Application of ADAM8 gene and / or ADAM8 protein in preparation of medicine for treating chronic sinusitis with nasal polyp and medicine

Abstract

The invention discloses application of an ADAM8 gene and / or ADAM8 protein in preparation of a medicine for treating chronic sinusitis with nasal polyp and the medicine, and relates to the technical field of biological medicines. According to the application of the ADAM8 gene and / or the ADAM8 protein in preparation of a medicine for treating chronic sinusitis with nasal polyp, the ADAM8 gene and / or the ADAM8 protein are / is used as a treatment target. The research finds that the gene and protein expression level of ADAM8 are obviously up-regulated in ECRSwNP and NECRSwNP and are mainly distributed in inflammatory cells of epithelium and below the epithelium, which prompts that ADAM8 may be a key pathogenic factor in CRSwNP pathogenesis. By specifically inhibiting the activity of ADAM8, nasal polyp-like lesions can be remarkably relieved, infiltration of eosinophilic granulocytes, neutrophil granulocytes and other inflammatory cells can be reduced, key inflammatory signal pathways can be inhibited, collagen deposition and metal matrix protease expression can be reduced, then inflammatory response and a tissue remodeling process can be effectively relieved, and the application of ADAM8 in treatment of nasal polyp-like diseases can be promoted. The scientificity and clinical application prospect of the ADAM8 as a treatment target are verified.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of ADAM8 gene and / or ADAM8 protein in the preparation of a drug for treating chronic rhinosinusitis with nasal polyps. BACKGROUND

[0002] Chronic rhinosinusitis with nasal polyps (CRSwNP) is a multifactorial, heterogeneous inflammatory disease, and its pathological features mainly include eosinophilic and neutrophilic infiltration and tissue remodeling. Common clinical manifestations include nasal congestion, increased nasal discharge, loss of smell, facial pain, and recurrent sinus infections. According to the degree of eosinophilic infiltration in the tissue, CRSwNP can be divided into eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP) and non-eosinophilic chronic rhinosinusitis with nasal polyps (NECRSwNP). ECRSwNP is characterized by type 2 inflammation and eosinophilic infiltration, while NECRSwNP is characterized by type 1 / 3 inflammation and neutrophilic infiltration. Recent studies have found that some patients exhibit mixed inflammation, which is characterized by the co-infiltration of eosinophils and neutrophils. Such patients usually have poor prognosis factors such as severe disease, poor treatment response, and high risk of recurrence.

[0003] The current clinical treatment methods include local or systemic glucocorticoids, functional endoscopic sinus surgery, and biological agent therapy in recent years. The surgery can temporarily relieve symptoms, but it is a traumatic treatment with the risk of complications and cannot cure the inflammation, and has a high recurrence rate, especially in patients with complex inflammation. The glucocorticoid treatment is more suitable for type 2 CRSwNP than type 2 CRSwNP, but drug resistance is also observed in the glucocorticoid treatment of type 2 CRSwNP, which can be partially explained by the presence of neutrophil inflammation in CRSwNP. In fact, corticosteroid treatment reduces eosinophilic inflammation, while neutrophilic inflammation remains unchanged or even increases, so the glucocorticoid treatment has limited effect on mixed CRSwNP. The biological agent that has emerged in recent years mainly targets type II inflammation-related targets such as IL-5, IL-4Rα and IgE, and is mainly used to intervene in CRSwNP dominated by eosinophilic inflammation. In fact, in patients with mixed eosinophilic-neutrophilic CRSwNP, even after treatment with anti-IL-5 biological agents, neutrophils are still significantly enriched in the nasal polyp tissue after the reduction of eosinophilic inflammation, and play an important role in the persistence and recurrence of the disease.

[0004] In summary, the prior art still has the following shortcomings: first, the current CRSwNP treatment methods have a single target and cannot effectively cover the mixed inflammation involving eosinophils and neutrophils; second, there is a lack of new targets that take into account multiple inflammation mechanisms and tissue remodeling regulation, resulting in limited treatment effect and high recurrence rate. In this context, new targets that regulate broad-spectrum inflammatory responses have become the focus of CRSwNP treatment research. SUMMARY

[0005] To solve the technical problems in the prior art, the embodiments of the present application provide an ADAM8 gene and / or ADAM8 protein for use in the preparation of a drug for treating chronic rhinosinusitis with nasal polyps and a drug. The technical solution is as follows:

[0006] The ADAM8 gene and / or ADAM8 protein for use in the preparation of a drug for treating chronic rhinosinusitis with nasal polyps, wherein the ADAM8 gene and / or ADAM8 protein serve as a therapeutic target.

[0007] Alternatively, the ADAM8 gene is up-regulated in the nasal polyp tissue, which includes epithelium and inflammatory cells; the ADAM8 protein promotes the infiltration of inflammatory cells, thereby aggravating the condition of chronic rhinosinusitis with nasal polyps, and participates in the tissue remodeling process of chronic rhinosinusitis with nasal polyps by regulating collagen deposition and degradation.

[0008] Use of an agent inhibiting expression of ADAM8 gene or an agent inhibiting activity of ADAM8 protein in preparation of a medicament for treating chronic rhinosinusitis with nasal polyps.

[0009] Optionally, the agent inhibiting expression of ADAM8 gene or the agent inhibiting activity of ADAM8 protein comprises BK-1361.

[0010] Optionally, the agent inhibiting expression of ADAM8 gene or the agent inhibiting activity of ADAM8 protein alleviates inflammatory response and tissue remodeling of chronic rhinosinusitis with nasal polyps.

[0011] A pharmaceutical composition for treating chronic rhinosinusitis with nasal polyps, comprising an agent inhibiting expression of ADAM8 gene or an agent inhibiting activity of ADAM8 protein.

[0012] Optionally, the agent inhibiting expression of ADAM8 gene or the agent inhibiting activity of ADAM8 protein comprises BK-1361.

[0013] Optionally, the agent inhibiting expression of ADAM8 gene or the agent inhibiting activity of ADAM8 protein alleviates inflammatory response and tissue remodeling of chronic rhinosinusitis with nasal polyps.

[0014] The technical scheme provided by the embodiments of the present application has at least the following beneficial effects:

[0015] The present application develops an intervention strategy taking ADAM8 as the core, which is expected to make up for the limitations of existing treatment schemes and provide new treatment ideas and targets for CRSwNP patients, especially those with mixed inflammatory phenotypes.

[0016] 1. The present application researches and finds that the gene and protein expression levels of ADAM8 are significantly up-regulated in ECRwNP and NECRSwNP, and mainly distributed in the epithelium and the inflammatory cells below it, suggesting that ADAM8 may be a key pathogenic factor in the pathogenesis of CRSwNP.

[0017] 2. The present application researches and shows that the expression level of ADAM8 is closely related to the infiltration of inflammatory cells such as eosinophils and neutrophils, tissue remodeling and disease severity, indicating that it has a key regulatory function in the inflammatory response and pathological progression of CRSwNP, and has potential application value for disease prognosis evaluation.

[0018] 3. The present application can significantly reduce the nasal polypoid lesions, reduce the infiltration of eosinophils, neutrophils and other inflammatory cells, inhibit the key inflammatory signaling pathways, reduce the expression of metal matrix proteases, and thus effectively alleviate the inflammatory response and tissue remodeling process, verifying the scientificity and clinical application prospect of ADAM8 as a therapeutic target. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] FIG. 1A is a diagram of differentially expressed genes of the nasal mucosa tissue of a patient with chronic rhinosinusitis with nasal polyps compared with the normal nasal mucosa tissue provided by embodiment 1 of the present application; FIG. 1B is a diagram of the mRNA expression level of ADAM family members including ADAM8 in the nasal mucosa tissue provided by embodiment 1 of the present application; FIG. 1C is a diagram of the expression site and level of ADAM8 analyzed by immunofluorescence staining of the nasal mucosa tissue provided by embodiment 1 of the present application;

[0021] FIG. 2A is a diagram of the correlation analysis of the expression amount of ADAM8 and the infiltration of inflammatory cells in the tissue provided by embodiment 2 of the present application; FIG. 2B is a diagram of the correlation analysis of the expression amount of ADAM8 and the clinical indicators of CRSwNP provided by embodiment 2 of the present application; FIG. 2C is a diagram of the analysis of the collagen type and content by picrosirius staining of the nasal mucosa tissue and the correlation analysis of the expression of ADAM8 provided by embodiment 2 of the present application;

[0022] FIG. 3 is a diagram of the experimental process of the mouse CRSwNP model and the intervention of ADAM8 inhibitor provided by embodiment 3 of the present application;

[0023] FIG. 4A is a diagram of the evaluation of polypoid structure and eosinophil infiltration by HE staining of the paraffin section of the mouse nasal mucosa provided by embodiment 4 of the present application; FIG. 4B is a diagram of the evaluation of neutrophil and total inflammatory cell infiltration by immunohistochemical staining of the paraffin section of the mouse nasal mucosa provided by embodiment 4 of the present application; FIG. 4C is a diagram of the evaluation of ADAM8 expression by immunohistochemical staining of the paraffin section of the mouse nasal mucosa provided by embodiment 4 of the present application;

[0024] FIG. 5Ais a venn diagram showing the differential gene results of different groups of mice provided in Embodiment 5 of the present application; FIG. 5B is a diagram of GO-BP and KEGG enrichment results of differential genes between different groups provided in Embodiment 5 of the present application. DETAILED DESCRIPTION

[0025] The technical solutions in the present application will be described below with reference to the accompanying drawings.

[0026] A Disintegrin and Metalloproteinase 8 (ADAM8) is a transmembrane protein belonging to the ADAM family, which has proteolytic activity and integrin regulation function. ADAM8 is widely expressed in immune cells such as eosinophils, neutrophils, macrophages, and airway epithelial cells. Although the role of ADAM8 in other airway diseases has been studied to some extent, there is no literature that clearly indicates the functional role of ADAM8 in CRSwNP, and there is no experimental research or clinical application report on improving CRSwNP disease by inhibiting the expression or activity of ADAM8.

[0027] BK-1361 is composed of six amino acids: arginine (R), leucine (L), serine (S), lysine (K), aspartic acid (D, D-form), and a second lysine (K). Its molecular structure forms a stable lactam ring structure through head-to-tail cyclization, improving its anti-enzymatic ability and conformational stability. For example, BK-1361 can be purchased from ProbeChem Company.

[0028] English name: BK-1361 (cyclo(RLsKDK)); molecular formula: C 31 H 57 N 11 O9;

[0029] CAS No.: 1975145-82-4; molecular weight: 727.865;

[0030] The chemical structural formula of BK-1361 is as follows:

[0031] .

[0032] In view of the problems of complex inflammatory subtypes, repeated disease conditions and limitations of existing treatment methods in CRSwNP, the present application aims to clarify the functional role of ADAM8 in CRSwNP, such as the mechanism of action in the process of inflammatory response, especially inflammatory cell infiltration and tissue remodeling, to evaluate the feasibility of ADAM8 as a potential therapeutic target, and to provide new theoretical basis and intervention strategy for precise treatment of CRSwNP.

[0033] To make the technical problems, technical solutions and advantages to be solved by the present application clearer, the following will be described in detail in conjunction with the drawings and specific embodiments.

[0034] Example 1: RNA sequencing and immunohistochemical detection of ADAM8 expression in CRSwNP

[0035] This example aims to analyze the expression characteristics of ADAM8 in different subtypes of chronic rhinosinusitis with nasal polyps (CRSwNP) by RNA sequencing and immunohistochemical methods.

[0036] Specific steps:

[0037] 1. The research subjects were patients who underwent endoscopic surgery in the Department of Rhinology of Beijing Tongren Hospital, and were divided into three groups: control group, ECRSwNP and NECRSwNP, wherein,

[0038] Inclusion criteria: CRSwNP patients were diagnosed according to the 2020 European Rhinosinusitis and Nasal Polyps Position Paper. ECRSwNP and NECRSwNP were defined according to the percentage of eosinophil infiltration in the nasal mucosa tissue in previous studies, i.e. randomly taking 5 high-power fields (400x), taking the average value, if the percentage of eosinophils in each high-power field accounted for more than 27% of the total number of inflammatory cells, it was defined as ECRSwNP, otherwise it was NECRSwNP.

[0039] Exclusion criteria: those with the following diseases: cystic fibrosis, primary immunodeficiency, aspirin-exacerbated respiratory disease, autoimmune disease and fungal sinusitis. Those who used steroids, immunomodulators and antibiotics within 4 weeks before surgery.

[0040] Matching conditions for the control group: middle turbinate and uncinate process tissue of patients with deviated nasal septum with obstructive sleep apnea (OSA) or bullous turbinate. Patients had no history of other sinus diseases and did not use steroids, immunomodulators and antibiotics within 4 weeks before surgery.

[0041] A total of 38 CRSwNP patients were finally included, including 20 ECRSwNP patients and 18 NECRSwNP patients; and 15 control group patients.

[0042] 2. Collect the nasal mucosa tissue of the control group, ECRSwNP and NECRSwNP.

[0043] The collection method is as follows: the nasal mucosa tissue of the ECRSwNP and NECRSwNP groups was collected from the nasal polyp tissue; the control group tissue was collected from the middle turbinate or uncinate process, and the sampling time was during the endoscopic surgery. All tissues were immediately frozen in liquid nitrogen after sampling and stored in a -80°C refrigerator until subsequent processing.

[0044] 3. After total RNA extraction, high-throughput RNA sequencing was performed.

[0045] The total RNA was extracted using an animal tissue / cell RNA extraction kit (Kangwei Century, China). High-throughput RNA sequencing was performed by Novogene (China).

[0046] Differentially expressed genes were analyzed by edgeR software, with the screening criteria of |log2FC|≥1 and P<0.05. The expression was standardized by FPKM (the number of fragments per kilobase of transcript per million mapped fragments).

[0047] 4. The expression site and level of ADAM8 were analyzed by immunofluorescence staining of nasal mucosa tissue, and the specific steps were as follows:

[0048] From the sequenced samples, specimens with paraffin-embedded tissues were selected for histological analysis. The preparation method of the nasal tissue paraffin section was as follows: after the nasal tissue sample was collected, it was immediately placed in 10% neutral formalin and fixed at room temperature for 16-32 hours (appropriately adjusted according to the size and type of the tissue), then the tissue was washed with 1xPBS. After dehydration by gradient ethanol (70% to 100%), the sample was transparently treated with xylene, and then embedded in paraffin according to the conventional histological process. After embedding, the tissue can be stored at room temperature under dry conditions. When slicing, the paraffin-embedded block was cut into 5±1 μm thick slices using a microtome, and the slices were spread on a water bath at 40-45°C, then attached to a SUPERFROST® PLUS glass slide and air-dried at room temperature overnight, and then baked at 60°C for 1 hour. Then, the glass slide was sequentially incubated in two fresh xylene for 5 minutes and two 100% ethanol for 2 minutes (during which the glass slide rack was moved up and down appropriately), and finally placed on absorbent paper and dried at 60°C for 5 minutes or until completely dry.

[0049] When performing ADAM8 immunofluorescence staining, the nasal tissue paraffin section was incubated with rabbit anti-ADAM8 antibody (Abcam, 1:100) overnight at 4°C in the dark, then stained with Alexa Fluor® 594-labeled goat anti-rabbit IgG (Zhongshanjinqiao, China) and counterstained with DAPI, and imaged using a confocal laser microscope (Olympus, Japan).

[0050] ImageJ (v2.14.0) was used for image quantitative analysis, and 4 fields of view were randomly selected under 400x magnification, the mean fluorescence intensity (MFI) was calculated and the average value was obtained.

[0051] Experimental results:

[0052] The experimental results are shown in FIGS. 1A-1C .

[0053] FIG. 1A Figure 2 is a plot of differentially expressed genes in nasal mucosa tissue of patients with chronic rhinosinusitis with nasal polyps compared to normal nasal mucosa tissue. From FIG. 1A As can be seen from Figure 2, there are 3114 significantly up-regulated genes, indicating that there are active transcriptional regulation and inflammatory response processes in the nasal polyp tissue. Among these significantly up-regulated genes, the reason why the inventors chose ADAM8 for further study is that ADAM8 is a transmembrane metalloproteinase, which has been confirmed to be involved in the regulation of immune cell infiltration, cell adhesion and matrix degradation in a variety of related diseases involving inflammation and tissue remodeling, such as asthma, chronic obstructive pulmonary disease (COPD) and pancreatic cancer. Previous studies have detected elevated levels of ADAM8 in the nasal secretions of patients with chronic rhinosinusitis with nasal polyps (CRSwNP), and it has been shown to be helpful for disease typing prediction and postoperative disease control evaluation, but the exact expression pattern, regulatory mechanism and functional role of ADAM8 in the pathogenesis of CRSwNP are still unclear.

[0054] FIG. 1B Figure 3 is a plot of mRNA expression levels of ADAM family members, including ADAM8, in nasal mucosa tissue. From FIG. 1B As can be seen from Figure 3, compared with the control group, the expression of multiple ADAM family members (including ADAM8, ADAM12, ADAM19 and ADAM33) in CRSwNP tissue was significantly up-regulated, among which the up-regulation of ADAM8 was the most significant (log2FC = 1.74, P < 0.0001). Further subtype analysis showed that the ADAM8 gene was most significantly up-regulated in ECRSwNP patients (log2FC = 2.08, P < 0.0001) and was also significantly up-regulated in NECRSwNP patients (log2FC = 1.22, P < 0.01).

[0055] FIG. 1C Figure 4 is a plot of immunofluorescence staining of nasal mucosa tissue to analyze the expression site and level of ADAM8. From FIG. 1CAs can be seen, the nucleus is stained blue by DAPI, showing the location of the nucleus; ADAM8 protein is detected by red fluorescence labeling, which is located in the epithelial layer of the nasal mucosa and sub-epithelial inflammatory cells. The results of staining under high magnification field (x400) showed that compared with the control group, the expression of ADAM8 in ECRSwNP group and NECRSwNP group was significantly increased. Specifically, the average fluorescence intensity of ADAM8 positive area in the epithelial layer increased from 1.454±0.2552 in the control group to 3.746±0.5032 in the ECRSwNP group and 3.118±0.3608 in the NECRSwNP group. The number of sub-epithelial inflammatory cells increased significantly from 5.972±1.057 in the control group to 52.50±6.136 in the ECRSwNP group and 47.14±7.873 in the NECRSwNP group.

[0056] FIGS. 1A-1C The above results suggest that in each subtype of CRSwNP, the ADAM8 gene and protein levels are significantly increased, which may be involved in the pathogenesis of CRSwNP.

[0057] Example 2: Analysis of the relationship between ADAM8 expression and the histopathological features and clinical severity of CRSwNP

[0058] To further explore the relationship between ADAM8 and the histopathological changes and clinical severity of CRSwNP, paraffin-embedded nasal tissue sections from Example 1 were used for histological analysis by H&E staining, immunohistochemistry, and Sirius red staining.

[0059] Hematoxylin and eosin (H&E) staining was used to identify eosinophils, plasma cells, lymphocytes, and other inflammatory cells. The H&E staining method is as follows:

[0060] The tissue sections were first baked in a 60°C oven for 3 minutes, then deparaffinated in xylene for 10 minutes each time. Hydrated with gradient ethanol (100%, 95%, 80%) for 2 minutes each step. The sections were immersed in hematoxylin staining solution for about 10 minutes, then rinsed with running water for 3 times. Differentiate with differentiation solution, gently move up and down on the hand-held rack for 2 times, then rinse with running water for 3 times. The sections were placed in ammonia water for 5 minutes to enhance nuclear staining, then rinsed with running water. Next, the sections were immersed in eosin staining solution for 5 minutes, then rinsed with running water for 3 times. Dehydrated with 80%, 95%, and absolute ethanol for 3-5 minutes each step. Finally, after treatment with transparent agent, the sections were mounted, and after the mounting agent dried, microscopic observation was performed.

[0061] Immunohistochemistry was used to detect MPO-positive neutrophils using rabbit anti-myeloperoxidase (MPO) antibody (Abcam, 1:1000). The immunohistochemical method is as follows:

[0062] After incubation in 60°C oven for 60 minutes, the sections were washed with PBS for 3 times (5 minutes each time) and then subjected to sodium citrate high pressure boiling antigen retrieval. After cooling to room temperature, the sections were washed with PBS again. Then the sections were incubated with 3% hydrogen peroxide for 10 minutes at room temperature, washed and blocked with goat serum for 20 minutes at room temperature. The diluted MPO primary antibody was added and incubated overnight at 4°C. The next day, the sections were washed with PBS and then added with enzyme-labeled goat anti-rabbit IgG polymer (Zhongshanjinqiao, China) and incubated for 1 hour at room temperature. The sections were washed with PBS again. Then DAB color developing solution was added and the staining was observed under a microscope and stopped with tap water. The nuclei were stained with hematoxylin for 3 minutes, washed with water, differentiated with hydrochloric alcohol for a few seconds, washed with water for 3 times, returned to blue with ammonia water and washed clean. Finally, the sections were dehydrated with gradient ethanol (80%, 95%, and anhydrous, each for 3 minutes), treated with transparency agent for 2 times (each for 3 minutes), mounted with neutral resin, and observed under a microscope after the mounting agent was dried.

[0063] The collagen deposition was evaluated by observing the Sirius red staining under bright field and polarized light. The method of Sirius red staining was as follows:

[0064] The staining was performed using a modified Sirius red staining kit (Solebao, China). The specific steps were as follows: the tissue sections were routinely deparaffinized to water. The iron hematoxylin staining solution was prepared before use, added dropwise and stained for 5 minutes, and then washed with distilled water for 10-20 seconds to remove the excess staining solution. The sections were washed with tap water for 5 minutes to return to blue, and then washed with distilled water for 3 times, each for 5-10 seconds. The sections were added dropwise with Sirius red staining solution for 10 minutes, and then quickly washed with distilled water to remove the excess staining solution. The sections were quickly dehydrated with series of ethanol starting from 75%, transparented with xylene, and mounted with neutral resin. The sections were observed under a microscope after the mounting agent was dried.

[0065] In the statistical analysis of H&E and immunohistochemistry, the number of cells or the optical density value (MOD) was analyzed by ImageJ software under 400 times magnification by randomly selecting 5 fields of view; the total collagen content was calculated by selecting 10 200 times magnified fields of view in each sample, and the average value of the proportion of positive staining area was taken. All histological staining was evaluated by two independent researchers who were blind to the grouping information to ensure the objectivity of the analysis results.

[0066] Experimental results:

[0067] The experimental results are shown in FIGS. 2A-2C .

[0068] FIG. 2A is a graph of the correlation analysis of the expression level of ADAM8 and the infiltration of inflammatory cells in the tissue. As can be seen from FIG. 2A , the expression level of ADAM8 is significantly positively correlated with the counts of eosinophils, neutrophils, plasma cells and total inflammatory cells.

[0069] The above results suggest that the increased expression of ADAM8 gene may promote the local infiltration of inflammatory cells, especially eosinophils and neutrophils, and thus participate in the regulation of tissue inflammatory response.

[0070] FIG. 2B is a graph of the correlation analysis of ADAM8 expression level and clinical indicators of CRSwNP. From FIG. 2B It can be seen from that the expression level of ADAM8 is positively correlated with the Lund-Mackay CT score and the nasal polyp score.

[0071] The above results show that the expression level of ADAM8 is related to the severity of CRSwNP, suggesting that it may be involved in the progression of the disease.

[0072] FIG. 2C is a graph of the analysis of collagen type and content in nasal mucosa tissue by Sirius red staining, and the correlation analysis of ADAM8 expression. From FIG. 2C It can be seen from that compared with the control group, the collagen content in ECRSwNP and NECRSwNP tissues is significantly reduced, and the thick orange type I collagen is almost undetectable. At the same time, this collagen reduction trend is significantly correlated with the increased expression of ADAM8.

[0073] The above results suggest that ADAM8 may participate in the tissue remodeling process of CRSwNP by regulating collagen deposition and degradation.

[0074] Example 3: Establishment of CRSwNP mouse model and ADAM8 targeting inhibition treatment model

[0075] 6-8 week old female BALB / c mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into a control group, a CRSwNP model group and an ADAM8 inhibitor treatment group, with 8 mice in each group.

[0076] The mice in the CRSwNP model group and the treatment group were established as a model by intranasal administration of a mixture of Aspergillus oryzae protease (AP, purchased from Sigma-Aldrich, USA) and Ovalbumin (OVA, purchased from Sigma-Aldrich, USA), with a dose of 2U AP + 75μg OVA dissolved in 20μL sterile PBS, 3 times a week for 12 weeks. The mice in the control group were given 20μL PBS by intranasal administration at the same time.

[0077] The mice in the treatment group were intraperitoneally injected with ADAM8 specific inhibitor BK-1361 (10μg / g (representing 10μg BK-1361 drug per 1g mouse body weight), purchased from Probechem Biochemical Reagent Co., Ltd., USA) from the 5th week, 3 times a week for 8 weeks, and the specific process is shown inFIG. 3 .

[0078] Example 4: Intervention effect of ADAM8 inhibitor on inflammatory cell infiltration in CRSwNP mice

[0079] In this experiment, the mice in the control group, CRSwNP model group and ADAM8 inhibitor treatment group of Example 3 were evaluated for inflammatory cell infiltration.

[0080] The preparation method of the mouse nasal mucosa paraffin section is as follows: after heart perfusion, the skin, soft tissue and mandible of the mouse head are cut off, the skull is placed in 4% paraformaldehyde for fixation for more than 24 hours, and decalcified in 5% nitric acid for 3 days. Take out the tissue, trim the target area with a scalpel and mark it, and put it into a dehydration box. During dehydration, the tissue is dehydrated in gradient alcohol as follows: 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, anhydrous ethanol for 130 minutes, anhydrous ethanol II for 30 minutes, then sequentially with alcohol benzene for 5-10 minutes, dimethylbenzene I for 5-10 minutes, dimethylbenzene II for 5-10 minutes. Then, the paraffin is immersed for three times, each for 1 hour. When embedding, the tissue is placed in the embedding frame, labeled, cooled into a block at -20°C and trimmed. The section thickness is 4um, the section is flattened in 40°C warm water, and then taken out to the glass slide, dried at 60°C and stored at room temperature.

[0081] 1. H&E staining was used to evaluate the polypoid lesions and eosinophilic infiltration in the nasal tissue. The method is as follows: the tissue section is first baked in a 60°C oven for 3 minutes, then deparaffinized in xylene for 10 minutes each time. Hydrated with gradient ethanol (100%, 95%, 80%) for 2 minutes each step. The section is immersed in hematoxylin staining solution for about 10 minutes, then washed with running water for 3 times. Differentiate with differentiation solution, gently move up and down on the hand-held rack for 2 times, then wash with running water for 3 times. The section is placed in ammonia water for 5 minutes to enhance nuclear staining, then washed with running water. Then, the section is immersed in eosin staining solution for 5 minutes, washed with running water for 3 times. Dehydrate with 80%, 95% and anhydrous ethanol for 3-5 minutes each step. Finally, after clearing agent treatment, mount the section, and after the mounting agent is dry, observe under a microscope.

[0082] 2. The number of total inflammatory cell infiltrates was detected by immunohistochemical staining of CD45 (Abeam, 1:1000). The method was as follows: after the slice was placed in a 60°C oven for 60 minutes, it was washed with PBS for 3 times (5 minutes each time), and then subjected to sodium citrate high-pressure boiling antigen repair. After cooling to room temperature, it was washed with PBS again. Then it was incubated with 3% hydrogen peroxide at room temperature for 10 minutes, and after washing, it was blocked with goat serum at room temperature for 20 minutes. After adding the diluted CD45 primary antibody, it was incubated at 4°C overnight. The next day, after washing with PBS, enzyme-labeled goat anti-rabbit IgG polymer (Zhongshanjingqiao, China) was added and incubated at room temperature for 1 hour, and then washed with PBS again. Then DAB developing solution was added, and the staining was observed under a microscope and terminated with tap water. The cell nuclei were stained with hematoxylin for 3 minutes, washed with water, differentiated with hydrochloric acid alcohol for a few seconds, washed with water for 3 times, returned to blue with ammonia water and washed clean. Finally, dehydrated with gradient ethanol (80%, 95%, anhydrous, 3 minutes each), treated with transparency agent twice (3 minutes each), mounted with neutral resin, and after the mounting agent was dried, observed under a microscope.

[0083] 3. The number of neutrophil infiltrates was evaluated by immunohistochemical staining of MPO (Abeam, 1:1000). The method was as follows: after the slice was placed in a 60°C oven for 60 minutes, it was washed with PBS for 3 times (5 minutes each time), and then subjected to sodium citrate high-pressure boiling antigen repair. After cooling to room temperature, it was washed with PBS again. Then it was incubated with 3% hydrogen peroxide at room temperature for 10 minutes, and after washing, it was blocked with goat serum at room temperature for 20 minutes. After adding the diluted MPO primary antibody, it was incubated at 4°C overnight. The next day, after washing with PBS, enzyme-labeled goat anti-rabbit IgG polymer (Zhongshanjingqiao, China) was added and incubated at room temperature for 1 hour, and then washed with PBS again. Then DAB developing solution was added, and the staining was observed under a microscope and terminated with tap water. The cell nuclei were stained with hematoxylin for 3 minutes, washed with water, differentiated with hydrochloric acid alcohol for a few seconds, washed with water for 3 times, returned to blue with ammonia water and washed clean. Finally, dehydrated with gradient ethanol (80%, 95%, anhydrous, 3 minutes each), treated with transparency agent twice (3 minutes each), mounted with neutral resin, and after the mounting agent was dried, observed under a microscope.

[0084] 4. ADAM8 expression was evaluated by immunohistochemical staining with ADAM8 (abcam, 1:1000). The method was as follows: after the section was baked in a 60°C oven for 60 minutes, it was washed with PBS for 3 times (5 minutes each time), and then subjected to sodium citrate high-pressure boiling antigen repair. After cooling to room temperature, it was washed with PBS again. Then it was incubated with 3% hydrogen peroxide at room temperature for 10 minutes, washed, and then blocked with goat serum at room temperature for 20 minutes. After adding the diluted ADAM8 primary antibody, it was incubated overnight at 4°C. The next day, after washing with PBS, enzyme-labeled goat anti-rabbit IgG polymer (Zhongshanjingqiao, China) was added and incubated at room temperature for 1 hour, and then washed with PBS again. Then DAB developing solution was added, and the staining was observed under a microscope and the color development was terminated with tap water. The cell nuclei were stained with hematoxylin for 3 minutes, washed with water, differentiated with hydrochloric acid alcohol for a few seconds, washed with water for 3 times, returned to blue with ammonia water and washed clean. Finally, dehydrated with gradient ethanol (80%, 95%, and anhydrous, each for 3 minutes), treated with transparency agent for 2 times (each for 3 minutes), mounted with neutral resin, and after the mounting agent was dried, observed under a microscope.

[0085] The cell number, subepithelial thickness, and optical density value (MOD) in the mouse group analysis were randomly selected under a high-power lens (400 times magnification) in 4 respiratory area fields, and analyzed using ImageJ software. All histological staining was blindly evaluated by two independent researchers who were unaware of the grouping information to ensure the objectivity of the analysis results.

[0086] Experimental results:

[0087] The experimental results are shown in FIGS. 4A-4C .

[0088] FIG. 4A Figure is a H&E staining of mouse nasal mucosa paraffin sections to evaluate the polyp-like structure and eosinophil infiltration. As can be seen from FIG. 4A , under a high-power lens, no polyp-like structure was observed in the control group, and the eosinophil count was 2.267±0.8327; while in the CRSwNP model group, obvious polyp-like lesions were observed, the number of polyps was 8.267±0.4522, and the eosinophil count increased significantly to 184.8±12.87. After treatment with the ADAM8 inhibitor, the number of polyp-like lesions decreased significantly to 5.600±0.2211, and the eosinophil count also decreased significantly to 85.47±10.56. The above results show that compared with the control group, the polyp-like lesions and eosinophil infiltration in the mouse nasal tissue of the CRSwNP model group increased significantly, proving that the CRSwNP model was successfully constructed. After treatment with the ADAM8 inhibitor, the polyp-like lesions and eosinophil infiltration decreased significantly, which supports the clinical value of ADAM8 as a potential therapeutic target.

[0089] FIG. 4BFigure 2 is a graph of the evaluation of neutrophil and total inflammatory cell infiltration in the mouse nasal mucosa paraffin section immunohistochemical staining. From the FIG. 4B As can be seen from the above, the number of neutrophil infiltrating cells under high power field, the control group was 17.87±1.611; the CRSwNP model group was 286.5±23.12; the ADAM8 inhibitor treatment group was 171.2±20.76. The number of CD45+ inflammatory cells, the control group was 50.93±4.568; the CRSwNP model group was 562.3±38.14; the ADAM8 inhibitor treatment group was 331.5±29.67. The above results of MPO and CD45 immunostaining showed that the number of neutrophils and total inflammatory cells in the CRSwNP model group was significantly increased, while the above cell infiltration was significantly reduced after ADAM8 inhibition, suggesting that inhibition of ADAM8 can effectively reduce the inflammatory response by inhibiting the infiltration of neutrophils and other inflammatory cells.

[0090] FIG. 4C Figure 3 is a graph of the evaluation of ADAM8 expression in the mouse nasal mucosa paraffin section immunohistochemical staining. From the FIG. 4C As can be seen from the above, when observing the immunohistochemical staining results of the mouse nasal epithelial tissue under high power microscope, the control group showed no obvious ADAM8 positive expression, the average optical density value of the epithelium was 0.003020±0.0003642, and the number of ADAM8 positive inflammatory cells under the epithelium was 12.40±3.682; while the CRSwNP model group showed significant ADAM8 positive expression, the average optical density value of the epithelium was 0.02086±0.001053, and the number of ADAM8 positive inflammatory cells under the epithelium was 148.8±21.72. After ADAM8 inhibitor treatment, the ADAM8 positive expression was significantly reduced, the average optical density value of the epithelium was reduced to 0.01142±0.002077, and the number of ADAM8 positive inflammatory cells under the epithelium was reduced to 58.40±4.632. The above results of ADAM8 immunohistochemistry showed that the expression of ADAM8 in the nasal epithelium of the control group mice was very low, and only a small amount of positive inflammatory cells existed under the epithelium. In the CRSwNP model group, ADAM8 was significantly up-regulated in the epithelial layer, accompanied by a large number of inflammatory cell infiltration. After treatment, the expression of ADAM8 in the epithelial layer and the inflammatory cells under the epithelium was significantly decreased. These results showed that ADAM8 was significantly increased in the CRSwNP model, participated in the pathogenesis of the disease, and the application of ADAM8 inhibitor not only inhibited the activity of ADAM8 but also effectively reduced the expression of ADAM8, thereby inhibiting the occurrence and development of CRSwNP.

[0091] In summary, the above results showed that ADAM8 played a key role in the pathogenesis of CRSwNP, and its targeted inhibition could significantly reduce the inflammatory response, which had potential therapeutic value.

[0092] Example 5: Transcriptome level validation of the regulatory effect of ADAM8 inhibition on mouse CRSwNP inflammation and tissue remodeling

[0093] In this example, the nasal mucosa tissue of the mice in Example 3 was collected, and the collection method was as follows: after heart perfusion, the skin, soft tissue and mandible of the mouse head were removed, the skull was cut along the median sagittal plane, the mouse nasal mucosa was scraped with forceps and placed in a cryogenic tube, then frozen in liquid nitrogen and stored in a -80°C refrigerator until subsequent processing.

[0094] After extracting total RNA, high-throughput RNA sequencing was performed. Among them, the total RNA was extracted by MJzol animal RNA isolation kit (Shanghai). High-throughput RNA sequencing was performed by Novogene (China) Co., Ltd. The differentially expressed genes were analyzed using edgeR, and the screening criteria were |log2FC|≥1 and P<0.05. Further GO and KEGG functional enrichment analysis was performed using clusterProfiler R package.

[0095] Experimental results:

[0096] The experimental results are shown in FIGS. 5A-5B .

[0097] FIG. 5A is a venn diagram showing the differentially expressed genes of mice in different groups. As can be seen from FIG. 5A , there are 3,679 differentially expressed genes in the CRSwNP model group compared with the control group, of which 1,519 are up-regulated; there are 1,150 differentially expressed genes in the ADAM8 inhibitor treatment group compared with the model group, of which 740 are down-regulated. There are 756 common genes between the two groups, of which 514 show a "treatment reversal effect", i.e. genes that are up-regulated in the model group and down-regulated in the treatment group.

[0098] FIG. 5B is a graph of GO-BP and KEGG enrichment results of differentially expressed genes between different groups. As can be seen from FIG. 5B , GO functional enrichment analysis shows that up-regulated genes in the CRSwNP model group are mainly enriched in GO-BP pathways such as "inflammatory response", "cytokine-mediated signal pathway", "positive regulation of cell migration", etc. The corresponding KEGG pathways include "cytokine-receptor interaction", "NF-κB signaling pathway" and "Th17 cell differentiation", suggesting that the CRSwNP model group has a highly pro-inflammatory microenvironment.

[0099] The down-regulated genes of the ADAM8 inhibitor treatment group were significantly enriched in the pathways of "inflammatory response", "extracellular matrix structure organization", "cell adhesion" and "migration", suggesting that ADAM8 plays an important regulatory role in the process of inflammatory cell infiltration and tissue remodeling. In addition, the NF-κB, TNF and Th17 signaling pathways were significantly inhibited, further verifying that ADAM8 inhibition can alleviate the inflammatory activation state of CRSwNP.

[0100] Further analysis of the 514 key genes down-regulated by the treatment group found that these genes were mainly enriched in the functional pathways of inflammatory response, cell migration positive regulation and extracellular matrix remodeling. In the extracellular matrix remodeling pathway, multiple metal matrix proteases (such as MMP3, MMP10, MMP12, MMP19) were significantly decreased in the treatment group, suggesting that ADAM8 may participate in ECM (extracellular matrix) remodeling by regulating MMPs. In addition, key inflammation-related genes such as NLRP10, ECM1, ITGB6, EDN1, ADAM9 and MMP3 were significantly down-regulated after ADAM8 inhibition, indicating that ADAM8 may regulate immune response, cell adhesion and chemotaxis through these targets, ultimately affecting the inflammatory response and tissue remodeling of CRSwNP.

[0101] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Use of ADAM8 gene and / or ADAM8 protein in the preparation of a drug for treating chronic rhinosinusitis with nasal polyps, characterized in that, The ADAM8 gene and / or ADAM8 protein as a therapeutic target.

2. Use according to claim 1, characterized in that, The ADAM8 gene expression is up-regulated in the nasal polyp tissues, including epithelium and inflammatory cells; the ADAM8 protein promotes the infiltration of inflammatory cells, thereby aggravating the condition of chronic rhinosinusitis with nasal polyps, and participates in the tissue remodeling process of chronic rhinosinusitis with nasal polyps by regulating collagen deposition and degradation.

3. Use of an agent inhibiting the expression of ADAM8 gene or an agent inhibiting the activity of ADAM8 protein in the preparation of a drug for treating chronic rhinosinusitis with nasal polyps.

4. Use according to claim 3, characterized in that, The agent inhibiting the expression of ADAM8 gene or the agent inhibiting the activity of ADAM8 protein includes BK-1361.

5. Use according to claim 3, characterized in that, The agent inhibiting the expression of ADAM8 gene or the agent inhibiting the activity of ADAM8 protein alleviates the inflammatory response and tissue remodeling of chronic rhinosinusitis with nasal polyps.

6. A pharmaceutical composition for treating chronic rhinosinusitis with nasal polyps, characterized by, The pharmaceutical composition includes the agent inhibiting the expression of ADAM8 gene or the agent inhibiting the activity of ADAM8 protein.

7. The pharmaceutical composition of claim 6, wherein, The agent inhibiting the expression of ADAM8 gene or the agent inhibiting the activity of ADAM8 protein includes BK-1361.

8. The pharmaceutical composition of claim 6, wherein, The agent inhibiting the expression of ADAM8 gene or the agent inhibiting the activity of ADAM8 protein alleviates the inflammatory response and tissue remodeling of chronic rhinosinusitis with nasal polyps.

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