Lactobacillus paragasseri and application thereof

Abstract

The invention discloses lactobacillus paragasseri, and the preservation number of the lactobacillus paragasseri is CGMCC (China General Microbiological Culture Collection Center) No.29973. The lactobacillus paragasseri provided by the invention can relieve inflammation and change the structure and composition of intestinal flora so as to effectively prevent, treat and relieve urticaria and symptoms thereof, and has important application value in the aspects of regulation of intestinal flora disorder caused by urticaria and prevention and treatment of 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 of between 1% and 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 medication 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 to be an immune-mediated inflammatory skin disease characterized by mast cell activation and release of inflammatory mediators. Autoantibodies against self-antigens, such as IgE or FcεRIα chain, activate mast cells mainly through FcεRI. Mast cell activation can be participated by a variety of immune cells, including dendritic cells, B cells, T cells, vascular endothelial cells, and inflammatory factors such as IL-4, IL-5, and IL-10. Therefore, it is urgent to further study the pathogenesis of CSU to explore new diagnostic tools and develop innovative treatment strategies. Purines are converted to uric acid through a series of enzymatic reactions, and reactive oxygen species (ROS) are generated during the conversion process. Purine disorders are associated with a variety of diseases, including hyperuricemia and gout, kidney stones, immunodeficiency, anemia, peripheral and central nervous system diseases, and myopathy. However, the potential relationship between chronic spontaneous urticaria (CSU) and purine metabolism disorders is still unclear.

[0004] Previous studies have shown that the intestinal flora plays a key role in human health, the development and maturation of the immune system, and resistance to pathogen invasion. An imbalance in the intestinal flora is associated with several immune-mediated allergic diseases, including atopic dermatitis, asthma, and urticaria. et al. found that the diversity of intestinal flora in children with atopic dermatitis was reduced, the relative abundance of Bifidobacterium and Lactobacillus decreased, while the proportions of Escherichia coli, Clostridium difficile and Staphylococcus aureus increased; Arrieta et al. found that infants at risk of asthma generally had intestinal microbial imbalance in the first 100 days after birth. Further observations found that compared with healthy children, the relative abundance of Lachnospira, Veillonella, Faecalibacterium and Roseburia in the intestines of children at risk of asthma was significantly reduced; Wang et al. found that the number of undetermined Enterobacteriaceae increased in the urticaria patient group, while the number of Bacteroidetes, Faecalibacterium, Bifidobacterium, Firmicutes and undetermined Ruminococcus decreased.

[0005] Using probiotics to regulate intestinal flora and alleviate metabolic disorders has become a new method for treating allergic diseases. Some clinical trials have shown that lactobacillus alone or in combination with bifidobacteria can reduce the severity of urticaria and improve the quality of life. Some studies have also found that probiotics combined with antihistamines can significantly relieve the itching and number of wheals of urticaria. Therefore, finding a probiotic for the treatment or combined treatment of urticaria has become a top priority. 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 provide a Lactobacillus paragaseri and uses thereof.

[0008] Technical solution:

[0009] A Lactobacillus paragasseri strain, whose deposit number is CGMCC No.29973.

[0010] The Lactobacillus paragaseri described in the present invention is deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration, the deposit address 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 breast milk of healthy women, identified as Lactobacillus paragasseri, genus Lactobacillus, and 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, and the transformed variants contain the basic functions and properties of the above-mentioned strains, and the transformed 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 variant strains obtained by continuous passage. The variants usually have the same or substantially the same gene sequence as the deposited strain, for example, at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% of the same 16S rDNA fragment as the deposited 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 preparation form, such as a freeze-dried preparation, a capsule preparation, a liquid preparation, a tablet preparation, a gel preparation, or an emulsion preparation. In one embodiment of the present disclosure, the Lactobacillus paragaseri is a liquid preparation freeze-dried preparation.

[0014] The inventors of the present disclosure verified the effect of the above-mentioned Lactobacillus paragaseri on urticaria by in vitro experiments and in vivo animal experiments. Therefore, another aspect of the present disclosure is to provide the use of the above-mentioned Lactobacillus paragaseri in preparing food, medicine or skin care medicine or food 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 food, medicine or skin care medicine or food for relieving 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 a feed additive for treating or preventing urticaria.

[0017] In the present disclosure, the above-mentioned Lactobacillus paragaseri can be used for preventing, treating, assisting in the treatment, and alleviating urticaria in humans. Similarly, it can also be used as a veterinary drug or feed additive for preventing, treating, assisting in the treatment, and alleviating urticaria in other mammals, for example, 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 medicine or food 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 ingested per day is ≥1×10 6 CFU / g.

[0020] Further, based on the research results disclosed in the present invention, another aspect of the present invention is to provide a pharmaceutical composition, food composition or skin care composition for preventing or treating urticaria, wherein the pharmaceutical composition, food composition or skin care composition contains the above-mentioned Lactobacillus paragaseri as its effective ingredient. In the above-mentioned pharmaceutical composition, food composition or skin care composition, the Lactobacillus paragaseri can be in a suitable preparation form, for example, a freeze-dried preparation, a capsule preparation, a liquid preparation, a tablet preparation, a gel preparation, and an emulsion preparation. In some embodiments of the present invention, the above-mentioned pharmaceutical composition and food composition can be in the form of oral ingestion, and the above-mentioned skin care composition can be in the form of external application.

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

[0022] In order to enhance the effect 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, foods or skin care products that have the effect 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. In other embodiments of the present disclosure, the food used in combination can be, for example, vitamins, health products containing Chinese medicine ingredients, health products containing minerals and trace elements, other probiotics, etc. In addition, in other embodiments of the present disclosure, the skin care products used in combination may be, for example, vitamins (such as nicotinic acid), hyaluronic acid, green tea polyphenols, oats, aloe vera, allantoin, food and medicine homologous substance extracts, Chinese medicine extracts, other postbiotics, etc. The above-mentioned other drugs, foods or skin care products that have the effects of preventing, treating, assisting in the treatment, and alleviating urticaria may be in any suitable form.

[0023] When used as a pharmaceutical composition, food composition or skin care product for humans, the amount of the Lactobacillus paragaseri contained 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] At the same time, another aspect of the present disclosure is to provide the use of the above-mentioned Lactobacillus paragaseri in the preparation of a drug or food, health product, or skin care product preparation for correcting purine metabolism disorders in the body. Further, in some embodiments of the present disclosure, the above-mentioned drug or preparation for correcting purine metabolism disorders in the body is a drug 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 to effectively prevent, treat and alleviate urticaria and its symptoms, and has important application value in regulating the intestinal flora and purine metabolism disorders caused by urticaria and preventing and treating urticaria.

[0027] Biodeposit Information:

[0028] Deposit number: CGMCC No.29973

[0029] Deposit date: March 8, 2024

[0030] Depository: China General Microbiology 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 The figure is a result diagram of degradation of uric acid (Uricacid), hypoxanthine (Hypoxanthine), adenosine (AMP), guanosine (GMP) and inosine (IMP) in the purine metabolic pathway by Lactobacillus subglabrata LG-1 in the embodiment 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 Lactobacillus subtilis LG-1 in the disclosed embodiments;

[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 disclosed embodiments;

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

[0038] Figure 7 This is a graph showing the results of the intervention of Lactobacillus subtilis LG-1 on the skin oxidoreductase levels of urticaria mice in 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 the inflammatory factors in urticaria mice treated with Lactobacillus subtilis LG-1 in the disclosed embodiment, wherein A is IL-4, B is TNF-α, C is IL-1β, and D is IL-10;

[0040] Fig. 9 The results of the intervention of Lactobacillus subtilis LG-1 on the levels of xanthine oxidase in the ileum (A) and serum (B) of urticaria mice in the disclosed embodiments are shown in FIG.

[0041] Fig.10 The results of the intervention of Lactobacillus subtilis LG-1 on the content of purine metabolites in plasma (A) and feces (B) of urticaria mice in the disclosed embodiments are shown in FIG.

[0042] Fig.11 1 is a graph showing the results of the α-diversity analysis of the intestinal flora of urticaria mice treated with Lactobacillus subtilis LG-1 in the disclosed embodiment, wherein A is the Chao 1 index; B is the richness index;

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

[0044] The present invention discloses a strain of Lactobacillus paragaseri having the function of relieving 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 it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0045] In the present invention, unless otherwise specified, the 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 "including" or its variations such as "comprising" or "including" etc. will be understood to include the stated elements or components without excluding other elements or other components. The terms "such as", "for example", etc. are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0046] I. Some of the terms used in the present invention are explained below.

[0047] The term "Lactobacillus" may 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 for reproduction.

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

[0049] The term "food" or "food composition" in the present disclosure refers to a functional food or functional food composition that has the function of preventing, treating, assisting in the treatment, and alleviating urticaria and its symptoms, such as beverages, health foods, multivitamins, etc. In some embodiments of the present disclosure, suitable additives may be added to the food or food composition, the effects of which are known and do not affect the properties of the Lactobacillus paragaseri as an effective ingredient. Examples of the additives include, but are not limited to, carbohydrates, flavoring agents, flavoring agents, preservatives, coloring agents, nutrients, excipients, etc.

[0050] The term "skin care product" or "skin care product composition" refers to a skin care product that has the function of protecting the normal skin. Its basic functions include, for example, beautifying and nourishing the skin, replenishing moisture, moisturizing, delaying aging, etc. In the embodiments of the present disclosure, in addition to the above basic functions, the skin care product or skin care product composition also has the effects of preventing, treating, assisting in the treatment, and relieving urticaria.

[0051] The term "feed additive" refers to a small amount or trace amount of substances added to feed in order to meet the nutritional needs of animals and improve feed utilization. These substances play an important role in strengthening the nutritional value of basic feed, improving animal production performance, ensuring animal health, saving 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 farmed animal for food or an experimental animal for experiment, such as a mouse.

[0052] II. The degradation effect of Lactobacillus paragaseri on uric acid, hypoxanthine, adenylic acid, guanylic acid or inosinic acid in the purine metabolic pathway.

[0053] 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 degradation effect on uric acid, hypoxanthine, adenylic acid, guanylic acid or inosinic acid in the purine metabolic pathway alone or independently. The specific research method is:

[0054] LG-1 was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 48h. Take 2mL of bacterial suspension, centrifuge at 4000rpm for 10min, wash three times with 1mL PBS, discard the supernatant, and suspend the bacterial precipitate in 750μL of uric acid, hypoxanthine, inosinic acid, adenylic acid, and guanylic acid solution, respectively, and incubate at 37°C for 60min. Subsequently, centrifuge at 4000rpm for 10min to obtain the supernatant. Add 100μL of 5% trifluoroacetic acid to the supernatant, mix thoroughly to prevent further reaction, filter with 0.22um filter membrane, take 10μL and inject into high performance liquid chromatograph for detection. The results are as follows Figure 1 The results showed that after 1 h of treatment, the degradation rates of uric acid, hypoxanthine, adenylic acid, guanylic acid and inosinic acid by the Lactobacillus paragaricei LG-1 strain isolated from breast milk were 22.8%, 10.94%, 23.74%, 44.7% and 27.53%, respectively.

[0055] III. Effects of Lactobacillus paragaseri on other intestinal flora species.

[0056] Based on the research disclosed herein, the present disclosure also provides a use of the above-mentioned Lactobacillus paragasseri LG-1 in the preparation of a product for increasing the relative abundance of Lactobacillus and reducing the relative abundance of Enterococcus and Aerococcus in the intestine. Furthermore, in certain embodiments of the present disclosure, the combination of the Lactobacillus paragasseri and an antihistamine drug can improve the effect of the drug on the richness of the intestinal microbial flora.

[0057] IV. Embodiment:

[0058] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0059] Example 1: Isolation, culture and identification of LG-1

[0060] 1. Preparation of culture medium:

[0061] Preparation of separation culture medium: The culture medium used for lactobacillus isolation and culture is MRS solid culture medium. The mass concentration of each component of MRS solid culture 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.

[0062] Preparation of culture medium for amplifying bacteria: The culture medium used for enriching 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.

[0063] 2. Isolation, identification and purine degradation ability assessment:

[0064] Twenty lactic acid bacteria isolated from breast milk were stored in the Institute of Food Science, Zhejiang Academy of Agricultural Sciences.

[0065] In order to evaluate the degradation ability of uric acid and hypoxanthine, all isolated strains were inoculated on MRS and cultured under anaerobic conditions at 37°C for 48h. Take 2mL of culture solution, centrifuge at 4000rpm for 10min, wash 3 times with 1mL PBS, resuspend in 750μL uric acid and hypoxanthine solution, incubate at 37°C for 60min, centrifuge at 4000rpm for 10min, and obtain supernatant. Add 100μL 5% trifluoroacetic acid (TFA) to the supernatant, mix thoroughly to prevent further degradation, filter and take 20μ into high performance liquid chromatography (HPLC) device. The HPLC conditions are as follows: CAPCELL PAK ADME S5 (4.6 mm inner diameter × 250 mm), mobile phase is 50 mmol / L ammonium acetate solution (pH 4.65), flow rate is 1 ml / min, column temperature is 40°C, wavelength is 254 nm, and elution time is 20 minutes. The pretreatment steps of plasma and fecal samples were 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. Vortex mixing was performed at 3000 rpm for 1 min, followed by sonication in an ice-water bath for 2 min, and the samples were centrifuged at 12000 rpm and 4°C for 10 min. For fecal samples, freeze drying was performed until dry. The powder of each sample was weighed into an Eppendorf tube, and 20 μl of 80% methanol was added per mg of powder. Homogenization was performed using a ball mill at 30 Hz for 2 min, three times, with the assistance of 2 metal beads, and then sonication in an ice-water bath for 5 min. The tube was centrifuged at 14000 rpm and 5°C for 20 min. 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 with a 0.22 μm filter. Finally, 10 μl of the sample solution was injected into the ADME column. The HPLC results are shown in Figure 1 As shown in Figure 1, the degradation rates of uric acid, hypoxanthine, adenylic acid, guanylic acid and inosinic acid by LG-1 strain were 22.8%, 10.94%, 23.74%, 44.7% and 27.53%, respectively. The 16S rRNA gene and whole genome sequencing of the strain were performed, and the obtained sequences were compared in the NCBI database and the phylogenetic tree analysis showed 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 ).

[0066] Example 2: Effects of Lactobacillus paragaseri on scratching behavior and tissue pathology in urticaria mice

[0067] 1. Establishment of mouse model:

[0068] 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.

[0069] 2) The normal control group was intraperitoneally injected with 0.1 ml PBS, and the other four groups of mice were intraperitoneally injected with a PBS suspension containing (0.1 mg ovalbumin, 100 mg aluminum hydroxide) for the first immunization on the first day, and the second immunization was performed on the 10th day, 0.1 ml each time. On the 8th day of the initial immunization, gavage was started, once a day, and the intervention continued for 21 days. The dosing schedule is shown in Table 1. On the 22nd day, mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital. Orbital blood was collected and divided into ordinary centrifuge tubes and EDTA anticoagulant tubes. After standing for 30 minutes, centrifuged at 3000r / min for 20 minutes to obtain serum and plasma. The liver, back skin, spleen and intestinal tissues of mice in each group were collected. After washing with physiological saline, all samples were stored in a -80°C refrigerator except for samples used for histopathology, which were stored in 4% paraformaldehyde.

[0070] Table 1 Dosage schedule

[0071]

[0072]

[0073] 2. Mouse behavioral assessment

[0074] 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. The degree of itching of mice in each group was observed and recorded within 30 minutes after the second immunization, including the scratching latency, scratching duration, and scratching times. 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. The results are shown in Figure 4 (AC) Compared with the control group, the mice in the urticaria group showed obvious scratching behavior. After intervention with LG-1, the scratching behavior of the urticaria mice was alleviated, and the number of scratching times and the duration of scratching were significantly reduced (P < 0.05). This indicates that Lactobacillus paragaseri can effectively alleviate the clinical symptoms of urticaria mice.

[0075] 3. Evaluation of the pathological condition of the back skin of mice

[0076] The skin tissue from the back of the mouse was fixed in 4% paraformaldehyde overnight, 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, slicing was performed and HE staining was performed, and the tissue was 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 for each mouse is the average of the total scores of the three pathological features of the three visual fields. See the results. Figure 5 A and Figure 5 The results of C and HE staining showed that the epidermis and dermis of the skin tissue of mice in the control group were intact and normal in morphology, without abnormal changes such as edema. Compared with the control group, the dermis of mice in the urticaria model group was significantly swollen, the capillaries of the skin tissue were dilated, and inflammatory cells were infiltrated. Compared with the model group, the pathological morphology of the skin tissue of mice in the lactobacillus intervention group, the positive drug group, and the combined drug group were improved to varying degrees, and the histological score was significantly reduced (P < 0.05).

[0077] Mast cells play a vital 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 the 3um slices with a series of ethanol water, use toluidine blue dye for 20-30 minutes, wash away the excess dye with running water, separate with 95% ethanol, dehydrate and make transparent, and seal with neutral gum. Select 3 different fields of view under an optical microscope to count mast cells. The results are shown in Figure 5 B and Figure 5 D. In this study, the number of mast cells and mast cell degranulation released by urticaria mice was significantly higher than that of normal control mice, indicating that urticaria mice had a significant allergic reaction. The number of mast cells and degranulation in mice treated with Lactobacillus paragaseri was significantly reduced (P < 0.05).

[0078] 4. ELISA to detect immunoglobulin E (IgE) content

[0079] After the mice are anesthetized, blood is collected from the eye sockets and centrifuged at 3000rpm for 20min to obtain serum. Take the kit out of the refrigerator in advance and return it to room temperature. Strictly follow the instructions to prepare various solutions required for the experiment. Add samples and wash the plates. Add enzyme-labeled antibodies and wash the plates. After incubation with colorimetric reagent, add 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 level of urticaria mice was 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 combined drug group were significantly reduced (P < 0.05).

[0080] 5. Oxidative Stress in Mouse Skin

[0081] The dorsal tissue was homogenized in cold physiological saline (10%, w / v), centrifuged at 3000 rpm and 4°C for 15 min, and the supernatant was retained. Oxidative stress indicators (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 Table 1. 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 (P<0.05), and there was no significant change in MDA and CAT. In the positive drug group, the MDA in the loratadine group was significantly decreased, and the CAT was significantly increased (P<0.05).

[0082] 6. Mouse Skin Inflammation Indicators

[0083] (1) Total RNA extraction: Take out the back skin tissue of the mouse from the -80℃ refrigerator, weigh it, and cut it into pieces. Add TransZol Up and RNA extractant in proportion and homogenize to fully lyse the tissue cells. Centrifuge at 4℃, 12000rpm for 15min, take the supernatant, and add an equal volume of isopropanol, mix by inverting, and let stand on ice for 15min. Centrifuge again and discard the supernatant. Add 950μL 75% ethanol to each tube, mix by inverting, and centrifuge again. After centrifugation, discard the supernatant, open the lid and dry (about 5min). Add 50μL DEPC water to each tube, pipette to dissolve the RNA, and obtain a total RNA solution.

[0084] (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°C.

[0085] (3) Reverse transcription: Based on the proposed 1ug RNA of each sample as a template, calculate the amount of RNA required for the reverse transcription process. Prepare a reaction mixture in an eight-tube strip according to the reverse transcription reaction system of 20 μL, as shown in Table 2 below.

[0086] Table 2 Reverse transcription reaction system

[0087] Grouping Treatment Total RNA Variable(1ug) TransScript Enzyme Mix RT / RI 10 μL RNase-free Water Variable gDNA Remover 1μL Total volume 20μL

[0088] Mix the above system evenly, put it into the PCR amplification instrument, and set the reaction steps: 50℃2min, 85℃15min. After the reaction is completed, the cDNA solution is obtained and stored at -20℃.

[0089] (4) qPCR: Prepare the qPCR reaction system in an eight-tube strip. The total volume of the qPCR reaction mixture in each well is 20ulμL, including: 10μL 2X SYBR Green qPCR Mix, 0.5μL each of forward primer and reverse primer, 1μL cDNA solution and 8μL double distilled water. Mix well after adding the sample and centrifuge. Set up 3 replicate wells for each sample. qPCR reaction program: 50℃2min, 95℃10min, and then perform 40 PCR cycles (95℃15s, 60℃1min). Finally, 95℃15s, 60℃1min to obtain the melting curve and terminate the reaction. The primers used are shown in Table 3 below.

[0090] Table 3 qPCR reaction primer list

[0091]

[0092] The results of qPCR detection of IL-4, TNF-α, IL-1β, and IL-10 mRNA expression levels in mouse back skin tissue are shown in 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 skin lesions of mice in the urticaria group were significantly upregulated (P < 0.05), and the expression level of TNF-α mRNA was upregulated, but there was no statistical significance (P > 0.05). After the use of lactobacillus or positive drugs or combined use, the expression levels of IL-1β, IL-4, IL-10, and TNF-α mRNA were significantly reduced (P < 0.05), but still higher than those in the control group. Our research results show that urticaria mice have inflammatory reactions, and lactobacillus can reduce the inflammatory response during the onset of urticaria.

[0093] 7. Xanthine oxidase detection

[0094] Serum samples were processed according to the guidelines of the kit manual (Beijing Box Biotechnology Co., Ltd., Beijing, China). The extracted colon tissue was weighed, and the subsequent steps were carried out according to the instructions in the kit manual. Specifically, the tissue block was added to the pre-cooled extraction solution at a ratio of 1:10, mechanically homogenized, centrifuged, and the supernatant was collected for measuring XOD activity. The corrected protein concentration was determined by the BCA (bicinchoninic acid) method. The results are shown in Fig. 9As 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 ).

[0095] 8. Detection of purine metabolism-related metabolites

[0096] High performance liquid chromatography was used to determine the contents of uric acid, hypoxanthine, inosinic acid, adenylic acid, and guanylic acid in plasma samples. The chromatographic conditions were as follows:

[0097] Chromatographic column: CAPCELL PAK ADME S5 (4.6mm id×250mm)

[0098] Column temperature: 40℃ Flow rate: 1mL / min

[0099] Mobile phase: 50mmol / L ammonium acetate solution (pH 4.65)

[0100] Elution time: 15min

[0101] Plasma / feces samples were 50ul / 50mg mixed with 200ul 0.08mol / L sulfuric acid solution and 100ul 10% sodium tungstate solution. The samples were vortexed at 3000rpm for 1min, sonicated in an ice water bath for 2min, and centrifuged at 12000rpm and 4°C for 10min. Then 120ul of the supernatant was transferred to a fresh Eppendorf tube with a 0.22μm filter. Finally, 10ul of the sample solution was injected into the ADME column.

[0102] The results are as follows Fig.10 As shown in the results, blood uric acid and fecal hypoxanthine in urticaria mice increased significantly, plasma GMP and fecal IMP decreased significantly, while treatment with Lactobacillus paragaseri or positive drugs inhibited the increase in uric acid and hypoxanthine concentrations. This indicates that urticaria mice have changes in purine metabolism, and treatment with Lactobacillus paragaseri normalizes purine metabolism, especially reducing uric acid and hypoxanthine levels.

[0103] Example 3: Effect of Lactobacillus paragaseri on the diversity and composition of intestinal flora

[0104] The intestinal flora of each group of mice obtained in Example 2 was subjected to 16S rRNA sequencing.

[0105] (1) DNA extraction from stool samples

[0106] The genomic DNA of the sample was extracted using the CTAB method. After testing the purity and concentration of the DNA, the DNA sample was diluted with 1 ng / μl of final concentration after dilution.

[0107] (2) Amplicon preparation

[0108] Using diluted genomic DNA as template and specific primers, High-Fidelity PCR Master Mix with GC Buffer, and high-efficiency high-fidelity enzymes amplify the V3-V4 region of the 16S rRNA gene. The PCR reaction mixture includes: 12.5ul PCR reaction complex containing high-fidelity DNA polymerase, 0.5ul forward primer, 0.5ul reverse primer, 1ul template DNA, 9.5ul ddH2O.

[0109] (3) Quantification and quality control of PCR products

[0110] Detect and purify the obtained PCR products. Measure the concentration of the PCR products, mix the samples in equal amounts according to the concentration, and after sufficient mixing, use 2% agarose gel electrophoresis to detect the PCR products, and use a gel recovery kit to recover the target bands.

[0111] (4) Sequencing library preparation and sequencing

[0112] The library was constructed using a library construction kit, and Qubit was used for preliminary quantification and qPCR for accurate quantification. After the library was qualified, NovaSeq6000 was used for sequencing.

[0113] (5) Bioinformatics analysis of sequencing data

[0114] After the offline data was split, it was intercepted and spliced ​​to obtain the original tags data. The original tags data was filtered to obtain high-quality tags data. Further screening was performed to remove possible contaminating sequences (such as host sequences, bacteriophages, etc.) to obtain valid data for final analysis. Based on sequence similarity (such as 97% similarity), the valid data was clustered into different ASVs, and each ASV represented a microbial species. Subsequently, the representative sequence of each ASV was aligned, annotated with 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 treatment groups.

[0115] 1. Analysis of α-diversity of mouse intestinal flora

[0116] The results are as follows Fig.11 As shown: LG-1 intervention had little effect on the diversity index, but it could increase Chao1 and richness index, which were significantly reduced by positive drug treatment. This indicates that LG-1 treatment increased community richness, while drug treatment decreased community richness. Combined treatment of LG-1 and positive drugs can improve the effect of drugs on the richness of intestinal flora.

[0117] 2. Analysis of mouse intestinal flora composition

[0118] The results are as follows Fig.12 As shown in A: At the genus level, treatment with Lactobacillus gasseri can change the composition of the intestinal microbiota of urticaria mice. Compared with the control group, the relative abundance of Aerococcus and Helicobacter in urticaria mice increased, and the genus Lactobacillus decreased significantly. Lactobacillus gasseri treatment can increase the relative abundance of Lactobacillus and Limosilactobacillus in urticaria mice. Conversely, the relative abundance of Aerococcus and Helicobacter decreased ( Fig.12 B). This indicates that the intervention of Lactobacillus can promote the recovery of intestinal flora composition.

[0119] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Lactobacillus paragasseri, whose 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 liquid preparation.

3. Use of Lactobacillus paragaseri as claimed in claim 1 or 2 in the preparation of food, medicine or skin care product for preventing or treating urticaria.

4. Use of Lactobacillus paragaseri as claimed in claim 1 or 2 in the preparation of food, medicine or skin care product for relieving urticaria symptoms.

5. Use of Lactobacillus paragaseri as claimed in claim 1 or 2 in the preparation of a feed additive for treating or preventing urticaria.

6. A pharmaceutical composition, food composition or skin care composition for preventing or treating urticaria, characterized in that: The pharmaceutical composition, food composition or skin care composition comprises the Lactobacillus paragaseri as claimed in claim 1 or 2 as an effective ingredient.

7. The pharmaceutical composition, food composition or skin care composition according to claim 6, characterized in that: The pharmaceutical composition, food composition or skin care composition further comprises at least one selected from the culture, lysate, extract or metabolite of the Lactobacillus paragaseri as its effective ingredient.

8. The pharmaceutical composition, food composition or skin care composition according to claim 6 or 7, characterized in that: The pharmaceutical composition comprises the Lactobacillus paragaseri and an antihistamine as its effective ingredients; Preferably, the antihistamine is one or more selected from diphenhydramine, promethazine, chlorpheniramine, cetirizine, loratadine, ebastine, desloratadine, levocetirizine, fexofenadine, cimetidine, ranitidine or famotidine.

9. The pharmaceutical composition, food composition or skin care composition according to claim 6, characterized in that: The content of Lactobacillus paragaseri in the composition is ≥1×10 6 CFU / g.

10. Use of Lactobacillus paragaseri as claimed in claim 1 or 2 in the preparation of a medicine or preparation for correcting purine metabolism disorder in an organism; Preferably, the drug or preparation for correcting the disorder of purine metabolism in the body is a drug or preparation for degrading uric acid, hypoxanthine, adenylic acid, guanylic acid or inosinic acid.

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