187, the cytokine levels tended to be that of the control group and decreased compared with the model group (decreased by about 23%, 55%, 51%, and 96% respectively). It is proved that XA-8187 can relieve the symptoms of allergic rhinitis by regulating the cytokine levels in the serum. In the results of IL-2 and IL-13, the performance of XA-8187 is even better than that of Lactobacillus paracasei GOLDGUT-Lpc969 (Lactobacillus paracasei GOLDGUT-Lpc969 is recorded in the patent application text with the publication number CN117511808A).

[0103] IL-6 and TNF-α have been proven to be positively correlated with inflammation in the body as pro-inflammatory factors. As Figures 16 to 17 shown, after OVA stimulation, the level of IL-6 in the serum of mice increased significantly (increased by about 170%), and TNF-α also showed an upward trend (increased by about 38%). After treatment with strain XA-8187, the levels of IL-6 and THF-α both decreased (decreased by about 51% and 36% respectively), indicating that XA-8187 can inhibit inflammation in mice and has a positive effect on the treatment of allergic rhinitis.

[0104] Example 4: Effect of Lactiplantibacillus plantarum XA-8187 on the secretion of histamine by anti-DNP-IgE-induced RBL-2H3 cells

[0105] 1. Experimental procedure

[0106] After taking out RBL-2H3 cells (purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences) from the liquid nitrogen tank, thaw them by moving and shaking in a 37°C water bath for 3 minutes to obtain thawed RBL-2H3 cells; wash the RBL-2H3 cells first with pre-warmed PBS buffer at 37°C, and then add 8 mL of cell culture medium (MEM medium containing 10% fetal bovine serum, % refers to volume ratio, MEM medium is purchased from Gibco, and fetal bovine serum is purchased from Solarbio), and culture at 5% (v / v) CO 2 , 37°C for 48 h until the logarithmic growth phase; adjust the density of RBL-2H3 cells in the logarithmic growth phase to 5×10 5 cells / mL with cell culture medium, then inoculate into a 24-well plate, 400 μL per well, that is, 2×10 5 cells / well.

[0107] Set a control well (CON) without adding any stimulant, and add 0.2 μg / mL anti-DNP-IgE (purchased from Sigma) to other wells, and stimulate at 5% (v / v) CO 2 , 37°C for 16 h; after the stimulation is over, wash three times with PBS buffer, replace with fresh MEM medium (purchased from Gibco), set a treated well without strain interaction (Treated) and a treatment well with strain XA-8187 interaction, add the strain (MOI = 10) and then interact at 5% (v / v) CO 2 , 37°C for 3 hours; after the interaction is over, add 1 μg / mL of DNP-HAS (purchased from 4ADI) to each well, and stimulate at 5% (v / v) CO 2 , 37°C for 15 min; after the stimulation is over, aspirate 100 μL of cell culture supernatant, add 20 μL of 1 M NaOH solution and 25 μL of reaction solution, and incubate in the dark at room temperature (25°C) for 4 min; after the incubation is over, add 10 μL of 3 M HCl solution to terminate the reaction; after terminating the reaction, use a multifunctional microplate reader to detect the fluorescence intensity (excitation wavelength is 355 nm, emission wavelength is 460 nm), and calculate the histamine level in each well through the formula: Relative Secretion = OD460 x / OD460 CON，avrg (where OD460 x is the fluorescence intensity of the sample at 460 nm, and OD460 CON，avrg is the average value of the fluorescence intensity of the CON group at 460 nm), and the experimental results are shown in Figure 18; wherein, the reaction solution is methanol (purchased from Sangon Biotech (Shanghai) Co., Ltd.) containing 1% (w / v, g / 100 mL) o-phthalaldehyde (purchased from Sigma).

[0108] 2. Experimental results

[0109] As the center of pruritus mediators, the histamine content is proportional to the severity of allergic symptoms. As Figure 18 shown, the histamine level secreted by the RBL-2H3 cells in the treatment group was significantly higher than that in the control group (increased by about 63%), and when treated with Lactiplantibacillus plantarum XA-8187, a significant decrease in histamine was observed (decreased by about 67%). Thus, Lactiplantibacillus plantarum XA-8187 can reduce the histamine concentration released by anti-DNP-IgE-induced RBL-2H3 cells, which further indicates that Lactiplantibacillus plantarum XA-8187 has a therapeutic effect on allergic diseases such as allergic rhinitis.

[0110] Example 5: Effect of Lactiplantibacillus plantarum LPC969 on the degranulation of anti-DNP-IgE-induced RBL-2H3 cells

[0111] 1. Experimental procedure

[0112] After taking out the RBL-2H3 cells (purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences) from the liquid nitrogen tank, thaw them in a 37°C water bath with shaking for 3 min to obtain thawed RBL-2H3 cells; wash the RBL-2H3 cells first with pre-warmed PBS buffer at 37°C, and then add 8 mL of cell culture medium (MEM medium containing 10% fetal bovine serum, % refers to volume ratio, MEM medium is purchased from Gibco, and fetal bovine serum is purchased from Solarbio), and culture at 5% (v / v) CO 2 2, 37°C for 48 h until the logarithmic growth phase; adjust the density of the RBL-2H3 cells in the logarithmic growth phase to 5×10 5 cells / mL with cell culture medium, and then inoculate them into a 24-well plate, 400 μL per well, that is, 2×10 5 cells / well.

[0113] Set a control well (CON) without adding any stimulant, and add 0.2 μg / mL anti-DNP-IgE (purchased from Sigma) to other wells, and culture at 5% (v / v) CO 2, Stimulate at 37°C for 16 h; after the stimulation ends, wash three times with PBS buffer, replace with fresh MEM medium (purchased from Gibco), set up positive control wells treated with 20 μg / mL anti-allergy drug azelastine hydrochloride (AZE, purchased from Macklin), wells of complete degranulation release treated with 0.1% (v / v) surfactant Triton X-100 (purchased from Sigma), and treatment wells interacting with strain XA-8187. Two groups with MOI of 10 and 1 were set up in the treatment wells (XA-8187 MO1 = 10, XA-8187 MOI = 1). After adding the strain, incubate in 5% (v / v) CO 2 , Interact at 37°C for 3 hours; after the interaction ends, add 1 μg / mL DNP-HAS (purchased from 4ADI) to each well, and incubate in 5% (v / v) CO 2 , Stimulate at 37°C for 15 min; after the stimulation ends, aspirate 25 μL of cell culture supernatant, add an equal volume of substrate solution, and incubate in 5% (v / v) CO 2 , React at 37°C for 1.5 min; after the reaction ends, add 200 μL of sodium carbonate-sodium bicarbonate buffer with a concentration of 0.1 M and a pH of 10.0 to terminate the reaction; after terminating the reaction, use a multifunctional microplate reader to detect the fluorescence intensity (excitation wavelength is 355 nm, emission wavelength is 405 nm) to evaluate the degree of cell degranulation (the higher the OD 405 value, the higher the degree of cell degranulation). The experimental results are shown in Figure 19 ; among them, the substrate solution is a sodium citrate solution with a concentration of 0.2 M and a pH of 4.5 containing 5 mM p-nitrophenyl-N-acetyl-β-d-glucosaminide (purchased from Sinopharm Chemical Reagent Co., Ltd.) and 5 mM p-nitrophenol-N-acetylglucosamine (NP-GlcNAc, purchased from Sinopharm Chemical Reagent Co., Ltd.).

[0114] 2. Experimental results

[0115] Inhibiting cell degranulation is an important treatment strategy for alleviating the symptoms of allergic rhinitis. As Figure 19 shown, the degranulation level of RBL-2H3 cells in the Triton X-100 treatment group after stimulation was significantly higher than that in the control group (OD 405 increased by about 0.4), while when treated with Lactiplantibacillus plantarum XA-8187, a significant decrease in the cell degranulation level was observed under the premise of MOI of 10 or 1 (OD 405Decreased by approximately 0.32 and 0.27 respectively). It can be seen that Lactiplantibacillus plantarum XA-8187 can significantly reduce the degree of degranulation of RBL-2H3 cells induced by anti-DNP-IgE, which further indicates that Lactiplantibacillus plantarum XA-8187 has a therapeutic effect on allergic diseases such as allergic rhinitis.

[0116] Example 6: Safety identification of Lactiplantibacillus plantarum XA-8187

[0117] The amino acid sequence encoded by the genome of strain XA-8187 was compared with the VFDB (Virulence Factors Database) database, and it was found that the sequence identity of potential virulence factors was all below 70%, and most of them had no clear function (the comparison results are shown in Table 1). The amino acid sequence encoded by the genome of strain XA-8187 was compared with the ARDB (Antibiotic Resistance Genes Database) database to analyze potential drug resistance genes, and it was found that the identity was all below 65% compared with the drug resistance gene sequences (the comparison results are shown in Table 2). The above results all indicate that strain XA-8187 has high safety.

[0118] At the same time, the drug resistance of XA-8187 was tested by AST (Antibiotic Sensitivity Test). The cells of XA-8187 were taken, transferred and spread evenly on a Brucella agar plate containing 5% (v / v) sheep blood (purchased from Qingdao Rishui Biotechnology) to create evenly distributed colonies. Antibiotic-containing discs were placed on the inoculated agar, and the plate was incubated at the optimal temperature of 37°C for 20 hours. After incubation, the formation of inhibition zones around each disc was observed. The results showed that XA-8187 was sensitive to 4 antibiotics including tetracycline, gentamicin, clindamycin and ampicillin (the test results are shown in Table 3). This result indicates that the growth of strain XA-8187 can be controlled by a variety of antibiotics, further indicating that the strain has high safety.

[0119] In addition, the hemolytic test of the strain is an important experimental method for evaluating the potential pathogenicity of the strain. By observing the hemolytic phenomenon of the strain on the blood plate, it can be judged whether the strain produces hemolysin, so as to evaluate its ability to destroy red blood cells. The hemolytic test of strain XA-8187 was carried out with reference to the literature "Jiao Yanping, Yu Ping, Zhao Di, Zhang Chunyu, Song Jia, & Tang Chun, etc. (2022). Safety evaluation and probiotic characteristics analysis of Lactiplantibacillus plantarum hcs03-001. Science and Technology of Food Industry, 43(5), 165-171." As Figure 20As shown, XA-8187 showed no hemolytic traces in the hemolytic test, further verifying the high safety of the strain.

[0120] Table 1 Virulence Factors Database Analysis (VFDB)

[0121]

[0122]

[0123] Table 2 Antibiotic Resistance Genes Database Analysis (ARDB)

[0124]

[0125] Table 3 XA-8187 Antibiotic Sensitivity Test Results (AST)

[0126] tetracycline erythromycin kanamycin gentamicin clindamycin chloramphenicol vancomycin ampicillin streptomycin TC EM KM GM CM CL VA AM SM 16 1.5 / 12 1.5 8 / 0.25 192 S R R S S I R S R 32 1 64 16 4 8 n.r 2 n.r

[0127] In Table 3, the unit of antibiotic concentration is μg / mL; *S: Sensitive; R: Resistant; I: Intermediate.

[0128] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum, characterized in that: The plant lactobacillus is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.32482.

2. Use of Lactobacillus plantarum in the preparation of a medicament for preventing and / or treating allergic diseases, characterized in that: The allergic diseases include allergic rhinitis.

3. The use according to claim 2, characterized in that The plant lactobacillus is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.32482.

4. The use according to claim 2 or 3, characterized in that The allergic disease is allergic rhinitis; the prevention and / or treatment of allergic rhinitis includes relieving the symptoms of allergic rhinitis, inhibiting the immune response caused by allergic rhinitis, inhibiting the inflammatory response caused by allergic rhinitis and / or repairing nasal mucosal damage caused by allergic rhinitis.

5. The use according to claim 4, characterized in that The relieving of the symptoms of allergic rhinitis includes reducing the frequency of nose scratching, sneezing and / or runny nose in patients with allergic rhinitis.

6. Application of Lactobacillus plantarum in the preparation of anti-inflammatory drugs.

7. The use according to claim 6, characterized in that The plant lactobacillus is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.32482.

8. The use according to claim 6 or 7, characterized in that The anti-inflammatory effect includes inhibiting the secretion of pro-inflammatory cytokines by immune cells, thereby inhibiting the inflammatory response.

9. A product, characterized in that The product contains the Lactobacillus plantarum according to claim 1.

10. A product according to claim 9, characterized in that The product comprises a food or a medicine.

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