Application of NTA10 inhibitor Remodelin in preparation of medicine for treating allergic asthma
By inhibiting the ac4C modification activity of NAT10 with Remodelin and blocking the M2 polarization pathway of macrophages, a liquid formulation was prepared for intraperitoneal injection, which solved the problem of insufficient targeting in the treatment of allergic asthma and achieved precise and efficient treatment of allergic asthma.
Patent Information
- Application Number
- CN202610186191.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-09
- Publication Date
- 2026-04-10
AI Technical Summary
Current treatments for allergic asthma lack targeted therapy, have limited efficacy for severe cases, and lack targeted therapies against the NAT10-macrophage M2 polarization pathway.
Remodelin was used as a NAT10 inhibitor. By binding to the catalytic domain of NAT10, it targeted and inhibited its ac4C modification activity, blocking the key signaling pathways of macrophage polarization induced by IL-4 and IL-13 signals. The product was prepared into a liquid dosage form for intraperitoneal injection.
It significantly inhibits M2 macrophage polarization, reduces airway smooth muscle cell proliferation and collagen fiber deposition, alleviates irreversible remodeling such as airway wall thickening and luminal narrowing, and improves the pathological state of allergic asthma.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of medicine, and particularly to the application of NTA10 inhibitor Remodelin in the preparation of a drug for treating allergic asthma. BACKGROUND
[0002] Allergic asthma is a chronic airway inflammatory disease with high incidence worldwide. The main clinical manifestations are recurrent airway spasm, excessive mucus secretion, airway hyperresponsiveness and reversible airflow limitation. According to statistics, the number of asthma patients worldwide has exceeded 300 million, and the prevalence of asthma in China is also showing a sustained upward trend. Especially in the context of urbanization and air pollution, the situation of asthma prevention and treatment is severe. Although existing treatment methods (such as inhaled glucocorticoids, long-acting β2 receptor agonists, leukotriene receptor antagonists, etc.) can control the symptoms of most patients, about 5% of patients still belong to severe asthma and have poor response to conventional treatment, which reveals that there is a significant clinical treatment gap in existing therapies in the face of severe asthma, and new action targets and treatment strategies are urgently needed.
[0003] In the pathogenesis of allergic asthma, the dysfunction of immune cells occupies a central position. Among them, macrophages, as the core effector cells of the airway mucosa immune system, the imbalance of their polarization state is a key pathogenic link. Macrophages are mainly divided into classical activated type (M1 type) and alternative activated type (M2 type), and the dynamic balance of the two directly regulates the occurrence and outcome of airway inflammation, and the enrichment of M2 macrophages is one of the core pathological features of allergic asthma, which is manifested as the up-regulation of markers such as mannose receptor (CD206), arginase 1 (Arg1), chitinase-like protein 3 (Ym1), etc. In the pathological microenvironment of allergic asthma, signals such as allergens (such as dust mites, pollen, pet dander) stimulation, alarmin (such as IL-33, TSLP) released by airway epithelial cell damage collectively drive macrophages to polarize to M2 type, leading to a large number of M2 macrophages enriched in the airway mucosa and lung tissue, and exacerbating the pathological process of asthma through multiple mechanisms: on the one hand, the Th2 type cytokines such as IL-4, IL-5, IL-13 secreted by M2 macrophages can directly induce the recruitment and activation of eosinophils, promote the release of toxic mediators such as eosinophil cationic protein and major basic protein by eosinophils, and damage the airway epithelial barrier; on the other hand, IL-4 and IL-13 can stimulate the proliferation of airway epithelial cells and goblet cells, leading to excessive mucus secretion and forming mucus plugs to block the airway; at the same time, the cytokines such as transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) secreted by M2 macrophages also accelerate the proliferation of airway smooth muscle cells and collagen deposition, causing irreversible airway remodeling such as airway wall thickening and lumen stenosis, and ultimately leading to airway hyperresponsiveness and permanent damage to lung function, so targeting the regulation of macrophage M2 polarization has become a key direction for the development of new asthma treatment drugs.
[0004] NAT10 (N-acetyltransferase 10) is the only known RNA N 4 The ac4C modification "writer" has been shown to play a key regulatory role in mRNA by introducing ac4C modifications in conserved sequences of target transcripts, regulating mRNA stability, nuclear export, and translation efficiency, and thus participating in various biological processes such as cell proliferation, differentiation, and inflammatory response. In recent years, the regulatory role of NAT10 in inflammatory diseases has gradually attracted attention. It is highly expressed in models of sepsis, rheumatoid arthritis, and inflammatory bowel disease, and its expression level is positively correlated with the severity of inflammation. Mechanism studies have confirmed that it can regulate the expression of inflammatory-related genes (such as TNF-α and IL-6) in macrophages through ac4C modification, and directly participate in the functional polarization process of macrophages. In the field of respiratory diseases, studies have shown that NAT10 is involved in airway epithelial cell injury repair and smooth muscle cell proliferation. Studies in the field of tumors have also confirmed that tumor cells can express or secrete NAT10 through their own expression or secretion, regulate macrophage M2 polarization in the tumor microenvironment of gastric cancer, intrahepatic cholangiocarcinoma, and esophageal squamous cell carcinoma through chemotactic factors and ac4C modification, and thus participate in tumor metastasis and proliferation. However, the specific regulatory mechanism of NAT10 in M2 polarization in macrophages and whether targeting NAT10 can improve the pathological phenotype of asthma are still unclear, and relevant research is still in a blank state. There is also a lack of asthma treatment drugs targeting the NAT10-macrophage M2 polarization pathway.
[0005] Remodelin is a small molecule inhibitor targeting NAT10 that can specifically inhibit its acetyltransferase activity by binding to the catalytic domain of NAT10, with good target action characteristics and biological safety. It has been confirmed that it can effectively inhibit the acetyltransferase activity of NAT10 in models of premature aging syndrome, various malignant tumors, and myocardial ischemia-reperfusion injury, and exhibit good anti-inflammatory, anti-fibrotic, and immunomodulatory effects. However, the potential of this inhibitor in the treatment of allergic asthma has not been explored, and there is currently no research to systematically evaluate whether Remodelin can improve airway inflammation, immune imbalance, and airway structural remodeling in asthma by inhibiting the NAT10-M2 macrophage polarization pathway. Therefore, the application of Remodelin in the treatment of asthma not only fills the research gap in targeting RNA epigenetic modification to regulate immune disorders in asthma, but also provides a new potential drug candidate for precise intervention in severe asthma, with important scientific significance and clinical translation value. SUMMARY
[0006] This invention aims to address the technical shortcomings of existing allergic asthma treatments, such as insufficient targeting and limited efficacy in severely ill patients. It provides a novel application for Remodelin, which systematically improves the pathological process of asthma by inhibiting the "Remodelin–NAT10–macrophage M2 polarization" regulatory axis. This fills the gap in targeted RNA epigenetic modification therapy for asthma and provides a precise and efficient treatment option for allergic asthma.
[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: the application of NAT10 inhibitor Remodelin in the preparation of drugs for treating allergic asthma.
[0008] In a preferred embodiment, the drug uses NAT10 as a molecular target for treating allergic asthma.
[0009] This application is based on the key driving role of NAT10 in macrophage M2 polarization. Using NAT10 as a molecular target, the drug Remodelin, which inhibits the function of NAT10, binds to the catalytic domain of NAT10 and targets and inhibits its ac4C modification activity. This effectively blocks the key signaling pathways of macrophage polarization induced by IL-4 and IL-13 signaling, thereby treating allergic asthma.
[0010] In vitro and in vivo experiments have confirmed that Remodelin can improve the pathological state of allergic asthma at multiple levels—it can significantly inhibit M2 macrophage polarization, while inhibiting the excessive production of serum total IgE to reduce the intensity of allergic reactions, reduce the proliferation of airway smooth muscle cells and collagen fiber deposition, and alleviate irreversible remodeling such as airway wall thickening and lumen narrowing.
[0011] This invention utilizes small interfering RNA (SRNA) technology to specifically knock down NAT10 in macrophages and CRISPR / Cas9 technology to specifically knock out NAT10, significantly inhibiting IL-4 / IL-13-induced macrophage M2 polarization. Related M2 markers were suppressed at both the mRNA and protein levels. Furthermore, downregulation of NAT10 expression inhibited phosphorylation of STAT6, a key signal transduction pathway in M2 polarization, revealing that the STAT6 signaling pathway may be involved in NAT10-regulated macrophage M2 polarization.
[0012] In the preferred embodiment, the drug has Remodelin as its sole active ingredient.
[0013] In the preferred embodiment, the drug is in the form of an injectable form.
[0014] In a preferred embodiment, the drug further comprises pharmaceutically acceptable excipients.
[0015] In a further preferred embodiment, the excipients include a diluent.
[0016] In a further preferred embodiment, the diluent is a mixture of DMSO, PEG300, Tween 80, and water in a volume ratio of 5:40:5:50. This formulation ensures complete drug dissolution and is suitable for safe in vivo administration.
[0017] In the preferred embodiment, the drug is administered via intraperitoneal injection.
[0018] The drug uses Remodelin as its core active ingredient and is formulated into a liquid dosage form with pharmaceutically acceptable excipients. Specifically, a mixed solution of 5% DMSO, 40% PEG300, 5% Tween80, and 50% water was used as a diluent to suit the liquid dosage form and intraperitoneal injection requirements. Other excipients were added as needed. The core objective was to improve the solubility, stability, and bioavailability of Remodelin, while ensuring that the osmotic pressure and pH value of the formulation met the biocompatibility requirements for intraperitoneal injection, thus reducing the risk of local irritation. This dosage form is specifically prepared as a sterile liquid, appearing as a clear and transparent solution free of insoluble particles, strictly adhering to the sterile and pyrogen-free quality standards for injectable preparations. Targeted treatment of allergic asthma is achieved through intraperitoneal injection. This administration method is convenient and has stable drug absorption efficiency. The drug can be rapidly absorbed by the body and targeted to lung tissue to exert its effect, balancing precise efficacy with ease of use. It is suitable for intervention during acute exacerbations of allergic asthma or maintenance treatment during chronic phases.
[0019] In an ovalbumin (OVA)-induced allergic asthma model animal, a therapeutically effective dose of 20 mg / kg was administered via intraperitoneal injection. The therapeutic effects were as follows: (1) Flow cytometry showed that after Remodelin intervention, the average fluorescence intensity of CD206 in mouse bronchoalveolar lavage fluid macrophages was significantly lower than that in the asthma model group; (2) ELISA detection confirmed that the serum total IgE level of mice treated with Remodelin decreased significantly, and the allergic reaction was relieved; (3) Histopathological examination: Hematoxylin-eosin staining (H&E staining) showed a significant reduction in inflammatory cell infiltration around the airways and blood vessels, a significant decrease in the inflammatory cell infiltration score, and an improvement in the degree of airway stenosis; periodic acid Schiff staining (PAS staining) revealed a significant reduction in goblet cell metaplasia of bronchial epithelium, a significant reduction in the amount of mucus secreted in the airway lumen, and an effective inhibition of mucus plug formation; Masson trichrome staining confirmed a significant reduction in the area of collagen fiber deposition in the airway wall and lung interstitium, and a significant improvement in the degree of lung tissue fibrosis, fully demonstrating the comprehensive therapeutic effect of Remodelin on airway inflammation, mucus hypersecretion, and airway remodeling in allergic asthma; (4) Molecular level detection showed that the transcriptional levels of M2 polarization markers such as Arg1, CD206, and Ym1 in lung tissue decreased synchronously.
[0020] This invention reveals for the first time the crucial role of the novel regulatory axis “Remodelin–NAT10–macrophage M2 polarization” in OVA-induced allergic asthma, with its core findings being: 1. Precise pathological targeting: This regulatory axis directly targets the core pathological link of OVA-induced asthma—macrophage M2 polarization. Remodelin precisely blocks the inflammatory amplification pathway by inhibiting NAT10 activity, avoiding widespread suppression of normal immune function throughout the body. 2. Systematic validation of efficacy: The therapeutic effect of Remodelin was clarified through the linkage validation at the cellular level (M2 polarization inhibition) and the animal level (asthma pathological improvement). The experimental data covered multiple dimensions including molecular markers, serum IgE levels and histopathology. 3. Clear dose-response: The effective dose range of Remodelin at the cellular and animal levels has been clearly defined, and no significant cytotoxicity or organ damage in animals has been observed at this concentration and dose, indicating controllable safety. 4. Targeted application scenarios: Specifically applicable to OVA-induced allergic asthma models, providing clear experimental evidence for the treatment of asthma patients with similar pathological mechanisms in subsequent clinical translation, filling the technological gap in targeted NAT10 therapy for asthma.
[0021] Furthermore, by inhibiting the function of the "Remodelin–NAT10–macrophage M2 polarization" regulatory axis, this invention can systematically improve airway inflammatory infiltration, immune imbalance and structural remodeling in OVA-induced allergic asthma, providing a new technical solution and experimental support for the precision treatment of allergic asthma.
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Breakthrough in Scientific Theory: This invention is the first to clearly define Remodelin as targeting NAT10, blocking the core pathological pathway of allergic asthma: "NAT10-macrophage M2 polarization." This fills the gap in the functional localization of NAT10 in the field of asthma, confirms its potential as a novel therapeutic target, provides a new immune regulatory explanation for the pathogenesis of allergic asthma, enriches the theoretical system of precision asthma treatment, and lays the theoretical foundation for the subsequent development of therapeutic strategies targeting immune cell polarization.
[0023] 2. Systematic and Profound Mechanism: This invention constructs a complete regulatory chain of "Remodelin - inhibition of NAT10 activity - inhibition of macrophage M2 polarization - alleviation of asthma pathological damage," and systematically verifies the mechanism of action from two core dimensions: cells and animals. It not only clarifies the regulatory effect of Remodelin on M2 polarization markers, but also, through in vitro and in vivo experiments, confirms that it can simultaneously improve multi-dimensional pathological phenotypes such as airway inflammatory infiltration and airway remodeling.
[0024] 3. Universality and Foresight of the Application Strategy: This invention has potential therapeutic value for different asthma populations, applicable to inflammation control during disease flare-ups and preventative intervention in high-risk groups, with a wide range of applications. Furthermore, the cross-disciplinary application of Remodelin, a NAT10 inhibitor already validated in other diseases, to asthma treatment provides an innovative approach to expanding the indications of existing small molecule inhibitors and offers a paradigmatic reference for subsequent drug development targeting related targets.
[0025] 4. Completeness and Reliability of the Technical Solution: This invention covers a technical solution encompassing "cell function verification - animal model verification," with rigorous experimental design and complementary data at each stage. Remodelin is readily available, its dosage form preparation process is conventional, the effective dosage and administration method are clearly defined, and the technical solution exhibits strong reproducibility, providing solid and reliable support for clinical translation and industrialization. Attached Figure Description
[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The figure shows the inhibitory effects of NAT10 knockdown, knockout, and remodelin intervention on macrophage M2 polarization; in the figure, Figure 1 Figure A shows the qPCR results for detecting NAT10 knockdown efficiency; Figure 1 Figure B shows the qPCR detection results after NAT10 knockdown and M2 induction. Figure 1 C is a Western blot result graph verifying the efficiency of NAT10 knockout; Figure 1 Figure D shows the qPCR results of macrophage-specific NAT10 knockout (Nat10-cKO) mice after M2 induction;Figure 1 E is the Western blot result of Nat10-cKO mice induced by M2; Figure 1 F shows the cytotoxicity test results of different concentrations of Remodelin; Figure 1 G represents the qPCR detection result after Remodelin treatment and M2 induction.
[0027] Figure 2 The image shows the effect of Nat10-cKO on improving lung damage in OVA-induced allergic asthma mice; among them, Figure 2 A is a schematic diagram of the construction and experimental process of the OVA-induced allergic asthma model; Figure 2 Figure B shows the qPCR detection results of lung tissue from asthmatic mice. Figure 2 C is a staining image of airway tissue from an asthmatic mouse. Figure 2 D represents the serological test results; Figure 2 E is a flow cytometry image.
[0028] Figure 3 This is a graph showing the effect of remodelin on improving OVA-induced allergic asthma in mice; where, Figure 3 A is a flowchart of the OVA-induced asthma model construction and Remodelin intervention experiment. Figure 3 B shows the results of flow cytometry and serological testing. Figure 3 C and Figure 3 D represents the qPCR detection results of lung tissue and bronchoalveolar lavage fluid from each group of mice; Figure 3 E shows the staining results of lung tissue from each group of mice. Detailed Implementation
[0029] The technical solution of the present invention will be further described and illustrated below through examples. All raw materials used in the examples are commercially available or prepared using conventional methods.
[0030] Example 1 In vitro mechanism verification of the inhibitory effect of NAT10 deficiency and Remodelin intervention on macrophage M2 polarization.
[0031] (1) Verification of the necessity of NAT10 in M2 polarization using siRNA knockdown technology: Specific siRNAs targeting mouse NAT10 were designed and synthesized, and transfected into wild-type (WT) mouse primary peritoneal macrophages using Lipofectamine RNAiMAX transfection reagent. A non-specific siRNA transfection group was set up as a negative control (si-NC). Forty-eight hours after transfection, the knockdown efficiency of NAT10 at the mRNA and protein levels was verified by qPCR and Western blot (results are shown in Figure 1). Figure 1(As shown in A); subsequently, M2 cell polarization was induced using a culture medium containing IL-4 and IL-13. RNA samples were collected at 0 h, 24 h, and 48 h during the polarization process, and the expression levels of key M2 polarization markers such as Arg1, Ym1, and Fizz1 in cells were detected by qPCR (results are shown in A). Figure 1 As shown in B).
[0032] (2) Confirmation of NAT10 function at the genetic level using gene knockout animal models: Western blot analysis confirmed the successful construction of the Nat10-cKO model (results are shown in Figure 1). Figure 1 (As shown in C), primary peritoneal macrophages from WT and Nat10-cKO mice were isolated, and M2 polarization was induced using IL-4 and IL-13. RNA samples were collected at 0 h, 24 h, and 48 h during the polarization process, and protein samples were collected sequentially at 0 h, 3 h, 6 h, and 9 h. The relative mRNA expression levels of a set of M2-related markers were detected using qPCR (results are shown in C). Figure 1 As shown in D), Western blot was used to detect the phosphorylation levels of Arg1 and STAT6 in cells (results are shown in D). Figure 1 E is shown.
[0033] (3) Remodelin cytotoxicity test and its intervention effect on M2 polarization: Primary peritoneal macrophages of WT mice were isolated and pretreated with 5 μM, 10 μM and 20 μM of the NAT10 inhibitor Remodelin for 2 h, respectively. An equal volume of DMSO solvent control group was set up. After pretreatment, M2 polarization of cells was induced by culture medium containing Remodelin, IL-4 and IL-13. After the samples were collected in sequence, the effect of Remodelin on cell viability within this concentration range was first evaluated by CCK-8 assay to confirm that it had no significant cytotoxicity, thus establishing the safe dosing concentration for subsequent functional studies (results are shown in the figure). Figure 1 (As shown in F); Subsequently, based on this safe concentration, cell samples were collected at multiple time points during the polarization induction process. The expression differences at the mRNA and protein levels were quantitatively confirmed by qPCR and Western Blot techniques, thereby demonstrating that Remodelin can effectively inhibit M2 polarization of macrophages in a concentration-dependent manner (results are shown in F). Figure 1 As shown in G).
[0034] Cellular experiment results as follows Figure 1 As shown, this study aims to verify the regulatory effect of NAT10 on macrophage M2 polarization and the intervention effect of Remodelin, providing in vitro evidence for subsequent in vivo experiments and drug applications.
[0035] (1) Verification of NAT10 knockdown efficiency and its impact on M2 polarization: such asFigure 1 As shown in Figure A, after transfecting macrophages with NAT10-specific siRNA, qPCR results confirmed a significant downregulation of NAT10 mRNA expression, with knockdown efficiency meeting experimental requirements. Further analysis of M2 polarization marker expression was performed, such as... Figure 1 As shown in Figure B, after NAT10 knockdown, the mRNA expression levels of M2 polarization markers Arg1, Ym1, and Fizz1 in macrophages induced by IL-4+IL-13 were significantly reduced, suggesting that NAT10 deficiency can inhibit the M2 polarization process in macrophages.
[0036] (2) The effect of Nat10-cKO on M2 polarization: such as Figure 1 As shown in Figure C, Western blot analysis confirmed the successful construction of the Nat10-cKO model. Figure 1 As shown in Figure D, after M2 induction in Nat10-cKO mice, qPCR detection showed that the mRNA expression levels of Arg1, Ym1, and Fizz1 were significantly lower than those in the WT group; Western blot analysis further confirmed this (e.g., ...). Figure 1 As shown in Figure E), the phosphorylation level of STAT6 in macrophages of the Nat10-cKO group was significantly downregulated, while the expression level of Arg1 protein was significantly downregulated, which together confirmed at the gene and protein levels that NAT10 deficiency can inhibit macrophage M2 polarization.
[0037] (3) Remodelin cytotoxicity detection and its intervention effect on M2 polarization: such as Figure 1 As shown in Figure F, the effect of different concentrations of Remodelin on macrophage viability was detected using the CCK8 assay. The results showed that treatment with 10 μM and 20 μM Remodelin had no significant cytotoxicity and could be used for subsequent intervention experiments. Figure 2 As shown in G, qPCR results showed that Remodelin downregulated the mRNA expression levels of Arg1, Ym1, and Fizz1 in M2-induced macrophages in a dose-dependent manner, confirming that Remodelin can inhibit macrophage M2 polarization by interfering with the expression of M2 polarization markers.
[0038] Example 2 The in vivo efficacy verification steps for Nat10-cKO on improving lung damage induced by OVA in mice with allergic asthma are as follows: S1, such as Figure 2As shown in A, 6-8 week old Nat10-cKO and WT mice were selected. Allergic asthma models were established by intraperitoneal injection of OVA + aluminum hydroxide combined with OVA nebulization challenge. During the sensitization period, aluminum hydroxide adjuvant was injected intraperitoneally on days 0, 7 and 14. During the challenge period, 1% OVA solution was nebulized for 30 min daily from days 21 to 27. On day 28, after anesthetizing the mice, lung tissue, bronchoalveolar lavage fluid and serum samples were collected for later use. S2. Rapidly separate the mouse lung tissues collected in step S1, cut a portion of the lung tissue into small pieces for subsequent testing; extract RNA from the lung tissue obtained in the experiment using the Trizol method, and detect the mRNA expression levels of M2 polarization-related markers and inflammatory factors in cells using qPCR technology (results are shown in Figure 1). Figure 2 (as shown in B) S3. Rapidly separate the left lung tissues of each group of mice collected in step S1. Fix the lung tissues in 4% paraformaldehyde fixative, and perform H&E staining to observe the number, distribution range, and density of inflammatory cells infiltrating the airways to determine the severity of airway inflammation. Masson staining and PAS staining are also performed to observe the collagen deposition area and degree of fibrosis around the airways and in the lung interstitium, as well as the degree of goblet cell proliferation in the bronchial epithelium and the amount of airway mucus secretion, thereby assessing the severity of airway remodeling in asthmatic mice (results are shown in Figure 1). Figure 2 (as shown in C) S4. Collect serum samples from each group of mice obtained in step S1. Using serological detection methods, strictly following the instructions of the detection kit, detect the IgE and OVA-IgE levels in the serum of each group of mice (results are shown below). Figure 2 (as shown in D); S5. Slowly perfuse the trachea of anesthetized mice in step S1 with pre-cooled sterile PBS to obtain bronchoalveolar lavage fluid. Repeat the lavage 2-3 times, collect the lavage fluid, centrifuge at 4 ℃, and retain the supernatant for subsequent analysis. Flow cytometry was used to detect relevant immune cell subsets. The bronchoalveolar lavage fluid cells obtained in the experiment were stained with FVD510 (live / dead dye), CD45, F4 / 80, CD11b, CD11c, CD170, and CD206. The expression level of CD206 in M2 macrophages in mouse bronchoalveolar lavage fluid was detected (results are shown in Figure 1). Figure 2 E is shown.
[0039] The animal experiment results of this embodiment are as follows: Figure 2 As shown, the Nat10-cKO mouse model further confirms the key role of NAT10 in the pathogenesis of allergic asthma, providing direct in vivo evidence for NAT10 as a therapeutic target for asthma.
[0040] (1) Detection of M2 polarization marker gene expression in lung tissue: such as Figure 2As shown in Figure B, qPCR results showed that, compared with the asthma model group, the mRNA expression levels of M2 polarization markers Arg1, Ym1, and Fizz1 in the lung tissue of Nat10-cKO mice were significantly reduced, confirming that Nat10 knockout can inhibit the M2 polarization process in lung tissue in vivo.
[0041] (2) Improvement in airway histopathological morphology: such as Figure 2 As shown in Figure C, H&E staining showed that the area of inflammatory cell infiltration around the airways of mice treated with Nat10-cKO was significantly reduced, and the airway inflammatory response was alleviated. Masson staining showed that the degree of collagen deposition around the airways in the Nat10-cKO group was lower than that in the model group, indicating that Nat10 knockout can improve pathological damage such as airway inflammation, mucus secretion, and airway remodeling in asthmatic mice. PAS staining showed that the degree of goblet cell proliferation in the bronchial epithelium and airway was reduced in the Nat10-cKO group, and the amount of mucus secretion was decreased.
[0042] (3) Serum IgE level detection: such as Figure 3 As shown in Figure D, serological tests revealed that the specific serum IgE level and total serum IgE level in the Nat10-cKO group mice were significantly lower than those in the asthma model group, indicating that Nat10 knockout can inhibit the allergic immune response in asthmatic mice.
[0043] (4) Detection of airway M2 macrophage levels: such as Figure 3 As shown in E, flow cytometry results showed that the expression level of CD206 in M2 macrophages in the bronchoalveolar lavage fluid of Nat10-cKO mice was significantly lower than that in the asthma model group, further confirming that NAT10 deficiency can improve lung damage in asthma by inhibiting the enrichment of airway M2 macrophages.
[0044] Example 3 The in vivo efficacy verification of remodelin on the improvement of OVA-induced allergic asthma in mice is as follows: S1. Animal model construction and intervention plan, the process is as follows: Figure 3 As shown in Figure A: WT mice were selected and divided into a normal control group, an asthma model group, and a Remodelin group. The asthma model group and the Remodelin group were sensitized by intraperitoneal injection of OVA + aluminum hydroxide (days 0, 7, and 14) combined with OVA nebulization challenge (days 21-27) to establish an allergic asthma model. The normal control group was treated with an equal volume of physiological saline. During the challenge period (days 21-27), 30 minutes before nebulization, the Remodelin group was administered the NAT10 inhibitor Remodelin by gavage, while the asthma model group received an equal volume of DMSO as a solvent control. After model establishment, lung tissue and bronchoalveolar lavage fluid were collected from the mice for subsequent tests. S2. Regulatory effects of Remodelin on M2 macrophages and humoral immunity in asthmatic mice: Bronchoalveolar lavage fluid (BAF) cells from each group of mice collected in step S1 were collected. Flow cytometry was used to detect relevant immune cell subsets. BAF cells were stained with FVD510 (live / dead dye), CD45, F4 / 80, CD11b, CD11c, CD170, and CD206 to detect the expression level of CD206 in M2 macrophages in mouse BAF. Simultaneously, serum samples were collected from each group of mice. Serological detection methods were used, strictly following the instructions of the detection kit, to detect the IgE levels in the serum of each group of mice (results are shown in the figure). Figure 3 (as shown in B) S3. Collect the lung tissues from each group of mice obtained in step S1 and grind them. Extract RNA from the lung tissues and bronchoalveolar lavage fluid obtained in the experiment using the Trizol method. Detect the mRNA expression levels of M2 polarization-related markers and inflammatory factors in cells using qPCR technology (results are shown in Figure 1). Figure 3 C and Figure 3 (as shown in D); S4. The left lung tissues of each group of mice collected in step S1 were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and then subjected to H&E staining to observe airway inflammatory infiltration and structural damage. Masson staining was used to assess the degree of airway remodeling in asthma model mice, focusing on the deposition of collagen fibers in the airway wall. PAS staining was used to assess bronchial goblet cell proliferation and mucus secretion in the lung tissue (results are shown in Figure 1). Figure 3 (as shown in E) Animal experiment results such as Figure 3 As shown, using an OVA-induced allergic asthma mouse model as the research subject, this study verified the therapeutic effect and related characteristics of Remodelin on asthma in vivo.
[0045] (1) Detection of airway M2 macrophage levels: such as Figure 3 As shown in Figure B, flow cytometry results indicated that the expression level of CD206 in M2 macrophages in the bronchoalveolar lavage fluid of mice treated with Remodelin was significantly lower than that in the asthma model group, demonstrating that Remodelin can inhibit the enrichment of M2 macrophages in the airways of asthmatic mice. Serological results showed that the serum IgE level in mice treated with Remodelin was significantly lower than that in the asthma model group, indicating that Remodelin can inhibit the humoral immune response disorder in asthmatic mice and alleviate allergic reactions.
[0046] (2) Detection of M2 polarization and anti-inflammatory related gene expression: such as As shown in C and D, qPCR results showed that Remodelin treatment significantly downregulated the mRNA expression levels of Ym1, Fizz1, Il-10, and Tgf-β1 in the lung tissue of asthmatic mice, and significantly reduced the mRNA expression levels of Arg1, Ym1, Il-10, and Tgf-β1 in bronchoalveolar lavage fluid. This molecularly confirms that Remodelin can inhibit local M2 polarization and inflammatory factor secretion in the airway.
[0047] (3) Histopathological morphological examination of airway tissue: such as As shown in E, the tissue staining results showed that: in H&E staining, the area of inflammatory cell infiltration around the airway in the Remodelin-treated group was significantly smaller than that in the asthma model group, and the airway inflammatory response was significantly reduced; in Masson staining, the degree of collagen deposition around the airway in the Remodelin group was significantly reduced compared with the model group, proving that Remodelin can effectively improve airway remodeling in asthmatic mice; in PAS staining, the degree of goblet cell proliferation in the bronchial airway was reduced and the amount of mucus secretion was decreased in the Remodelin group.
[0048] It should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. The application of NAT10 inhibitor Remodelin in the preparation of drugs for treating allergic asthma.
2. The application according to claim 1, characterized in that, The drug uses NAT10 as a molecular target for the treatment of allergic asthma.
3. The application according to claim 1, characterized in that, The drug described herein has Remodelin as its sole active ingredient.
4. The application according to claim 1, characterized in that, The drug is in the form of an injectable injection.
5. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.
6. The application according to claim 5, characterized in that, The excipients include a diluent.
7. The application according to claim 6, characterized in that, The diluent is a mixture of DMSO, PEG300, Tween80 and water in a volume ratio of 5:40:5:
50.
8. The application according to claim 1, characterized in that, The drug is administered via intraperitoneal injection.