Application of Lycium barbarum glycopeptide in the preparation of drugs for treating allergic airway inflammatory diseases

Abstract

The present invention discloses the use of Lycium barbarum glycopeptide in the preparation of a drug for treating allergic airway inflammatory diseases. Lycium barbarum glycopeptide or a drug mixed with one or at least two pharmaceutically acceptable carriers is used to prepare a drug for treating allergic airway inflammatory diseases, and the drug can play a good prevention, intervention and / or relief effect on allergic airway inflammatory diseases, and the drug also has the characteristics of small side effects and low drug resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to the use of a wolfberry glycopeptide in the preparation of a drug for treating allergic airway inflammatory diseases. Background Art

[0002] Allergic asthma and rhinitis are the most common allergic airway inflammatory diseases, and the two are often referred to as "same airway, same disease". In the past few decades, the incidence of these two diseases has increased year by year. Allergic rhinitis and asthma affect more than 300 million people worldwide and are major public health problems. They bring great burdens to patients themselves, society and families.

[0003] At present, clinical treatment mainly uses antihistamines, glucocorticoids and IgE monoclonal antibodies (expensive) to improve or relieve allergic rhinitis. Although the treatment of allergic rhinitis has achieved certain efficacy, it has not significantly reduced the incidence of allergic rhinitis. And with the increase in the number of patients with allergic rhinitis, clinical drugs can no longer meet the needs of all patients, resulting in limitations in the clinical treatment of allergic rhinitis. In addition, the current diagnostic method of specific IgE antibodies used in clinical practice has certain limitations. Therefore, studying the sensitization mechanism of Artemisia and related diagnostic indicators will help people prevent Artemisia allergies in advance, provide new clinical diagnostic ideas, and seek new and effective treatment methods, which is the focus of current research on allergic airway inflammatory diseases. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a use of Lycium barbarum glycopeptide in the preparation of a drug for treating allergic airway inflammatory diseases.

[0005] The present invention is achieved by using wolfberry glycopeptide in preparing medicine for treating allergic airway inflammatory diseases.

[0006] Preferably, the allergic airway inflammatory disease is allergic asthma and / or rhinitis.

[0007] Preferably, the allergic airway inflammatory disease is allergic asthma and / or rhinitis caused by allergy to Artemisia annua pollen.

[0008] Preferably, said treatment includes prevention, intervention and / or alleviation.

[0009] Preferably, the drug further contains one or at least two pharmaceutically acceptable carriers; the carrier is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant and a lubricant.

[0010] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: the wolfberry glycopeptide of the present invention is used to prepare a drug for treating allergic airway inflammatory diseases, which can have a good prevention, intervention and / or relief effect on allergic airway inflammatory diseases, and the drug also has the characteristics of small side effects and low drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a modeling scheme in an embodiment of the present invention;

[0012] Figure 2 is the statistical result of the mouse nasal mucosal thickness in the embodiment of the present invention;

[0013] Figure 3 is the lung inflammation scoring result in the embodiment of the present invention;

[0014] Figure 4 is the result of the test on the level of Artemisia annua pollen-specific IgE in the embodiment of the present invention;

[0015] Figure 5 The flow cytometry method in the present invention is used to detect the percentage of eosinophils in the lungs;

[0016] Figure 6 The flow cytometry method in the present invention is used to detect the percentage of Th1 cells in the spleen of mice;

[0017] Figure 7 In the embodiment of the present invention, flow cytometry was used to detect the proportion of Th2 cells in the spleen of mice;

[0018] Figure 8 The flow cytometry method in the present invention is used to detect the percentage of Th17 cells in the spleen of mice;

[0019] Fig. 9 The percentage of Treg cells in mouse spleen detected by flow cytometry in the embodiment of the present invention. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] In the embodiment of the present invention, Artemisia annua pollen is used as an allergen to sensitize Balb / c mice, to construct an Artemisia annua pollen allergic airway inflammation model mouse, and at the same time, the mice are intervened by oral administration of Lycium barbarum glycopeptide, and immunological indicators are detected.

[0022] 1. Experimental process

[0023] One month before the establishment of the allergic airway inflammation model, mice were gavaged with 200 μL, 2 mg / mL, 10 mg / mL, and 50 mg / mL Lycium barbarum glycopeptide solution (solvent: deionized water) every day until the model was completed. Mice in the control group and model group were given PBS instead of Lycium barbarum glycopeptide every day (Lycium barbarum glycopeptide was purchased from Tianren Bioengineering Co., Ltd., Zhongning, Ningxia). The model group and the model + Lycium barbarum glycopeptide group were sensitized by intraperitoneal injection of 100 μL of Artemisia annua antigen solution (the solution consisted of 30 μg Artemisia annua allergen protein and 1 mg aluminum hydroxide solvent, the Artemisia annua allergen protein was a mixed antigen extracted from Artemisia annua pollen, purchased from Beijing Xinhualian Union Pharmaceutical Co., Ltd.) on the 31st, 38th and 45th days; nasal drops were performed every day from the 52nd to the 58th day, wherein the Artemisia annua pollen antigen extract (purchased from Beijing Xinhualian Union Pharmaceutical Co., Ltd.) was diluted with deionized water to 0.5 mg / mL and then bilaterally dripped, 10 μL per side; the control group and the control + LBP group were sensitized and dripped with PBS using the same method and dose. After the end of the nasal drops on the 58th day, the allergic airway inflammation model caused by Artemisia annua pollen was successfully established ( Figure 1 ). On day 59, the mice were euthanized and samples were collected.

[0024] Grouping:

[0025] Con: blank control group, Con-low dose: low-dose (2 mg / mL) glycopeptide control group, Con-middle dose: medium-dose (10 mg / mL) glycopeptide control group, Con-high dose: high-dose (50 mg / mL) glycopeptide control group, APE Model: Artemisia annua model group, APE-low dose: low-dose (2 mg / mL) glycopeptide gavage model group, APE-middle dose: medium-dose (10 mg / mL) glycopeptide gavage model group, APE-high dose: high-dose (50 mg / mL) glycopeptide gavage model group.

[0026] 2. Experimental Results

[0027] 1. Statistics of mouse nasal mucosal thickness

[0028] Measurement of mucosal thickness by software [1] The statistical results are as follows Figure 2 As shown, Lycium barbarum glycopeptide can significantly reduce the thickness of the nasal mucosa of model mice and alleviate the level of edema.

[0029] 2. Pulmonary inflammation score

[0030] Two independent investigators performed histopathological evaluation and scored inflammation in a double-blind manner. [2]Among them, 0 represents no inflammation; 1 represents occasional inflammatory cell entanglement; 2 represents inflammatory cells with 1 to 2 cell layers around the bronchi or blood vessels; 3 and 4 represent inflammatory cells with a medium layer of 3 to 5 cells or more than 5 layers, respectively.

[0031] The experimental results are as follows Figure 3 As shown, Lycium barbarum glycopeptide can significantly reduce the level of inflammatory cell infiltration in the lungs of mice and alleviate lung inflammation.

[0032] 3. Detection of Artemisia annua pollen-specific IgE level

[0033] The serum samples were centrifuged at 3000g for 10 minutes at 4°C, and the supernatant was used to measure the IgE level. 2.5μg / mL Artemisia annua coating solution was coated on the ELISA plate and incubated overnight at 4°C. The serum was diluted 1:100 and the HRP-labeled IgE antibody was diluted 1:500. The colorimetric solution was added and allowed to stand at room temperature for 5 minutes until the liquid in the well turned blue. Then, the ELISA stop solution was added and the absorbance was measured at 450nm. [3] .

[0034] The experimental results are as follows Figure 4 As shown, ELISA was used to detect the level of Artemisia annua pollen-specific IgE in mouse serum. The test results showed that the sIgE of model mice gavaged with Lycium barbarum glycopeptide was significantly decreased compared with that of model mice, indicating that the severity of allergic airway inflammation was alleviated.

[0035] 4. Flow cytometry to detect the proportion of eosinophils in the lungs [4]

[0036] The left lung tissue of the mouse was ground into a single cell suspension, and red blood cell lysis buffer was added to lyse the red blood cells. The concentration of the cell suspension was adjusted to 1×10 7 Take 100 μL of the treated cell suspension, add 0.3 μL Percp-cy5.5-labeled anti-mouse CD45 antibody, 1.25 μL FITC-labeled anti-mouse CD11c antibody, 1.25 μL APC-labeled anti-mouse CD170 antibody, incubate at 4°C in the dark for 30 min, wash twice with staining buffer by centrifugation, resuspend the cells with 300 μL paraformaldehyde, and then detect EOS cells (CD45+CD11c-CD170+) by flow cytometry.

[0037] The experimental results are as follows Figure 5 As shown, Lycium barbarum glycopeptide can significantly reduce the proportion of eosinophils in the lungs of mice and alleviate lung inflammation.

[0038] 5. Flow cytometry detection of the proportion of Treg, Th1, Th2, and Th17 cells in mouse spleen [5]

[0039] Mouse spleen single cells were prepared, ground and passed through a 70 μm cell strainer, and suspended in PBS containing 2 mm EDTA and 5% fetal bovine serum (FBS). Red blood cell lysis buffer (BioLegend) was then used to lyse the red blood cells. After lysis, the cells were counted under a microscope and the cell concentration was adjusted to 10 7 Order of magnitude. Take 300 μL of the treated spleen cell suspension and transfer it to a 48-well plate, add ionomycin and phorbol lipids and place it in a 37°C incubator for 5 hours. Take 100 μL of the cell suspension and add anti-mouse CD4-FITC and anti-mouse CD3-APC on ice in the dark for 30 minutes, then add permeabilization solution and anti-mouse IFN-γ-PerCP-Cy5.5, anti-mouse IL-4-PE, anti-mouse BV421-IL-17A while permeabilizing and staining, incubate at room temperature for 30 minutes, wash with staining buffer, resuspend the cells in 300 μL of paraformaldehyde and analyze the percentage of Th1 cells, Th2 cells, and Th17 using flow cytometer Attune NxT4 (Thermo Fisher Scientific, USA). Anti-mouse CD4-FITC and anti-mouse CD3-APC were added to the cell suspension and incubated on ice in the dark for 30 min. Foxp3 fixation working solution (biolegend, San Diego, CA, USA) was added, followed by anti-mouse CD25-PE-cy7 and anti-mouse Foxp3-PE (biolegend, San Diego, CA, USA) according to the instructions of the reagents. After washing with staining buffer, the cells were resuspended in 300 μL of paraformaldehyde and CD25 in lymphocytes was analyzed by flow cytometry. high FOXP3 + Treg cells. All flow cytometry results were analyzed using Flowjo 10.8.

[0040] The experimental results are as follows Figures 6 to 9 As shown, allergic airway inflammation is mediated by Th2 cells. The proportion of Th2 cells in the spleen of the model group mice gavaged with Lycium barbarum glycopeptide decreased, Th1 cells increased, and Th17 cells decreased. The changes in the proportions of these three immune cells can alleviate allergic airway inflammation.

[0041] 3. Experimental Conclusion

[0042] After oral administration of Lycium barbarum glycopeptide, the model mice underwent changes in morphological and immunological indicators, and airway inflammation was alleviated.

[0043] References:

[0044] [1]Dendrobiumnobileprotects against ovalbumin-induced allergicrhinitis by regulating intestinal flora and suppressing lung inflammation.

[0045] [2]Gu-Ben-Fang-Xiao Decoction Ameliorated Murine Asthma in RemissionStage by Modulating Microbiota-Acetate-Tregs Axis.

[0046] [3]Rosae multiflorae fructus extract and its four active componentsalleviate ovalbumin-induced allergic inflammatory responses via regulation ofTh1 / Th2 imbalance in BALB / c rhinitis mice.

[0047] [4]Polymorphonuclear myeloid-derived suppressor cells attenuateallergic airway inflammation by negatively regulating group 2innate lymphoidcells.

[0048] [5]Elevated granulocytic myeloid-derived suppressor cells are closely related with elevation of Th17 cells in mice with experimental asthma, TheImmuno-Modulatory Activities of Pentaherbs Formula on Ovalbumin-InducedAllergic Rhinitis Mice via the Activation of Th1 and Treg Cells andInhibition of Th2 and Th17 Cells.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of Lycium barbarum glycopeptide in the preparation of a drug for treating allergic airway inflammatory diseases, wherein the allergic airway inflammatory diseases are allergic asthma caused by Artemisia annua pollen allergy and / or rhinitis caused by Artemisia annua pollen allergy.

2. The use according to claim 1, characterized in that The medicine further contains one or at least two pharmaceutically acceptable carriers; the carrier is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant and a lubricant.

Images