Application of cycloastragenol in preparation of medicine for preventing and treating epithelial barrier injury

Abstract

The invention discloses application of cycloastragenol in preparation of a medicine for preventing and treating epithelial barrier injury, and relates to the technical field of biomedicines.The cycloastragenol is adopted for intervention, so that organoid is remarkably protected from being damaged by house dust mites, the morphological structure and function of the organoid are maintained, and stability of a lumen is kept; the trans-epithelial permeability is detected through fluorescein isothiocyanate-glucan, and it is observed that cycloastragenol can enhance the integrity of an epithelial barrier. The cycloastragenol can maintain the integrity of the Caco-2 intestinal organ chip epithelial barrier damaged by the cytokine mixture. According to the cycloastragenol, expression of Occludin and ZO-1 in lung tissue of a dermatophagoides pteronyssinus-induced epithelial barrier impaired mouse model is remarkably improved, and epithelial barrier injury is improved; cycloastragenol does not generate liver and kidney toxicity in vivo. The cycloastragenol is proved to be capable of effectively repairing the epithelial barrier injury, and a new medicine preparation thought is provided for related research on prevention and treatment of the epithelial barrier injury.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of cycloastragenol in preparing a drug for preventing and treating epithelial barrier damage. Background Art

[0002] Epithelial barrier damage is an important pathological feature of inflammatory diseases, especially in respiratory and intestinal diseases. As the connection point between genes and the environment, abnormalities in its structure or function often trigger or aggravate inflammatory damage. Modern medicine has proposed the theory of "epithelial barrier damage", that is, when exposed to allergens, the epithelial barrier is damaged, resulting in increased epithelial permeability, thereby exacerbating the inflammatory response. At this time, tight junction proteins such as tight junction protein-1 (ZO-1) and occludin will be damaged. Previous studies have found that the expression of tight junction proteins in the epithelium of patients with respiratory diseases is significantly reduced, suggesting that their barrier structure has been damaged. However, existing treatments mostly focus on symptom control, and lack effective protection strategies for epithelial barrier damage. Therefore, there is an urgent need to develop a safe and effective therapy to repair epithelial barrier damage.

[0003] Cycloastragenol (CAG) is an active ingredient extracted from the traditional Chinese medicine Astragalus, which belongs to the aglycone skeleton of saponin compounds. It has multiple pharmacological effects such as antioxidant, anti-inflammatory, and anti-fibrosis. However, the role of cycloastragenol in epithelial barrier protection has not been fully studied and applied. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a new drug use of cycloastragenol, that is, the use of cycloastragenol in the preparation of drugs for preventing and treating epithelial barrier damage. The present invention finds that cycloastragenol increases the expression of tight junction proteins and strengthens the tight junctions between cells, thereby protecting the epithelial barrier, and then effectively reduces the surrounding inflammatory response, preventing and treating epithelial barrier damage inflammatory diseases caused by it.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The cycloastragenol of the present invention maintains the morphological structure and function of airway organoids, maintains the stability of the lumen, and can enhance the integrity of the epithelial barrier. In addition, cycloastragenol can maintain the integrity of the intestinal epithelial barrier. Cycloastragenol can also significantly increase the expression of tight junction proteins Occludin and ZO-1 in HDM-induced lung tissue without producing liver and kidney toxicity.

[0007] The cycloastragenol (English name: Cycloastragenol, molecular formula C 30 H 50O5, CAS registration number: 78574-94-4), has the structure of formula (I), and is commercially available.

[0008]

[0009] The present invention provides the use of cycloastragenol in preparing medicines for preventing and / or treating diseases related to epithelial barrier damage.

[0010] In some specific embodiments of the present invention, cycloastragenol is used as a single effective ingredient, or cycloastragenol is used in combination with other effective ingredients.

[0011] In some specific embodiments of the present invention, cycloastragenol significantly protects organoids from damage by house dust mites, maintains their morphological structure and function, and keeps the lumen stable.

[0012] In some embodiments of the present invention, cycloastragenol significantly enhances the integrity of the epithelial barrier.

[0013] In some specific embodiments of the present invention, cycloastragenol significantly increases the expression of tight junction proteins Occludin and ZO-1 in mouse lung tissue.

[0014] In some specific embodiments of the present invention, cycloastragenol does not produce liver and kidney toxicity in vivo.

[0015] In some specific embodiments of the present invention, an in vitro model of airway epithelial barrier damage is constructed by using organoids derived from human bronchial epithelial cells induced by house dust mites.

[0016] In some specific embodiments of the present invention, the in vitro model of airway epithelial barrier damage is constructed by culturing ALI derived from human bronchial epithelial cells induced by house dust mites.

[0017] In some specific embodiments of the present invention, an in vitro model of intestinal epithelial barrier damage is constructed by a Caco-2 intestinal organ chip induced by a cytokine mixture.

[0018] In some specific embodiments of the present invention, the in vivo model of airway epithelial barrier damage is constructed by inducing BALB / c mice with house dust mites.

[0019] The human bronchial epithelial cell line of the present invention is NHBE.

[0020] In some specific embodiments of the present invention, diseases associated with epithelial barrier damage include:

[0021] (I) Respiratory system diseases. In some specific embodiments of the present invention, the respiratory system diseases include allergic rhinitis, asthma, and chronic obstructive pulmonary disease.

[0022] (II) Intestinal inflammatory diseases In some specific embodiments of the present invention, the intestinal inflammatory disease includes inflammatory bowel disease (IBD).

[0023] Beneficial effects of the present invention:

[0024] The present invention provides the use of cycloastragenol in the preparation of drugs for preventing and treating epithelial barrier damage. The intervention of cycloastragenol significantly protects organoids from damage by house dust mites, maintains their morphological structure and function, and maintains the stability of the lumen; the transepithelial permeability is detected by fluorescein isothiocyanate (FITC)-dextran, and it is observed that cycloastragenol can enhance the integrity of the epithelial barrier. In addition, cycloastragenol can maintain the integrity of the epithelial barrier of the Caco-2 intestinal organ chip damaged by a cytokine mixture. In addition, cycloastragenol significantly increases the expression of tight junction proteins Occludin and ZO-1 in the lung tissue of a mouse model of epithelial barrier damage induced by house dust mites, and improves epithelial barrier damage; cycloastragenol does not produce hepatotoxicity in vivo. The present invention confirms that cycloastragenol can effectively repair airway and intestinal epithelial barrier damage, and provides a new idea for drug preparation for related research on preventing and treating airway and intestinal epithelial barrier damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The number, morphology and structure of organoids in different treatment groups under an optical microscope on the 21st day in Example 1 (100×), scale bar: 100 μm.

[0026] Figure 2 These are the results of FITC-Dextran transepithelial permeability in different treatment groups in Example 1; wherein A is a representative optical and fluorescence microscopy image (200×) of the organoid paracellular flux assay on day 21, scale bar: 50 μm; B is the quantitative statistics of FITC-Dextran transepithelial permeability.

[0027] Figure 3 It is the result of airway epithelial barrier permeability in different treatment groups of airway epithelial cell air-liquid interface (ALI) culture in Example 2; wherein A is transmembrane electrical resistance (TEER); and B is the quantitative statistics of FITC-Dextran transepithelial permeability.

[0028] Figure 4 These are the results of intestinal epithelial barrier permeability in different treatment groups based on the Caco-2 intestinal organ chip in Example 3; wherein A is the transmembrane electrical resistance (TEER); and B is the quantitative statistics of FITC-Dextran transepithelial permeability.

[0029] Figure 5The figures are the expressions of epithelial barrier-related Occludin and ZO-1 in different treatment groups in Example 4; A is the protein blot bands of Occludin and ZO-1; B is the protein expression of ZO-1 / GAPDH and Occludin / GAPDH; C is the immunohistochemical staining results of ZO-1 and Occludin (100×), scale: 100 μm; D is the positive area of ​​ZO-1 and Occludin.

[0030] Figure 6 The following are the pathological results of liver and kidney tissues of mice in different treatment groups in Example 4; A is a representative image of liver tissue H&E staining; B is a representative image of kidney tissue H&E staining (200×). Scale bar: 50 μm. DETAILED DESCRIPTION

[0031] The present invention discloses the use of cycloastragenol in the preparation of drugs for preventing and treating epithelial barrier damage. Those skilled in the art can refer to the content of the present invention and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described in the present invention without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0032] The cycloastragenol (CAG) described in the embodiment of the present invention has the following appearance: white powder; purity: 98%; packaging: 1 mg; storage conditions: 3 years / -20°C / powder.

[0033] The present invention uses house dust mite (HDM) to intervene in the organoid model established by the normal human bronchial epithelial cell line NHBE, ALI culture and BALB / c mouse model, and the Caco-2 intestinal organ chip epithelial injury model induced by a cytokine mixture, and uses CAG treatment to observe the repair effect of CAG on airway and intestinal epithelial barrier damage. The results show that CAG can maintain the morphological structure and function of airway organoids, maintain the stability of the lumen, and enhance the integrity of the epithelial barrier. In addition, CAG can maintain the integrity of the epithelial barrier of the Caco-2 intestinal organ chip damaged by a cytokine mixture. In addition, CAG significantly increases the expression of tight junction proteins Occludin and ZO-1 in HDM-induced lung tissue, and does not produce hepatotoxicity and renal toxicity. The results confirm that CAG has the effect of repairing epithelial barrier damage.

[0034] In the embodiments of the present invention, CAG is preferably used as a single ingredient in the preparation of a drug for preventing and treating epithelial barrier damage.

[0035] The raw materials and reagents used in the preparation of the drug for preventing and treating epithelial barrier damage using the cycloastragenol (CAG) of the present invention can all be purchased commercially.

[0036] The present invention uses house dust mite (HDM) intervention in the organoid model established by normal human bronchial epithelial cell line NHBE, ALI culture and BALB / c mouse model, and cytokine mixture-induced Caco-2 intestinal organ chip epithelial injury model, and CAG treatment. The present invention will be further described in conjunction with the embodiments and drawings for a more convenient understanding of the present invention. Figure 1-6 n = 4 for each group of organoids. n = 6 for each group of mice. Standard errors are means ± SEMs. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001 vs. model group.

[0037] The present invention will be further described below in conjunction with embodiments:

[0038] Example 1: CAG intervention in HDM-induced organoid epithelial barrier injury model

[0039] Normal human bronchial epithelial cell line NHBE was purchased from the American Type Culture Collection (ATCC) and cultured in all-trans-retinoic acid (ATRA)-differentiation medium containing 5% extracellular matrix (ECM) (BEBM and DMEM were prepared in a volume ratio of 1:1, hereinafter referred to as AO medium). The culture conditions were 37°C and 5% CO2 concentration. Preparation of AO medium: Mix 250mL BEBM and 250mL DMEM, and add BEGM SingleQuot except retinoic acid (RA) and triiodothyronine (T3) to make differentiation medium. When used, add 3μL of ATRA to each 5mL of differentiation medium, freshly prepare it to a concentration of 15ng / mL, and add 5% ECM to use as airway organoid (AO) medium.

[0040] 1. Experimental Methods

[0041] 1.1 HDM-induced epithelial barrier injury model and CAG intervention

[0042] After culturing for 18 days, medium containing HDM (optimal concentration: 200 μg / mL) was added and stimulated for 3 days to prepare an epithelial barrier injury model. The experiment was randomly divided into 6 groups, with 7 wells in each group, as follows:

[0043] 1) Control group: No additional intervention was performed except for the replacement of the culture medium during the culture process.

[0044] 2) 200 μg / mL HDM group: On day 18, AO medium containing 200 μg / mL HDM was added and stimulated for 3 days.

[0045] 3) CAG 0.03 μM group and CAG 0.11 μM group: AO medium containing 0.03 μM and 0.11 μM CAG was added from the 2nd day, and the medium was changed every other day until the 18th day.

[0046] 4) 200μg / mL HDM+CAG 0.03μM group and 200μg / mL HDM+CAG 0.11μM group: AO medium containing 0.03μM and 0.11μM CAG was added from day 2, and the medium was changed every other day until day 18. On day 18, AO medium containing 200μg / mL HDM was added and stimulated for 3 days.

[0047] And on the 21st day, the six groups were photographed under a light microscope.

[0048] 1.2 Fluorescein isothiocyanate (FITC)-dextran to detect transepithelial permeability

[0049] Three replicate wells were set up in each group. AO medium containing 0.5 mg / mL FITC-Dextran 4kDa was prepared in advance. 250 μL of the original medium was taken out from each well of the 96-well plate, and 250 μL of AO medium containing 0.5 mg / mL FITC-Dextran was added. The plates were incubated in a 37°C incubator with 5% CO2 for 24 h. After the incubation, at least 25 different organoid photos were taken in each group using a confocal microscope. The images were processed using FIJI (ImageJ, v2.3.0) software, and the background intensity at 3 locations in each image was randomly measured to obtain the average value. The intensity of the lumen of the organoid was then measured, and the ratio of the lumen intensity value to the background intensity mean was used to represent the intensity.

[0050] 2. Experimental results

[0051] 2.1 CAG can protect organoids from HDM damage

[0052] In the preliminary experiments of cytotoxicity test of human bronchial epithelial cells and air-liquid interface culture, we finally determined that the HDM intervention concentration at the organoid level was 200μg / mL, and the CAG administration concentration was 0.11μM and 0.03μM. We observed through microscopy that after 3 days of HDM intervention, the organoids showed a significant decrease in number, disappearance of the lumen, and shrinkage of the morphology. The 0.11μM CAG intervention group did not change under the microscope, but the 0.03μM CAG intervention was able to partially reverse this situation, so that the number and morphology of the organoids remained basically unchanged, and the stability of the lumen was maintained ( Figure 1 ).

[0053] 2.2 CAG can maintain the integrity of the epithelial barrier in organoid models

[0054] We used FITC-Dextran transepithelial permeability assay and confocal fluorescence microscopy to observe that HDM intervention significantly increased the permeability of the epithelial barrier, suggesting that HDM induced damage to the epithelial barrier. However, intervention with 0.11μM CAG and 0.03μM CAG tended to reduce the permeability of the epithelial barrier ( Figure 2 ), further confirming the repair effect of CAG on epithelial barrier damage.

[0055] Example 2: CAG intervention in HDM-induced airway epithelial ALI epithelial barrier injury model

[0056] Normal human bronchial epithelial cell line NHBE was purchased from the American Type Culture Collection ATCC, and the cells were inoculated into the Transwell chamber. ATRA-containing differentiation medium (BEBM and DMEM prepared in a volume ratio of 1:1) was used for ALI culture of airway epithelium. The preparation method of differentiation medium was as follows: BEGM SingleQuot was added to 500mL BEBM, and then 500mL DMEM was added to make differentiation medium. 200μL of upper chamber medium and 600μL of lower chamber medium. The culture conditions were 37°C and 5% CO2 concentration.

[0057] 1. Experimental Methods

[0058] 1.1 HDM-induced ALI epithelial barrier injury model and CAG intervention

[0059] On the second day of culture, 0.11 μM CAG and 0.03 μM CAG were added to the corresponding culture medium for intervention. After culture for 18 days, medium containing HDM (optimal concentration: 200 μg / mL) was added and stimulated for 3 days to prepare an epithelial barrier injury model. The experiment was randomly divided into 6 groups, with similar organ experiments in each group and intervention as above.

[0060] 1.2 Transmembrane resistance TEER detection

[0061] Gently wash the upper chamber with preheated PBS to remove floating cells and culture medium residues. Sterilize the electrodes and connect the EVOM2 transmembrane resistance meter to the STX2 electrode. Immerse one electrode in the upper chamber and place the other in the lower chamber, making sure that the electrode does not touch the cell layer to avoid mechanical damage to the cells. Start the EVOM2 device, read the transmembrane resistance (TEER) value stably, and record the measurement results. To obtain accurate data, repeat the measurement 3 times and take the average value.

[0062] 1.3 FITC-Dextran detection of ALI transepithelial permeability

[0063] Three replicate wells were set up in each group. A culture medium containing 2 mg / mL FITC-Dextran 4kDa was prepared in advance, and 200 μL of the original culture medium was taken out from each well of the upper chamber of the 24-well plate. After the upper chamber of the cells was gently washed with preheated PBS, 200 μL of preheated culture medium containing 2 mg / mL FITC-Dextran was added, and the lower chamber was replaced with the normal medium, and incubated in a 37°C incubator with 5% CO2 for 24 hours. After the incubation, 50 μL of culture medium was collected from the lower chamber, and the fluorescence intensity of the sample was measured with a fluorescence spectrophotometer. By comparing with the standard curve, the amount of FITC-Dextran that passed through was calculated to evaluate the permeability of the epithelial barrier.

[0064] 2. Experimental results

[0065] 2.1 CAG can maintain the integrity of the epithelial barrier in airway epithelial ALI culture

[0066] We observed that after 1 day of HDM intervention, the transmembrane resistance TEER was significantly reduced, and the intervention of 0.03 μM CAG could significantly restore the increased TEER value ( Figure 3 A).

[0067] 2.2 CAG can reduce the permeability of the airway epithelial barrier in airway epithelial ALI culture

[0068] We confirmed through FITC-Dextran transepithelial permeability assay that HDM intervention significantly increased airway epithelial permeability, while 0.11μM CAG and 0.03μM CAG intervention tended to reduce its permeability ( Figure 3 B) indicates that CAG repairs airway epithelial barrier damage.

[0069] Example 3: CAG intervention in the Caco-2 intestinal organ chip epithelial barrier injury model induced by cytokine mixture

[0070] Using Mimetas' intestinal organ chip platform, Caco-2 cells were inoculated into the microfluidic channel of the chip. The culture medium was DMEM culture medium containing 10% fetal bovine serum. The culture conditions were 37°C and 5% CO2 concentration. After 8 days of culture, Caco-2 cells will form a tight monolayer epithelium with complete barrier function.

[0071] 1. Experimental Methods

[0072] 1.1 Cytokine mixture-induced intestinal epithelial barrier injury model in intestinal organ chip and CAG intervention

[0073] On the second day of culture, 0.11 μM CAG and 0.03 μM CAG were added to the corresponding culture medium. After culturing for 8 days, a cytokine mixture was prepared, and its concentration was determined by the results of the previous preliminary experiment, which was 50 ng / ml TNF-α, 50 ng / ml IFN-γ and 50 ng / ml IL-1β. The prepared cytokine mixture was added to the upper chamber of the intestinal organ chip to ensure that the cytokines could evenly contact the Caco-2 cell monolayer and stimulate for 3 days to simulate the intestinal epithelial barrier damage microenvironment. The experimental groups were as above.

[0074] 1.2 Transmembrane resistance TEER detection

[0075] After the cells were kept in a horizontal position for 30 min, they were treated with Organo The instrument software reads the transmembrane electrical resistance (TEER) value and records the measurement results.

[0076] 1.3 Fluorescein isothiocyanate (FITC)-dextran to detect transepithelial permeability

[0077] Three replicate wells were set up for each group. A culture medium containing 2 mg / mL FITC-Dextran 4kDa was prepared in advance, the original culture medium in the upper and lower chambers was aspirated, and the upper and lower chambers of the chip were gently rinsed with preheated PBS. Then, 50 μL of preheated culture medium containing 2 mg / mL FITC-Dextran was added to the upper chamber, and the lower chamber was replaced with the normal medium. The cells were incubated in a 37°C incubator with 5% CO2 for 24 hours. After the incubation, 50 μL of culture medium was collected from the lower chamber, and the fluorescence intensity of the sample was measured with a fluorescence spectrophotometer. By comparing with the standard curve, the amount of FITC-Dextran that passed through was calculated, and the permeability of the epithelial barrier was evaluated.

[0078] 2. Experimental results

[0079] 2.1 CAG can maintain the integrity of the intestinal epithelial barrier

[0080] We observed that after 3 days of cytokine mixture intervention, transmembrane resistance TEER was significantly reduced, and intervention with 0.03 μM CAG could significantly restore the increased TEER value ( Figure 4 A).

[0081] 2.2CAG can reduce the permeability of the intestinal epithelial barrier

[0082] We confirmed through FITC-Dextran transepithelial permeability assay that the cytokine mixture intervention significantly increased the airway epithelial permeability, while the intervention of 0.11μM CAG and 0.03μM CAG had a tendency to reduce its permeability ( Figure 4 B), suggesting that CAG repairs intestinal epithelial barrier damage.

[0083] Example 4: CAG intervention in HDM-induced airway epithelial barrier injury mouse model

[0084] 1. Experimental Methods

[0085] 1.1 Construction of HDM-induced airway epithelial barrier injury mouse model

[0086] The mice were randomly divided into 3 groups: normal control (N), model (M), and CAG intervention (C). There were 5 mice in each group. Except for group N, the mice in the other two groups were sensitized by intranasal dripping of 0.4 mL of PBS solution containing 10 μg HDM on day 0. From day 7, the mice were challenged by intranasal dripping of 0.4 mL of PBS solution containing 20 μg HDM, once a day for 5 consecutive days. Group N was sensitized and challenged with PBS instead.

[0087] 1.2 Drug intervention

[0088] According to the drug dose conversion formula between humans and mice in "Modern Pharmacology Research Methods", that is, mouse dose (mg / kg) = 9.01 (conversion coefficient) * human dose (mg / kg), combined with relevant literature review, the dosage of CAG is set at: 125 mg / kg. CAG powder was added to the prepared 0.5% CMC-Na, stirring while adding, and after all added, ultrasonic instrument was used to dissolve for 10 minutes, and fresh suspension was re-prepared before each intervention. From the 7th day, mice in groups N and M were gavaged with 0.2 mL of 0.5% CMC-Na suspension, while mice in group C were gavaged with 0.2 mL of 125 mg / kg CAG-0.5% CMC-Na suspension, once a day for 5 consecutive days.

[0089] 1.3 Expression detection and localization of epithelial barrier-related proteins

[0090] Occludin and ZO-1 are two key proteins that maintain tight junctions of the epithelial barrier. Their expression can reflect whether the structure and function of the epithelial barrier are intact and whether the epithelial barrier is damaged. Therefore, we used Western Blot and immunohistochemistry to detect the expression and localization of Occludin and ZO-1.

[0091] 1.6 Pathological examination of liver and kidney tissues

[0092] The collected left liver and left kidney sections were fixed in formalin for 24 h, then embedded in paraffin and cut into 4 μm sections, followed by hematoxylin-eosin (H&E) staining for evaluation.

[0093] 2. Experimental results

[0094] 2.1CAG can improve the integrity of the epithelial barrier

[0095] At the protein level, HDM stimulation significantly reduced the expression of two tight junction proteins, Occludin and ZO-1, while the application of CAG was able to restore them to near normal levels (at least P < 0.05) ( Figure 5 AD).

[0096] 2.4 CAG does not cause liver and kidney toxicity in mice

[0097] We observed that in the N and C groups, the liver structures were relatively complete and clear, the hepatocytes were polyhedral in shape, and the hepatocyte cords were neatly arranged in a radial pattern ( Figure 6 A). In addition, the renal structures of the three groups were relatively complete and clear, with intact glomeruli, uniform cystic cavities, and clear boundaries of renal tubules ( Figure 6 B) The results indicate that administration of CAG does not cause structural damage to the liver and kidneys.

Claims

1. Application of cycloastragenol in the preparation of drugs for preventing and treating epithelial barrier damage.

2. The use according to claim 1, characterized in that: The cycloastragenol is used as a single active ingredient, or the cycloastragenol is used in combination with other active ingredients. The cycloastragenol increases the expression of tight junction proteins and strengthens the tight junctions between cells, thereby protecting the epithelial barrier.

3. The use according to claim 1, characterized in that: The epithelial barrier damage-related disease is at least one of the following (I)-(II): (I) Respiratory diseases, including allergic rhinitis, asthma, and chronic obstructive pulmonary disease; (II) Intestinal inflammatory diseases, including inflammatory bowel disease.

4. The use according to claim 1, characterized in that: At least one of the following (I)-(II): Preparation of medicines for protecting against damage by house dust mites, maintaining their morphological structure and function, and keeping the lumen stable; Preparation of a medicament for enhancing the integrity of the epithelial barrier; Preparation of drugs for increasing the expression of tight junction proteins Occludin and ZO-1 in lung tissue.

5. The use according to claim 1, characterized in that: The in vitro model of airway epithelial barrier damage was constructed by organoids derived from human bronchial epithelial cells induced by house dust mites, and the human bronchial epithelial cell line was NHBE; the specific construction method is as follows: Normal human bronchial epithelial cell line NHBE was cultured with ATRA-differentiation medium containing 5% ECM at 37°C and 5% CO2. After culturing for 18 days, medium containing house dust mites was added at a concentration of 200 μg / mL and stimulated for 3 days to prepare an epithelial barrier damage model.

6. The use according to claim 1, characterized in that: The in vitro model of airway epithelial barrier injury was constructed by culturing ALI derived from human bronchial epithelial cells induced by house dust mites, wherein the human bronchial epithelial cell line was NHBE; the specific construction method is as follows: Normal human bronchial epithelial cell line NHBE was inoculated into Transwell chambers and ALI culture of airway epithelium was performed using ATRA-differentiation medium, 200 μL of upper chamber medium and 600 μL of lower chamber medium, at 37°C and 5% CO2 concentration. After culturing for 18 days, medium containing house dust mites was added at a concentration of 200 μg / mL and stimulated for 3 days to prepare an epithelial barrier injury model.

7. The use according to claim 1, characterized in that: The in vitro model of intestinal epithelial barrier damage was constructed by a Caco-2 intestinal organ chip induced by a cytokine mixture; the specific construction method is as follows: Using the intestinal organ chip platform, Caco-2 cells were inoculated into the microfluidic channel of the chip. The culture medium was DMEM medium containing 10% fetal bovine serum. The culture conditions were 37°C and 5% CO2 concentration. After culturing to the 8th day, a cytokine mixture of 50ng / ml TNF-α, 50ng / ml IFN-γ and 50ng / ml IL-1β was prepared and added to the upper chamber of the intestinal organ chip to ensure that the cytokines could evenly contact the Caco-2 cell monolayer and stimulate for 3 days to simulate the intestinal epithelial barrier damage microenvironment.

8. The use according to claim 1, characterized in that: The in vivo model of airway epithelial barrier damage was established by inducing BALB / c mice with house dust mites. The specific construction method is as follows: BALB / c mice were sensitized by intranasal dripping of 0.4 mL of PBS solution containing 10 μg of house dust mites on day 0, and challenged by intranasal dripping of 0.4 mL of PBS solution containing 20 μg of house dust mites starting from day 7, once a day for 5 consecutive days.

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