Lactobacillus paragaseri and its application

Abstract

The present invention discloses a Lactobacillus paragasseri, which has a deposit number of CGMCC No. 29973. The Lactobacillus paragasseri provided by the present invention can reduce inflammation and change the structure and composition of intestinal flora, thereby effectively preventing, treating and alleviating urticaria and its symptoms. It has important application value in regulating intestinal flora disorders caused by urticaria and preventing and treating urticaria.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular, to a Lactobacillus paragaseri and an application thereof in preparing a preparation for preventing and treating urticaria. Background Art

[0002] Urticaria, also known as wheals or skin edema, is a skin disease caused by mast cell activation, which leads to dilation of small blood vessels in the skin and mucous membranes and increased permeability. This lesion disappears within 24 hours but can recur. Urticaria is common in all ages and races, with a global incidence ranging from 1% to 30%, with a higher incidence in women. Because it is not an infectious disease, it is not contagious. Based on medical history and physical examination, urticaria can be divided into spontaneous urticaria and induced urticaria, and spontaneous urticaria can be divided into acute and chronic. Antihistamines are generally used clinically for the symptomatic treatment of urticaria. Although second-generation antihistamines are currently the first-line clinical drug for the treatment of acute or chronic urticaria, nearly 50% of patients still do not experience significant improvement in symptoms after taking regular doses or 4 times the dose of second-generation antihistamines.

[0003] The pathogenesis of urticaria is complex and is considered an immune-mediated inflammatory skin disease characterized by mast cell activation and the release of inflammatory mediators. Autoantibodies against self-antigens, such as IgE or FcεRI α chain, activate mast cells primarily through FcεRI. Mast cell activation can be mediated by multiple immune cells, including dendritic cells, B cells, T cells, vascular endothelial cells, and inflammatory cytokines such as interleukin (IL)-4, IL-5, and IL-10. Therefore, in-depth research on the pathogenesis of chronic spontaneous urticaria (CSU) is urgently needed to explore new diagnostic tools and develop innovative treatment strategies. Purines are converted to uric acid through a series of enzymatic reactions, generating reactive oxygen species (ROS). Purine disorders are associated with a variety of diseases, including hyperuricemia and gout, nephrolithiasis, immunodeficiency, anemia, peripheral and central nervous system diseases, and myopathy. However, the potential relationship between chronic spontaneous urticaria (CSU) and impaired purine metabolism remains unclear.

[0004] Previous studies have shown that the gut microbiota plays a key role in human health, the development and maturation of the immune system, and resistance to pathogen invasion. Imbalances in the gut microbiota are associated with several immune-mediated allergic diseases, including atopic dermatitis, asthma, and urticaria. For example, Björkstén et al. found that children with atopic dermatitis had reduced gut microbial diversity, with decreased relative abundance of Bifidobacterium and Lactobacillus, while the proportions of Escherichia coli, Clostridium difficile, and Staphylococcus aureus increased. Arrieta et al. found that infants at risk for asthma generally exhibited gut microbial imbalances during the first 100 days of life. Further observation revealed that compared with healthy children, children at risk for asthma had significantly lower relative abundances of Lachnospira, Veillonella, Faecalibacterium, and Roseburia in their guts. Wang et al. found that patients with urticaria had increased numbers of unidentified Enterobacteriaceae and decreased numbers of Bacteroidetes, Faecalibacterium, Bifidobacterium, Firmicutes, and unidentified Ruminococci.

[0005] Using probiotics to regulate the intestinal flora and alleviate metabolic disorders has become a new approach for treating allergic diseases. Some clinical trials have shown that Lactobacillus, alone or in combination with Bifidobacterium, can reduce the severity of urticaria and improve quality of life. Other studies have found that combining probiotics with antihistamines can significantly reduce the pruritus and number of wheals associated with urticaria. Therefore, the search for a probiotic for the treatment or combination therapy of urticaria has become a pressing issue. Summary of the Invention

[0006] Technical issues solved:

[0007] One aspect of the present disclosure is to address the problem in the prior art of lack of new probiotics for the prevention, treatment, auxiliary treatment and symptom relief of urticaria, and to provide a Lactobacillus paragaseri and uses thereof.

[0008] Technical solution:

[0009] A Lactobacillus paragaseri ( Lactobacillus paragasseri ), its deposit number is CGMCC No.29973.

[0010] The Lactobacillus paragaseri described in the present disclosure is deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC), the deposit address of which is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 8, 2024, and the deposit number is CGMCC NO. 29973.

[0011] The urticaria described in the present disclosure is isolated from the breast milk of healthy women and is identified as Lactobacillus spp. Lactobacillus ), Lactobacillus paragaseri species ( Lactobacillus paragasseri ), named LG-1.

[0012] In order to further improve the functions of the above-mentioned strains, in some embodiments of the present invention, the above-mentioned strains can be transformed using conventional methods in the prior art. The modified variants contain the basic functions and properties of the above-mentioned strains, and the modified variants are also considered to be included in the protection scope of the present invention. At the same time, the variants also include strain variants with natural or spontaneous genetic changes and mutant strains obtained by continuous passage. The variants usually have the same or substantially the same gene sequence as the preserved strain, for example, at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical 16S rDNA fragments to the preserved strain. Similarly, these variants are also considered to be included in the protection scope of the present invention.

[0013] In the present disclosure, the Lactobacillus paragaseri can be stored or used in a suitable formulation, such as a lyophilized formulation, a capsule formulation, a liquid formulation, a tablet formulation, a gel formulation, or an emulsion formulation. In one embodiment of the present disclosure, the Lactobacillus paragaseri is a liquid lyophilized formulation.

[0014] The inventors of the present disclosure have verified the effect of the above-mentioned Lactobacillus paragasseri on urticaria through in vitro and in vivo animal experiments. Therefore, another aspect of the present disclosure is to provide the use of the above-mentioned Lactobacillus paragasseri in the preparation of a drug for preventing or treating urticaria.

[0015] Meanwhile, another aspect of the present disclosure is to provide the use of the above-mentioned Lactobacillus paragaseri in the preparation of a medicine for alleviating urticaria symptoms.

[0016] Meanwhile, another aspect of the present disclosure is to provide the use of the above-mentioned Lactobacillus paragaseri in the preparation of veterinary drugs for treating or preventing urticaria.

[0017] In the present disclosure, the Lactobacillus paragaseri can be used to prevent, treat, assist in the treatment of, or alleviate urticaria in humans. Similarly, it can also be used as a veterinary drug to prevent, treat, assist in the treatment of, or alleviate urticaria in other mammals, such as pets (cats, dogs, etc.), cattle, sheep, horses, and other livestock.

[0018] In the present disclosure, the above-mentioned Lactobacillus paragaseri may be living bacteria and / or dead bacteria, and metabolites thereof.

[0019] When used as a drug for humans, the dosage of the above-mentioned Lactobacillus paragaseri can be adjusted according to individual conditions. Generally, in some embodiments of the present disclosure, the total amount of the above-mentioned Lactobacillus paragaseri taken per day is ≥1×10 6 CFU / g.

[0020] Furthermore, based on the research findings of the present disclosure, another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating urticaria, comprising the aforementioned Lactobacillus paragaseri as its active ingredient. In the pharmaceutical composition, the Lactobacillus paragaseri can be in a suitable formulation, such as a lyophilized formulation, a capsule formulation, a liquid formulation, a tablet formulation, a gel formulation, or an emulsion formulation. In some embodiments of the present disclosure, the pharmaceutical composition can be taken orally.

[0021] In some embodiments of the present disclosure, the pharmaceutical composition may further comprise at least one selected from the culture, lysate, extract, or metabolite of Lactobacillus paragaseri as its active ingredient. The culture, lysate, extract, or metabolite has the same or similar efficacy as that of Lactobacillus paragaseri.

[0022] In order to enhance the effects of prevention, treatment, adjuvant therapy, and relief of urticaria, in some embodiments of the present disclosure, the above-mentioned Lactobacillus paragaseri can also be used in combination with other drugs that have the effects of prevention, treatment, adjuvant therapy, and relief of urticaria. In some embodiments of the present disclosure, the drug used in combination can be an antihistamine. Further, in certain embodiments of the present disclosure, the antihistamine can be one or more selected from diphenhydramine, promethazine, chlorpheniramine, cetirizine, loratadine, ebastine, desloratadine, levocetirizine, fexofenadine, cimetidine, ranitidine, or famotidine.

[0023] When used as a pharmaceutical composition for humans, the amount of the Lactobacillus paragaseri can be adjusted according to individual conditions. Generally, in some embodiments of the present disclosure, the total amount of the Lactobacillus paragaseri taken per day is ≥1×10 6 CFU / g.

[0024] Another aspect of the present disclosure provides the use of the aforementioned Lactobacillus paragaseri in the preparation of a medicament for correcting purine metabolism disorders. Furthermore, in some embodiments of the present disclosure, the aforementioned medicament or preparation for correcting purine metabolism disorders is a medicament or preparation that degrades uric acid, hypoxanthine, adenylic acid, guanylic acid, or inosinic acid.

[0025] Beneficial effects:

[0026] The Lactobacillus paragaseri provided by the present disclosure can reduce inflammation and change the structure and function of the intestinal flora, thereby effectively preventing, treating and alleviating urticaria and its symptoms, and has important application value in regulating the intestinal flora and purine metabolism disorders caused by urticaria and in preventing and treating urticaria.

[0027] Biodeposit Information:

[0028] Accession number: CGMCC No. 29973

[0029] Deposit date: March 8, 2024

[0030] Depository: China General Microbiology Culture Collection Center, CGMCC, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0031] Classification name: Lactobacillus paragaseri BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph showing the results of degradation of uric acid, hypoxanthine, adenosine monophosphate (AMP), guanylate monophosphate (GMP), and inosine monophosphate (IMP) in the purine metabolic pathway by Lactobacillus subtilis LG-1 in the embodiments of the present disclosure;

[0033] Figure 2 A SNP phylogenetic tree diagram of Lactobacillus subglabrata LG-1 constructed based on whole genome sequencing data in the embodiments of the present disclosure;

[0034] Figure 3 This is a scanning electron microscope image of the secondary Grignard lactic acid rod LG-1 in the embodiment of the present disclosure;

[0035] Figure 4 This is a graph showing the results of the scratching behavior evaluation of urticaria mice treated with LG-1 in the embodiments of the present disclosure;

[0036] Figure 5 This is a diagram showing the evaluation results of the back skin pathological condition of urticaria mice treated with Lactobacillus subtilis LG-1 in the embodiments of the present disclosure;

[0037] Figure 6 This is a graph showing the results of the intervention of LG-1 on the IgE content in urticaria mice in the examples disclosed herein;

[0038] Figure 7 This is a graph showing the results of the intervention of Lactobacillus subtilis LG-1 on the skin oxidoreductase levels in urticaria mice according to the disclosed embodiment, wherein A is superoxide dismutase, B is malondialdehyde, and C is catalase;

[0039] Figure 8 This is a graph showing the qPCR results of inflammatory factors in mice with urticaria treated with Lactobacillus subtilis LG-1 in the embodiment of the present disclosure, wherein A represents IL-4, B represents TNF-α, C represents IL-1β, and D represents IL-10;

[0040] Figure 9Graph showing the results of the intervention of Lactobacillus subtilis LG-1 on xanthine oxidase levels in the ileum (A) and serum (B) of urticaria mice in the examples of the present disclosure;

[0041] Figure 10 Graph showing the results of the intervention of Lactobacillus subtilis LG-1 on the levels of purine metabolites in the plasma (A) and feces (B) of urticaria mice in the examples disclosed herein;

[0042] Figure 11 : This is a graph showing the α-diversity analysis results of the intestinal flora of urticaria mice treated with Lactobacillus subtilis LG-1 in the embodiments of the present disclosure, wherein A is the Chao 1 index; B is the richness index;

[0043] Figure 12 Graph showing the analysis results of intestinal flora composition in urticaria mice treated with Lactobacillus subtilis LG-1 in the disclosed embodiments, wherein A is a relative abundance graph of flora at the genus level, and B is a graph showing characteristic bacterial genera with significant differences. DETAILED DESCRIPTION

[0044] The present invention discloses a strain of Lactobacillus paragaseri having the function of alleviating urticaria and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. It is obvious that relevant persons can modify or appropriately change and combine the content described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0045] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "comprise" or variations thereof such as "comprises" or "comprising" will be understood to include the elements or components recited without excluding other elements or other components. The terms "such as" and "for example" are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0046] I. The following is an explanation of some of the terms used in this invention.

[0047] The term "Lactobacillus" can be used to refer not only to the specific Lactobacillus species of the present invention itself, but also more broadly to the culture supernatant or cell-free culture filtrate derived from the culture medium in which the specific Lactobacillus species of the present invention has been cultured. In certain embodiments, in the case of administration of the Lactobacillus species itself, the cells are viable and reproducibly grown.

[0048] The term "lyophilized preparation" refers to probiotics or compositions containing probiotics obtained by freeze-drying. Such freeze-drying methods are well known to those skilled in the art. Generally, the moisture content of the lyophilized preparation should be less than 5%, preferably less than 3%.

[0049] The term "feed additive" refers to substances added to feed in small or trace amounts to meet the nutritional needs of animals and improve feed utilization. These substances play an important role in enhancing the nutritional value of basic feed, improving animal production performance, ensuring animal health, reducing feed costs, and improving meat quality. In some embodiments of the present disclosure, the animal can be a mammal, including a pet. It can be a domesticated animal for food or a laboratory animal used in experiments, such as mice.

[0050] II. Degradation of uric acid, hypoxanthine, adenylate, guanylate, or inosinate in the purine metabolic pathway by Lactobacillus paragaseri.

[0051] In one embodiment of the present disclosure, the inventors conducted further research on the Lactobacillus paragaseri described in the present disclosure and found that it has a degrading effect on uric acid, hypoxanthine, adenylate, guanylate, or inosinate in the purine metabolic pathway, either alone or independently. The specific research method is as follows:

[0052] LG-1 was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 48 hours. Take 2 mL of bacterial suspension, centrifuge at 4000 rpm for 10 minutes, wash three times with 1 mL of PBS, discard the supernatant, and suspend the bacterial pellet in 750 μL of uric acid, hypoxanthine, inosinic acid, adenylic acid, and guanylic acid solution, respectively, and incubate at 37°C for 60 minutes. Subsequently, centrifuge at 4000 rpm for 10 minutes to obtain the supernatant. Add 100 μL of 5% trifluoroacetic acid to the supernatant, mix thoroughly to prevent further reaction, filter with a 0.22 um filter membrane, and inject 10 μL into a high performance liquid chromatograph for detection. The results are as follows. Figure 1 The results showed that after 1 hour of treatment, the degradation rates of uric acid, hypoxanthine, adenylate, guanylate and inosine by the Lactobacillus paragaricea LG-1 strain isolated from breast milk were 22.8%, 10.94%, 23.74%, 44.7% and 27.53%, respectively.

[0053] III. Effects of Lactobacillus paragaseri on other gut microbiota species.

[0054] Based on the research disclosed herein, the present disclosure also provides a method of treating Lactobacillus paragaseri ( Lactobacillus paragasseri LG-1 ) in the preparation of increasing intestinal lactic acid bacteria ( Lactobacillus ) and reduce cocci ( Enterococcus) and Aerococcus. Furthermore, in certain embodiments of the present disclosure, the combination of Lactobacillus paragaseri and antihistamines can improve the effect of drugs on the richness of intestinal microbial flora.

[0055] IV. Examples:

[0056] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0057] Example 1: Isolation, culture and identification of LG-1

[0058] 1. Preparation of culture medium:

[0059] Preparation of isolation culture medium: The culture medium used for the isolation and culture of lactobacilli is MRS solid medium. The mass concentration of each component of MRS solid medium is as follows: peptone 10g, beef extract powder 8g, yeast extract powder 4g, glucose 20g, potassium dihydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g, agar 14g, Tween 80 1g. The pH value is adjusted to 6.5±0.2 at room temperature and dissolved in 1L distilled water.

[0060] Preparation of culture medium for amplification of bacteria: The culture medium used for enrichment of lactobacilli is MRS broth medium. The mass concentration of each component of MRS broth medium is as follows: peptone 10g, beef extract powder 8g, yeast extract powder 4g, glucose 20g, potassium dihydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g. The pH value is adjusted to 5.7±0.2 at room temperature and dissolved in 1L distilled water.

[0061] 2. Isolation, identification and purine degradation ability assessment:

[0062] Twenty lactic acid bacteria strains isolated from breast milk were stored in the Institute of Food Science, Zhejiang Academy of Agricultural Sciences.

[0063] To evaluate their ability to degrade uric acid and hypoxanthine, all isolates were inoculated on MRS plates and cultured anaerobically at 37°C for 48 hours. Two mL of culture medium was centrifuged at 4000 rpm for 10 minutes, washed three times with 1 mL of PBS, and resuspended in 750 μL of uric acid and hypoxanthine solutions, respectively. The supernatant was incubated at 37°C for 60 minutes and centrifuged at 4000 rpm for 10 minutes. The supernatant was then added with 100 μL of 5% trifluoroacetic acid (TFA) and mixed thoroughly to prevent further degradation. After filtration, 20 μL of the supernatant was loaded onto a high-performance liquid chromatography (HPLC) apparatus. HPLC conditions were as follows: CAPCELL PAK ADME S5 (4.6 mm i.d. × 250 mm), mobile phase: 50 mmol / L ammonium acetate (pH 4.65), flow rate: 1 mL / min, column temperature: 40°C, wavelength: 254 nm, elution time: 20 minutes. Plasma and fecal samples were pretreated as follows: For plasma samples, 50 μl was mixed with 200 μl of 0.08 mol / L sulfuric acid solution and 100 μl of 10% sodium tungstate solution. The samples were vortexed at 3000 rpm for 1 minute, then sonicated in an ice-water bath for 2 minutes. The samples were then centrifuged at 12,000 rpm and 4°C for 10 minutes. Fecal samples were freeze-dried to dryness. The powder of each sample was weighed into an Eppendorf tube, and 20 μl of 80% methanol was added per mg of powder. The samples were homogenized using a ball mill at 30 Hz for 2 minutes three times, using two metal beads, followed by sonication in an ice-water bath for 5 minutes. The tubes were centrifuged at 14,000 rpm and 5°C for 20 minutes. The supernatant was concentrated by vacuum centrifugation and redissolved in an equal volume of water. 120 μl of the supernatant was transferred to a fresh Eppendorf tube and filtered through a 0.22 μm filter. Finally, 10 μl of the sample solution was injected into the ADME column. The HPLC results were as follows: Figure 1 As shown: The degradation rates of uric acid, hypoxanthine, adenylate, guanylate, and inosinate by the LG-1 strain were 22.8%, 10.94%, 23.74%, 44.7%, and 27.53%, respectively. Full-length 16S rRNA gene and whole-genome sequencing of the strain were performed. Comparison of the resulting sequences with the NCBI database and phylogenetic tree analysis revealed that the LG-1 strain was most closely related to Lactobacillus paragaseri (see Figure 2 Scanning electron microscopy showed that LG-1 had a typical morphology of Lactobacillus paragaseri (see Figure 3 ).

[0064] Example 2: Effects of Lactobacillus paragaseri on scratching behavior and histopathology in urticaria mice

[0065] 1. Establishment of mouse model:

[0066] 1) Thirty BALB / c mice were randomly divided into normal control group (NC), model group (Model), Lactobacillus group (LG-1), positive drug group (Loratadine), and Lactobacillus combined with positive drug group (LG-1+Loratadine), with 6 mice in each group.

[0067] 2) The normal control group received an intraperitoneal injection of 0.1 ml of PBS. Mice in the other four groups received a primary immunization on day 1 and a second immunization of 0.1 ml each containing a PBS suspension (0.1 mg ovalbumin and 100 mg aluminum hydroxide) on day 10. Oral gavage began on day 8 after the initial immunization and continued daily for 21 consecutive days. The dosing schedule is shown in Table 1. On day 22, mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital. Orbital blood was collected and aliquoted into standard centrifuge tubes and EDTA-anticoagulant tubes. After 30 minutes of rest, the blood was centrifuged at 3000 rpm for 20 minutes to obtain serum and plasma. Liver, dorsal skin, spleen, and intestinal tissues were also collected from each group. After washing with normal saline, all samples were stored at −80°C, except for samples used for histopathology, which were stored in 4% paraformaldehyde.

[0068] Table 1 Dosage schedule

[0069]

[0070] 2. Mouse behavioral assessment

[0071] Within 12 hours after the second immunization, except for the mice in the normal control group, all mice showed symptoms such as lethargy, scratching, eye swelling, and jelly-like stool, indicating that the model was successfully established. Within 30 minutes after the second immunization, the degree of itching of mice in each group was observed and recorded, including the scratching latency, scratching duration, and number of scratching. Scratching behavior refers to scratching the head with the front paws, scratching the trunk with the hind paws, and biting all parts of the body with the mouth. Figure 4 (AC) Compared with the control group, mice in the urticaria group showed obvious scratching behavior. After LG-1 intervention, the scratching behavior of urticaria mice was alleviated, with both the number of scratching and the duration of scratching significantly reduced (P < 0.05). This suggests that Lactobacillus paragaseri can effectively alleviate the clinical symptoms of urticaria mice.

[0072] 3. Evaluation of mouse back skin pathology

[0073] The skin tissue from the back of the mouse was taken, fixed in 4% paraformaldehyde overnight, and then dehydrated with 80%, 90%, 95% and anhydrous ethanol in a gradient manner, made transparent in xylene, and embedded in wax. The embedded wax block was fixed on a microtome, sectioned, and stained with HE and sealed with neutral gum. The pathological changes of the skin tissue, including inflammation, edema, and capillary dilation, were observed under a microscope and scored. Scoring criteria: 0 points: no edema; no capillary dilation; no inflammatory cell infiltration; 1 point: mild edema; mild capillary dilation; mild inflammatory cell infiltration; 2 points: moderate edema; moderate capillary dilation, moderate inflammatory cell infiltration; 3 points: severe edema; severe capillary dilation, severe inflammatory cell infiltration. The histological score of each mouse is the average of the total scores of the three pathological characteristics of the three visual fields. See the results. Figure 5 A and Figure 5 C and HE staining revealed that the epidermis and dermis of the control mice were intact and morphologically normal, with no abnormalities such as edema. Compared with the control group, the urticaria model group exhibited significant dermal swelling, dilated capillaries, and inflammatory cell infiltration. Furthermore, compared with the model group, the skin histopathological morphology of mice in the Lactobacillus intervention group, the positive drug group, and the combination drug group all improved to varying degrees, with significant reductions in histological scores (P < 0.05).

[0074] Mast cells play a crucial role in the pathogenesis of urticaria. Toluidine blue staining can be used to determine the infiltration and degranulation of mast cells in skin tissue. After washing 3um sections with a series of ethanol water, stain with toluidine blue solution for 20-30 minutes, wash away the excess stain with running water, separate with 95% ethanol, dehydrate and make transparent, and seal with neutral gum. Select three different fields of view under an optical microscope to count mast cells. The results are shown in Figure 5 B and Figure 5 In this study, the number of mast cells released and mast cell degranulations in urticaria mice was significantly higher than that in normal control mice, indicating that the urticaria mice underwent a significant allergic reaction. Treatment with Lactobacillus paragaseri significantly reduced the number of mast cells and degranulations in mice (P < 0.05).

[0075] 4. ELISA to detect immunoglobulin E (IgE) content

[0076] After the mouse is anesthetized, blood is collected from the eye socket, centrifuged at 3000 rpm for 20 minutes, and serum is obtained. Remove the kit from the refrigerator in advance and return to room temperature. Strictly follow the instructions to prepare all the solutions needed for the experiment. Add the sample and wash the plate. Add the enzyme-labeled antibody and wash the plate. After incubation with the colorimetric reagent, add the stop solution, mix well, and immediately measure the OD450 value. Calculate the concentration of IgE in the serum based on the standard curve. Results are shown in Figure 6Compared with the normal control group, the IgE levels of urticaria mice were significantly increased (P < 0.01). Compared with the urticaria model group, the serum IgE levels of the Lactobacillus group, the positive drug group, and the combination drug group were significantly decreased (P < 0.05).

[0077] 5. Oxidative Stress in Mouse Skin

[0078] Dorsal tissue was homogenized in cold physiological saline (10%, w / v) and centrifuged at 3000 rpm at 4°C for 15 minutes, and the supernatant was retained. Oxidative stress markers (superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA)) in skin tissue were measured according to the instructions of the kit (Beijing Bioson Biotechnology Co., Ltd., Beijing, China). The results are shown in Figure 2. Figure 7 As shown in the figure, compared with the normal control group, the MDA content in the skin tissue of urticaria mice was significantly increased, and SOD and CAT were significantly decreased (P<0.01). Compared with the model group, the SOD level in the skin tissue of the Lactobacillus group mice was significantly increased.

[0079] (P<0.05), MDA and CAT had no significant changes. In the positive drug group, MDA in the loratadine group was significantly reduced.

[0080] CAT was significantly increased (P<0.05).

[0081] 6. Mouse Skin Inflammation Indicators

[0082] (1) Total RNA extraction: Remove the dorsal skin tissue of the mouse from the -80°C freezer, weigh it, and chop it into small pieces. Add TransZol Up and RNA extractant in proportion and homogenize to fully lyse the tissue cells. Centrifuge at 4°C, 12,000 rpm for 15 minutes. Take the supernatant and add an equal volume of isopropanol. Mix by inverting and let it stand on ice for 15 minutes. Centrifuge again and discard the supernatant. Add 950 μL of 75% ethanol to each tube, mix by inverting and centrifuge again. Discard the supernatant after centrifugation and open the lid to dry (about 5 minutes). Add 50 μL of DEPC water to each tube and pipette to dissolve the RNA to obtain a total RNA solution.

[0083] (2) RNA concentration determination: Place the total RNA solution on ice, take 1 μL and test it on the Nanodrop machine, record the total RNA concentration, and store the remaining sample back at -80℃.

[0084] (3) Reverse transcription: Calculate the amount of RNA required for reverse transcription based on the 1 μg RNA template used for each sample. Prepare a 20 μL reverse transcription reaction mixture in eight tube strips, as shown in Table 2.

[0085] Table 2 Reverse transcription reaction system

[0086]

[0087] Mix the above system thoroughly and place it in a PCR amplifier. Set the reaction steps to: 50°C for 2 minutes, 85°C for 15 minutes. After the reaction is complete, the cDNA solution is obtained and stored at -20°C.

[0088] (4) qPCR: Prepare the qPCR reaction system in an eight-well tube strip. The total volume of the qPCR reaction mixture in each well is 20 ul μL, including: 10 μL of 2X SYBR Green qPCR Mix, 0.5 μL each of forward primer and reverse primer, 1 μL of cDNA solution, and 8 μL of double-distilled water. After adding the sample, mix well and centrifuge. Set up three replicate wells for each sample. The qPCR reaction program is as follows: 50℃ for 2 min, 95℃ for 10 min, and then perform 40 PCR cycles (95℃ for 15 s, 60℃ for 1 min). Finally, perform 95℃ for 15 s and 60℃ for 1 min to obtain the melting curve and terminate the reaction. The primers used are shown in Table 3 below.

[0089] Table 3 qPCR reaction primer list

[0090]

[0091] The expression levels of IL-4, TNF-α, IL-1β, and IL-10 mRNA in the back skin tissue of mice were detected by qPCR. Figure 8 The experimental results showed that compared with the control group, the expression levels of IL-1β, IL-4, and IL-10 mRNA in the lesions of mice in the urticaria group were significantly increased (P < 0.05). The expression level of TNF-α mRNA was increased, but not statistically significant (P > 0.05). After treatment with Lactobacillus, positive drugs, or combined treatment, the expression levels of IL-1β, IL-4, IL-10, and TNF-α mRNA were significantly decreased (P < 0.05), but still higher than those in the control group. Our results indicate that mice with urticaria experience an inflammatory response, and Lactobacillus can alleviate the inflammatory response during the development of urticaria.

[0092] 7. Xanthine Oxidase Assay

[0093] Serum samples were processed according to the guidelines in the kit manual (Beijing Box Biotechnology Co., Ltd., Beijing, China). The extracted colon tissue was weighed, and subsequent steps were performed according to the instructions in the kit manual. Specifically, the tissue pieces were added to the pre-cooled extraction solution at a ratio of 1:10, mechanically homogenized, and centrifuged, and the supernatant was collected for measurement of XOD activity. The protein concentration was corrected by the BCA (bicinchoninic acid) method. The results are shown in Figure 2. Figure 9 As shown in Figure 2, the levels of xanthine oxidase (XOD) in the serum and ileum of mice in the urticaria model group were elevated, and returned to the levels of the control group after intervention with LG-1 or loratadine ( Figure 7 ).

[0094] 8. Detection of purine metabolism-related metabolites

[0095] The levels of uric acid, hypoxanthine, inosinic acid, adenylic acid, and guanylic acid in plasma samples were determined using high performance liquid chromatography (HPLC). The chromatographic conditions were as follows:

[0096] Chromatographic column: CAPCELL PAK ADME S5 (4.6 mm id × 250 mm)

[0097] Column temperature: 40°C Flow rate: 1 mL / min

[0098] Mobile phase: 50 mmol / L ammonium acetate solution (pH 4.65)

[0099] Elution time: 15 min

[0100] Plasma / feces samples (50 μl / 50 mg) were mixed with 200 μl of 0.08 mol / L sulfuric acid solution and 100 μl of 10% sodium tungstate solution. The samples were vortexed at 3000 rpm for 1 minute, sonicated in an ice-water bath for 2 minutes, and centrifuged at 12000 rpm and 4°C for 10 minutes. Subsequently, 120 μl of the supernatant was transferred to a fresh Eppendorf tube fitted with a 0.22 μm filter. Finally, a 10 μl aliquot of the sample solution was injected onto the ADME column.

[0101] The results are as follows Figure 10 As shown, uric acid and fecal hypoxanthine levels in urticaria mice increased significantly, while plasma GMP and fecal IMP decreased significantly. However, treatment with Lactobacillus paragaseri or positive drugs suppressed the increase in uric acid and hypoxanthine concentrations. This suggests that purine metabolism is altered in urticaria mice, and treatment with Lactobacillus paragaseri normalizes purine metabolism, particularly by reducing uric acid and hypoxanthine levels.

[0102] Example 3: Effect of Lactobacillus paragaseri on the diversity and composition of intestinal flora

[0103] The intestinal flora of each group of mice obtained in Example 2 was subjected to 16S rRNA sequencing.

[0104] (1) DNA extraction from stool samples

[0105] The genomic DNA of the samples was extracted using the CTAB method. After testing the purity and concentration of the DNA, the DNA samples were diluted to a final concentration of 1 ng / μl.

[0106] (2) Amplicon preparation

[0107] Using diluted genomic DNA as a template, amplify the V3-V4 region of the 16S rRNA gene using specific primers, Phusion® High-Fidelity PCR Master Mix with GC Buffer, and a high-efficiency, high-fidelity enzyme. The PCR reaction mixture includes: 12.5 μl of PCR reaction complex containing a high-fidelity DNA polymerase, 0.5 μl of forward primer, 0.5 μl of reverse primer, 1 μl of template DNA, and 9.5 μl of ddH2O.

[0108] (3) Quantification and quality control of PCR products

[0109] Detect and purify the resulting PCR product. Measure the concentration of the PCR product and mix equal amounts according to the concentration. After thorough mixing, use 2% agarose gel electrophoresis to detect the PCR product and use a gel recovery kit to recover the target band.

[0110] (4) Sequencing library preparation and sequencing

[0111] Libraries were constructed using a library construction kit, and preliminary quantification was performed using Qubit and precise quantification by qPCR. Once qualified, the libraries were sequenced using the NovaSeq6000.

[0112] (5) Bioinformatics analysis of sequencing data

[0113] The offline data were split, intercepted, and spliced ​​to obtain the original tag data. The original tag data were filtered to obtain high-quality tag data. Further screening was performed to remove possible contaminating sequences (such as host sequences, bacteriophages, etc.) to obtain the final valid data for analysis. Based on sequence similarity (such as 97% similarity), the valid data were clustered into different ASVs, and each ASV represented a microbial species. The representative sequence of each ASV was then aligned, annotated to specific species information, and the abundance of each species was counted. At the same time, alpha diversity and beta diversity were calculated for ASVs. Principal coordinates analysis (PCoA) was performed using the R programming language (Version 2.15.3), and the linear discriminant analysis (LDA) effect size (LEfSe) method was used to identify biomarkers with statistical differences between the treatment groups.

[0114] 1. Analysis of α-diversity of mouse intestinal flora

[0115] The results are as follows Figure 11 As shown, LG-1 treatment had minimal effects on diversity indices but increased Chao1 and richness indices, which were significantly reduced by positive drug treatment. This suggests that LG-1 treatment increased community richness, while drug treatment decreased it. Combining LG-1 with positive drug treatment can ameliorate the effects of drugs on gut microbial richness.

[0116] 2. Analysis of mouse intestinal flora composition

[0117] The results are as follows Figure 12 As shown in A: At the genus level, Lactobacillus gasseri treatment can change the composition of the intestinal microbiota of urticaria mice. Compared with the control group, the relative abundance of Aerococcus and Helicobacter increased, while Lactobacillus decreased significantly in urticaria mice. Lactobacillus gasseri treatment can increase the relative abundance of Lactobacillus and Limosilactobacillus in urticaria mice. Conversely, the relative abundance of Aerococcus and Helicobacter decreased ( Figure 12 B). This indicates that the intervention of Lactobacillus can promote the recovery of intestinal flora composition.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Lactobacillus paragaseri ( Lactobacillus paragasseri ), its deposit number is CGMCC No.29973.

2. The Lactobacillus paragaseri according to claim 1, characterized in that The Lactobacillus paragaseri is in the form of a freeze-dried preparation.

3. Use of the Lactobacillus paragaseri according to claim 1 or 2 in the preparation of a medicine for treating urticaria.

4. Use of the Lactobacillus paragaseri according to claim 1 or 2 in the preparation of a medicine for alleviating urticaria symptoms.

5. Use of the Lactobacillus paragaseri according to claim 1 or 2 in the preparation of veterinary drugs for treating urticaria.

6. A pharmaceutical composition for treating urticaria, characterized in that: The pharmaceutical composition comprises the Lactobacillus paragaseri according to claim 1 or 2 as its active ingredient, and the content of Lactobacillus paragaseri in the composition is ≥1×10 6 CFU / g.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition comprises the Lactobacillus paragaseri and an antihistamine as effective ingredients.

8. The pharmaceutical composition according to claim 7, characterized in that The antihistamine drug is one or more selected from diphenhydramine, promethazine, chlorpheniramine, cetirizine, loratadine, ebastine, desloratadine, levocetirizine or fexofenadine.

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