Application of a medical cell CMU-Pb-L5 in the preparation of drugs for preventing and treating allergic asthma

Abstract

The invention discloses an application of a medical cell CMU-Pb-L5 in the preparation of a drug for preventing and treating allergic asthma, and belongs to the field of microbial technology. The invention discloses an application of a medical cell CMU-Pb-L5 in the preparation of a drug for preventing and treating allergic asthma. The intake of the medical cell CMU-Pb-L5 is expected to regulate the immune response of patients with allergic asthma, establish a more balanced intestinal flora, and make these patients react more mildly to inhaled allergens, reduce the damage caused by inflammation, and has great potential application prospects in the preparation of drugs for preventing and treating allergic asthma.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically to an application of a medical cell CMU-Pb-L5 in the preparation of a drug for preventing and treating allergic asthma. Background Art

[0002] Allergic asthma (BA) is a chronic airway inflammation involving multiple cells, especially mast cells, eosinophils and T lymphocytes. In susceptible individuals, this inflammation can cause recurrent wheezing, shortness of breath, chest tightness and cough. In the past decade, the prevalence and mortality of asthma have been on the rise. There are about 100 million asthma patients worldwide, and it has become a chronic disease that seriously threatens public health. At present, traditional drug treatments for BA mainly include glucocorticoids, β-receptor agonists and leukotriene receptor antagonists, but the use of these drugs is usually accompanied by adverse side effects, so finding a reliable alternative therapy has become an urgent issue.

[0003] At the same time, the use of probiotics as an alternative is increasing worldwide. Some probiotic strains have been shown to have immunomodulatory properties. The intake of probiotics is expected to regulate the immune response of BA patients, establish a more balanced intestinal flora, and make these patients react more mildly to inhaled allergens; and reduce the damage caused by inflammation. Based on this, the use of probiotics is currently a popular strategy for the adjuvant treatment of BA.

[0004] Therefore, providing a medical cell CMU-Pb-L5 for use in the preparation of drugs for the prevention and treatment of allergic asthma is an issue that needs to be urgently addressed by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides an application of medical cells CMU-Pb-L5 in the preparation of drugs for preventing and treating allergic asthma.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A use of a medical cell CMU-Pb-L5 in the preparation of a drug for preventing and treating allergic asthma, wherein the preservation number of the medical cell CMU-Pb-L5 is CGMCC No. 25653 (see patent number 202311240999.2).

[0008] Furthermore, the medical cell CMU-Pb-L5 is used in the preparation of a drug for inhibiting allergic asthma mediated by immunoglobulin E.

[0009] Furthermore, the medical cell CMU-Pb-L5 is used in the preparation of drugs for inhibiting the secretion of pro-inflammatory factors.

[0010] Furthermore, the pro-inflammatory factor is IFN-γ.

[0011] Furthermore, the medical cell CMU-Pb-L5 is used in the preparation of immunoglobulin E clearance drugs for allergic asthma patients.

[0012] Furthermore, the medical cell CMU-Pb-L5 is used in the preparation of a drug for inhibiting clinical symptoms of allergic asthma.

[0013] Furthermore, the medical cell CMU-Pb-L5 is a bacterial suspension.

[0014] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses an application of a medical cell CMU-Pb-L5 in the preparation of a drug for preventing and treating allergic asthma. The medical cell CMU-Pb-L5 is a strain of Lactobacillus paracasei isolated from the oral cavity of a healthy person and can be prepared into a drug for preventing and treating allergic asthma. The medical cell CMU-Pb-L5 has a significant ability to inhibit immunoglobulin E-mediated allergic asthma inflammation, effectively reduce the inflammatory symptoms caused by allergic asthma, and promote the repair of cell tissue damage caused by allergic asthma. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0016] Figure 1 The accompanying drawings are the results of ELISA testing of IFN-γ and IgE in each group of serum of the present invention;

[0017] Among them, A: IFN-γ; B: IgE;

[0018] Figure 2 The attached figure is a HE staining result diagram of each group of the present invention; the scale bar is 100 μm;

[0019] Among them, A is the PBS group; B is the OVA group; C is the OVA+CMU-Pb-L5 intervention group;

[0020] Figure 3 The attached figure is a diagram of the PAS staining results of each group of the present invention; the scale bar is 100 μm;

[0021] Among them, A is the PBS group; B is the OVA group; C is the OVA+CMU-Pb-L5 intervention group. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] (1) Establishment of allergic asthma mouse model

[0025] C57BL / 6 mice were raised under SPF conditions, with a temperature of 22-26°C and a relative humidity of 50%-60%. Each time, 20 μg of OVA (ovalbumin) + 2 mg of aluminum hydroxide + 200 μl of saline solution was injected into the mouse peritoneal cavity, once every 2 days, for a total of 14 days, for a total of 7 injections. On the 15th day, 200 μg of OVA was dissolved in 20 μl of PBS and dripped into the mouse anterior nostril using a micropipette, 20 μL / side, dripped on both sides, once every 1 day, for 7 consecutive days.

[0026] (2) Experimental mouse grouping

[0027] The experimental mice were divided into 3 groups: PBS group, OVA group, and OVA+CMU-Pb-L5 intervention group. In the PBS group, normal C57BL / 6 mice were treated with PBS by intragastric administration; in the OVA group, mice treated with OVA intervention model were treated with PBS by intragastric administration; in the OVA+CMU-Pb-L5 intervention group, mice treated with OVA intervention model were treated with CMU-Pb-L5 suspension by intragastric administration. The concentration of CMU-Pb-L5 was 10 9 CFU / mL. The volume of each intragastric administration solution was 0.2 mL. The intervention lasted for 28 days, once a day.

[0028] Preparation of CMU-Pb-L5 bacterial suspension: Cryopreserved medical cells CMU-Pb-L5 were inoculated into a test tube containing 5 mL of MRS liquid culture medium, and then placed in a carbon dioxide incubator for activation for 24 h. The concentration of CMU-Pb-L5 was adjusted to 10 9 CFU / mL. Take 1×10 9 The bacterial solution of CMU-Pb-L5 with a concentration of 2% (v / v) was inoculated into 15 mL of MRS broth and cultured for 24 h. The precipitate obtained by centrifugation was washed three times with saline and then the concentration of the bacterial solution was adjusted to 1×10 9 After inactivation in a 70°C constant temperature water bath for 1 h, inactivated medical cells CMU-Pb-L5 were obtained and used in the intervention experiment.

[0029] (3) Collection and processing of mouse serum and lung tissue

[0030] After treatment, serum was collected from each group: mice were anesthetized with 10% chloral hydrate, the mice were fixed, the eyeballs were removed, the whole blood that flowed out was collected, and the blood was centrifuged at 3000rpm for 5min. The clear supernatant was taken as the serum of each group of mice. After blood was collected from the eyeballs, the skin was cut open from the lower abdomen of the mice, and the chest of the mice was cut to expose the lung tissue, which was removed and placed in physiological saline and stored at 4°C.

[0031] Example 2 Detection of the effect of medical cells CMU-Pb-L5 on inflammatory factors in allergic asthma

[0032] The kit used was an enzyme-linked immunosorbent assay kit (IL-4, IgE), which was purchased from Jiubang Biotechnology.

[0033] (1) Soak the microplate: Add 300 μl of 1× washing solution and let it soak for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper. After washing, use the microplate immediately and do not let it dry.

[0034] (2) Add standard: Add 100 μl of 2-fold diluted standard to the standard wells. Add 100 μl of standard dilution to the blank wells.

[0035] (3) Adding samples: Take the mouse serum samples obtained in Experimental Example 1 (150 μl per sample) as samples and add them to the wells.

[0036] (4) Add detection antibody: Add 50 μl of diluted detection antibody (1:100 dilution) to each well (the antibody used is the antibody provided with the kit). Ensure that steps (2), (3), and (4) are added continuously without interruption. The addition process should be completed within 15 minutes.

[0037] (5) Incubation: Seal the plate with a sealing film. Oscillate at 100-300 rpm (to ensure that the solution in each well does not spill and is fully mixed), and incubate at room temperature (25°C ± 3°C) for 1.5 hours.

[0038] (6) Washing: Discard the liquid, add 300 μl of washing solution to each well, and wash the plate 6 times. After each wash, pat the plate dry on absorbent paper.

[0039] (7) Enzyme incubation: Add 100 μl of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well.

[0040] (8) Incubation: Seal the plate with a new sealing film. Oscillate at 100-300 rpm (to ensure that the solution in each well does not spill and is fully mixed), and incubate at room temperature (25°C ± 3°C) for 30 minutes.

[0041] (9) Washing: Repeat step (6).

[0042] (10) Add substrate for color development: Add 100 μl of the color development substrate TMB to each well, protect from light, and incubate at room temperature (25°C ± 3°C) for 5-30 min.

[0043] (11) Add stop solution: Add 100 μl of stop solution to each well. The color changes from blue to yellow. If the color appears green or the color change is obviously uneven, gently tap the plate frame to mix thoroughly.

[0044] (12) Detection reading: Within 30 minutes, dual-wavelength detection was performed using an ELISA reader to measure the OD value at the maximum absorption wavelength of 450 nm.

[0045] (13) Analyze the detected data. Figure 1 As shown in the figure, it was found that the IFN-γ and IgE in the serum of the OVA group mice were significantly higher than those in the PBS group, indicating that the OVA model was relatively successful; after the CMU-Pb-L5 intervention treatment, the IFN-γ and IgE levels in the serum of the OVA+probiotic group mice decreased, indicating that after the intervention of probiotics, the IFN-γ and IgE levels in the blood of the mice decreased and the symptoms were relieved.

[0046] Example 3 Detection of the effect of medical cells CMU-Pb-L5 on the pathological morphology of lung tissue in mice with allergic asthma

[0047] (1) Specimen collection and fixation

[0048] The mouse lung tissue obtained in Example 1 (3) was fixed with 4% paraformaldehyde fixative for 2 h; the tissue was then dehydrated with gradient alcohol (50%-75%-85%-95%-95%-100%-100%) for 1 h each; then transparentized in xylene twice, each time for 5 min; then immersed in soft wax twice, each time for 50 min; then immersed in hard wax twice, each time for 50 min, and finally embedded in paraffin; after paraffin embedding, the specimen was cut into 4 μm slices using a microtome.

[0049] (2) HE staining

[0050] The slices obtained in Experimental Example 3 (1) were immersed in xylene for 5 minutes, then immersed in anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol solutions for 3 minutes, rinsed with tap water for 1 minute, dried on the slides, and immersed in pure water. Subsequently, the slices were stained with hematoxylin for 5 minutes, rinsed with running water, differentiated with 1% hydrochloric acid alcohol for 1 second, returned to blue with running water for 5 minutes, stained with eosin for 30 seconds, and then immersed in 75% ethanol, 85% ethanol, 95% ethanol and anhydrous ethanol solutions for 1 minute, baked in an oven at 60°C for 5 minutes, and finally sealed with a neutral resin.

[0051] The results are as follows Figure 2 As shown, the shape of the alveoli and trachea in the PBS group did not change much by HE staining; the OVA group showed airway narrowing, increased mucus secretion, and severe airway and vascular inflammation; the OVA+CMU-Pb-L5 intervention group alleviated the inflammation and reduced mucus secretion.

[0052] (3) PAS staining

[0053] The slices obtained in Experimental Example 3 (1) were placed in xylene twice, each for 10 minutes. Then, they were placed in anhydrous ethanol twice, each for 3 minutes; then, they were placed in 90% ethanol and 70% ethanol for 3 minutes each, and the slides were washed clean with tap water. After washing the slides with water, the periodic acid solution that had been equilibrated to room temperature was taken out (operation in the dark), and 200 μL of 0.5% periodic acid solution was added to each sample. After reacting in a wet box in the dark for 10 minutes, the periodic acid solution was removed, and the slides were immersed in water with occasional shaking, and washed for 5 minutes. 200 μL Schiff reagent (purchased from Biyuntian) was added to each sample, and the slides were placed in a 37°C oven in the dark for staining for 1 hour. The staining solution was removed, and the slides were immersed in water with occasional shaking, and washed for 5 minutes. Hematoxylin staining was performed for 60 seconds, and then rinsed with water until the floating color was washed away. PBS blueing was performed for 10 seconds. 70% ethanol for 5 seconds, 90% ethanol for 10 seconds, and anhydrous ethanol for 10 seconds. Place in a 65°C oven to dry for 5 minutes, then place in xylene for 1 minute and seal the slides (gum + xylene 1:1). Score and analyze the PAS-stained sections.

[0054] PAS score. Ten areas were selected from each slice, and 10 fields of view were randomly selected from each area to perform PAS score on PAS-positive goblet cells: 0 points, PAS-positive goblet cell percentage <5%; 1 point, PAS-positive goblet cell percentage greater than or equal to 5% and less than 25%; 2 points, PAS-positive goblet cell percentage greater than or equal to 25% and less than 50%; 3 points, PAS-positive goblet cell percentage greater than or equal to 50% and less than 75%; 4 points, PAS-positive goblet cell percentage greater than or equal to 75%. Finally, the average value of each group of data was sorted out to serve as the PAS staining score of the group.

[0055] Specific as Figure 3 As shown in Table 1, the PBS group had fewer positive goblet cells, only a few areas; the OVA group had relatively more positive goblet cells; and the OVA+CMU-Pb-L5 intervention group had improved symptoms after the intervention. The specific scores are shown in Table 1.

[0056] Table 1 PAS results of each group

[0057]

[0058] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A use of medical cells CMU-Pb-L5 in the preparation of drugs for preventing and treating allergic asthma, characterized in that: The deposit number of the medical cell CMU-Pb-L5 is CGMCC No.25653.

2. The use according to claim 1, characterized in that: The medical cell CMU-Pb-L5 is a bacterial suspension.

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