Application of interleukin 33 antibody in preparation of medicine for relieving asthma exacerbation

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of an interleukin 33 antibody in preparation of a medicine for relieving asthma aggravation, in particular to application of the interleukin 33 antibody in preparation of a medicine for relieving asthma aggravation induced by ozone exposure. According to the application disclosed by the invention, comprehensive research experiments are adopted, the intervention treatment of the IL-33 antibody is implemented by constructing an asthma model and utilizing an ozone-induced asthma exacerbation model, and the pathophysiological action of the IL-33 antibody in relieving ozone-induced asthma exacerbation is comprehensively disclosed. Experimental results show that the IL-33 antibody can relieve the conditions of asthma mouse lung dysfunction, lung tissue inflammatory cell infiltration and immune cell imbalance in lung tissue caused by ozone exposure. The invention provides a new perspective for the treatment of asthma, and provides a scientific basis and practical value for developing a novel and effective asthma prevention and treatment strategy. The traditional Chinese medicine composition is expected to improve the living quality of asthma patients and reduce health risks caused by environmental pollutants, and has important clinical significance and social value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of interleukin-33 antibody in the preparation of drugs to alleviate asthma exacerbations, and more specifically to the use of interleukin-33 antibody in the preparation of drugs to alleviate asthma exacerbations induced by ozone exposure. Background Technology

[0002] Existing technologies disclose that asthma is a chronic respiratory disease characterized by immune imbalance, and its pathological basis involves the complex interactions of numerous immune cells and cytokines. Typical clinical symptoms of the disease usually manifest as airway hyperresponsiveness (AHR), airway remodeling, and inflammatory responses, accompanied by wheezing, shortness of breath, cough, and chest tightness. According to statistics, in the past decade, more than 30 million new asthma patients have been diagnosed globally each year, and approximately 500,000 people have died from it. The shadow of asthma looms over 1% to 18% of the world's population.

[0003] Clinical studies show that asthma has diverse clinical phenotypes and different molecular pathogenesis mechanisms, mainly including Th2-type allergic asthma induced by allergens and non-Th2-type asthma caused by factors such as air pollution. The pathogenesis of asthma is complex. Th2-type asthma is characterized by an increase in the number of Th2 cells, eosinophils, and activated mast cells, and treatment primarily relies on glucocorticoids. However, clinical observations have found that over 40% of asthma patients primarily present with neutrophilic inflammation, and 10% to 15% of patients with severe asthma respond poorly to glucocorticoid treatment or exhibit some degree of resistance. Asthma is currently incurable, and under the influence of various triggering factors, symptoms can worsen rapidly, even becoming life-threatening. It is generally believed in the field that the occurrence and development of asthma is the result of the combined effects of genetic and environmental factors.

[0004] With the intensification of urbanization and global warming, ozone pollution has become an increasingly serious problem. Ozone, as a highly oxidizing inhalable gas, has a particularly significant impact on respiratory health. Globally, over 92% of the population is chronically exposed to unhealthy ozone levels. Recent environmental epidemiological studies have repeatedly emphasized that ozone exposure is closely related to various adverse health outcomes in vulnerable groups such as those with asthma. Ozone exposure may not only induce asthma attacks and exacerbate existing conditions but may also lead to asthma-related deaths. Toxicological studies have revealed that ozone exposure can cause airway epithelial damage, airway inflammation, and alveolar structural damage, leading to abnormal lung function. Animal experiments have shown that ozone exposure can increase the proportion of Th2 and Th17 cells in the lungs of mice and significantly increase the levels of Th2 and Th17-related cytokines in bronchoalveolar lavage fluid (BALF), suggesting that ozone exposure may exacerbate asthma by affecting the differentiation of Th2 and Th17 cells and the release of their cytokines.

[0005] Interleukin 33 (IL-33) is a cytokine that plays a crucial role in asthma and other allergic diseases. Belonging to the IL-1 family, it is primarily expressed by epithelial cells and released in response to tissue damage or stress. As an alarmist, IL-33 activates the innate immune system, particularly through interactions with ILCs and dendritic cells, triggering inflammatory responses. Furthermore, IL-33 promotes the differentiation of Th2 cells and the activation of Th2 and ILC2 cells. Cytokines secreted by these cells (such as IL-4, IL-5, and IL-13) play a vital role in the pathogenesis of asthma, enhancing airway inflammation and hyperresponsiveness.

[0006] Given the current insufficient understanding of asthma exacerbations caused by air pollution and the lack of effective interventions, this invention aims to utilize ozone, a typical air pollutant, to construct a mouse model of asthma exacerbated by ozone exposure, and to provide scientific evidence that IL-33 antibodies can alleviate ozone exposure-induced asthma exacerbations. This invention will provide a clearer theoretical basis for IL-33-based asthma treatment strategies, offer important scientific evidence for the prevention and control of ozone air pollution, and provide strong support for future directions in asthma treatment. Summary of the Invention

[0007] The purpose of this invention is to provide new uses for interleukin-33 antibodies in pharmaceutical manufacturing, based on the current state of the technology, specifically the use of interleukin-33 antibodies in the preparation of drugs to alleviate asthma exacerbations, and especially the use of interleukin-33 antibodies in the preparation of drugs to alleviate asthma exacerbations induced by ozone exposure.

[0008] This invention employs a comprehensive research approach, constructing an animal model of asthma to simulate the exposure of asthmatic individuals to ozone pollution and evaluating the efficacy of IL-33 antibodies in alleviating asthma symptoms. Results show that the use of IL-33 antibodies can reduce asthma exacerbations induced by ozone exposure. The interleukin-33 antibody of this invention can be used to prepare drugs for reducing asthma exacerbations, particularly those induced by ozone exposure.

[0009] Specifically, this invention employs the following comprehensive research experiments.

[0010] 1) Asthma model construction and ozone exposure experiment:

[0011] 2) Lung function test:

[0012] 3) Lung tissue collection experiment:

[0013] 4) Lung tissue pathological examination:

[0014] 5) IgE and IL-33 content detection experiment:

[0015] 6) Allergen sensitization, challenge, ozone exposure, and antibody intervention experiments:

[0016] 7) Flow cytometry detection:

[0017] Comprehensive research results show that this invention fully reveals the pathophysiological role of IL-33 antibody in alleviating ozone-induced asthma exacerbation. Experimental results indicate that treatment with IL-33 antibody can significantly reduce pulmonary dysfunction, infiltration of inflammatory cells in lung tissue, and imbalance of immune cells in lung tissue in ozone-exposed asthmatic mice.

[0018] More specifically, the present invention is implemented using the following technical solution:

[0019] This invention first utilizes ovalbumin (OVA) to construct an animal model of asthma. Six-week-old asthma-sensitive mice (BALB / c) were sensitized by intraperitoneal injection of OVA on days 0 and 12, and subsequently challenged by intranasal instillation of OVA from days 18 to 26. Control mice received the same amount of control reagent.

[0020] Asthmatic mice and control mice were randomly assigned to receive exposure. The study included a filtered air control group, an ozone group, an OVA + filtered air group, and an OVA + ozone group. Mice in the ozone group and the OVA + ozone group were exposed to controlled concentrations of ozone for specific time periods, while the filtered air control group and the OVA + filtered air group were exposed to clean air. The effects of ozone exposure on the pathophysiology of asthmatic mice were comprehensively assessed by measuring pulmonary ventilation function, airway responsiveness, lung histopathological changes, and pulmonary inflammatory markers. In an asthma model and an ozone-induced asthma exacerbation model, mice were administered IL-33 antibody or an equivalent amount of isotype control antibody from day 23 to 26. The role of IL-33 antibody in alleviating ozone-induced asthma exacerbations was investigated using pulmonary function tests, lung histopathological examination, and lung flow cytometry.

[0021] Experimental results showed that short-term ozone exposure significantly enhanced airway hyperresponsiveness in asthmatic mice, exacerbated pulmonary inflammation and lung tissue damage, and led to excessive release of IL-33. IL-33 antibody treatment significantly alleviated ozone-induced pulmonary dysfunction, inflammatory cell infiltration in lung tissue, and the imbalance in the proportion of ILC2, Th2, and Th17 cells in lung tissue. These findings reveal the crucial role of IL-33 in ozone-induced asthma exacerbation and confirm that IL-33 antibody treatment effectively improves asthmatic lung function by restoring immune balance and reducing inflammatory cell infiltration in lung tissue.

[0022] This invention provides a novel treatment strategy through the use of IL-33 antibody intervention. Results show that it significantly reduces lung inflammation, airway hyperresponsiveness, and pulmonary dysfunction caused by ozone exposure, effectively alleviating asthma symptoms. This invention provides a new use for interleukin-33 antibodies in the preparation of drugs to alleviate asthma exacerbations, particularly in the preparation of drugs to reduce ozone-induced asthma exacerbations. Furthermore, this invention provides a clearer theoretical basis for IL-33-based asthma treatment strategies, offers important scientific evidence for the prevention and control of ozone air pollution, and provides strong support for future directions in asthma treatment.

[0023] Furthermore, this invention provides a new perspective on asthma treatment through the application of IL-33 antibodies, and also offers potential treatment ideas for other respiratory diseases caused by environmental pollution. The invention has broad application prospects, not only applicable to asthma patients, but also providing new possibilities for the treatment of other respiratory diseases. With the acceleration of global urbanization and the increasing severity of environmental pollution problems, the implementation of this invention has significant practical implications for mitigating the impact of environmental pollution on human health. Attached Figure Description

[0024] Figure 1 This is the experimental result of ozone exposure aggravating lung function in asthmatic mice.

[0025] Figure 2 The results show that ozone exposure exacerbates lung inflammation and pathological damage in asthmatic mice.

[0026] Figure 3 This is the experimental result of ozone exposure disrupting the immune homeostasis of asthmatic mice.

[0027] Figure 4 These are experimental results regarding the level of IL-33 protein in lung tissue.

[0028] Figure 5 This is the experimental result that IL-33 antibody significantly alleviates lung dysfunction in ozone-exposed asthmatic mice.

[0029] Figure 6 The results show that IL-33 antibody significantly alleviates lung tissue inflammation and damage in ozone-exposed asthmatic mice.

[0030] Figure 7 This is an experimental result showing that IL-33 antibody intervention can rebalance the ozone-induced pulmonary immune imbalance. Detailed Implementation

[0031] Example 1

[0032] 1) Asthma model construction and ozone exposure experiment:

[0033] An animal model of asthma was established using ovalbumin (OVA) and mice were exposed to filtered air (FA) and ozone (O3). Six-week-old mice, after one week of acclimatization, were randomly divided into four groups: a filtered air control group, an ozone group (O3), an OVA + filtered air group (OVA), and an OVA + ozone group (O3 + OVA). Mice in the OVA and O3 + OVA groups were sensitized by intraperitoneal injection of 100 μg of OVA (emulsified with aluminum hydroxide) on days 0 and 12. From day 18 to day 26, asthma was induced by daily nasal instillation of 50 μg of OVA. The filtered air control group and the O3 group received intraperitoneal injection of the same dose of aluminum hydroxide and nasal instillation of an equal volume of physiological saline.

[0034] From days 24 to 26, mice in the O3 group and the O3+OVA group were exposed to 2 ppm of O3 from 1 p.m. to 4 p.m. Meanwhile, the filtered air control group and the OVA group were exposed to FA (air-filtered oxygen). Ozone concentration in each chamber was continuously measured and automatically recorded using an ozone detection device.

[0035] 2) Lung function test:

[0036] The day after the last exposure to FA or O3, mice were anesthetized with sodium pentobarbital (50 mg / kg body weight). The mice were then endotracheally intubated, and their trachea was connected to a computer-controlled ventilator. The respiratory rate was set to 90 breaths / min, and the expiratory-to-inspiratory time ratio was set to 1.5:1. Subsequently, pulmonary function tests were performed using the AniRes2005 pulmonary function system. Recorded parameters included forced vital capacity (FVC) and forced expiratory volume in 0.1 seconds (FEV1). 0.1 Peak expiratory flow (PEF) and expiratory flow rate were measured. To assess airway hyperresponsiveness (AHR), methacholine (MCH) was administered intravenously at concentration gradients of 0.025, 0.05, 0.1, and 0.2 mg / kg body weight every 5 minutes, and inspiratory airway resistance (R) was measured. L ), expiratory airway resistance (Re) and pulmonary dynamic compliance (Cdyn).

[0037] 3) Lung tissue collection experiment:

[0038] The lung tissue was processed in three parts. One part of the lung tissue was fixed with 4% paraformaldehyde for subsequent histopathological analysis; another part was used for flow cytometry analysis. The remaining lung tissue samples were finally preserved at -80°C for further in-depth analysis.

[0039] 4) Lung tissue pathological examination:

[0040] Lung tissue was fixed in 4% paraformaldehyde for 48 hours, then embedded in paraffin and cut into 5 μm thick sections. Hematoxylin-Eosin (H&E) staining was used to assess lung injury, including edema, congestion, hemorrhage, alveolar deformity, interalveolar thickness, and macrophage infiltration. In addition, Alcian Blue Periodic Acid Schiff (AB-PAS) staining was used to assess goblet cell proliferation, and Congo Red staining was used to determine the degree of eosinophil infiltration.

[0041] 5) IgE and IL-33 content detection experiment:

[0042] This experiment used enzyme-linked immunosorbent assay (ELISA) kits (3005 and 3010, Chondrex; M3300, R&D Systems) to measure serum total IgE and OVA-specific IgE, as well as lung tissue IL-33 levels.

[0043] 6) Allergen sensitization, challenge, ozone exposure, and antibody intervention experiments:

[0044] Six-week-old mice were randomly divided into four groups: OVA sensitization plus clean air exposure plus control antibody group (OVA+FA+IgG), OVA sensitization plus ozone exposure plus control antibody group (OVA+O3+IgG), OVA sensitization plus clean air exposure plus IL-33 antibody group (OVA+FA+Anti-IL-33), and OVA sensitization plus ozone exposure plus IL-33 antibody group (OVA+O3+Anti-IL-33). All mice were sensitized by intraperitoneal injection of 100 micrograms of OVA (emulsified with aluminum hydroxide) on days 0 and 12. From day 18 to day 26, asthma provocation was performed by intranasal instillation of 50 micrograms of OVA daily.

[0045] From 1 p.m. to 4 p.m., mice in the OVA+O3+IgG and OVA+O3+Anti-IL-33 groups were exposed to 2 ppm ozone. Simultaneously, the OVA+FA+IgG and OVA+FA+Anti-IL-33 groups were exposed to FA. At 10 a.m. on days 23-26, each mouse was intraperitoneally injected with 3.6 micrograms of either Anti-IL-33 or IgG.

[0046] 7) Flow cytometry detection:

[0047] After rinsing lung tissue with pre-cooled PBS to remove blood, the lung tissue was finely minced using sterile surgical scissors. Subsequently, it was digested for 30 minutes at 37°C using DuPont modified medium containing 1X trypsin and 0.05% MEDTA. After digestion, fetal bovine serum was added to stop the reaction. Finally, the mixture was filtered through a 70μm filter to obtain a lung single-cell suspension. The single-cell suspension was transferred to 96-well plates and incubated for 30 minutes using different fluorescein-conjugated antibodies. For surface-stained cells, they were fixed using PBS containing 1% paraformaldehyde. For nuclear staining, surface-stained cells were first fixed and infiltrated, then stained using a nuclear transcription factor staining kit, and finally detected using a BD LSR Fortessa instrument.

[0048] like Figure 1 As shown, A illustrates the experimental protocol for establishing the asthma model and ozone exposure; B shows the ozone concentrations in the FA chamber and O3 exposure chamber during exposure; CE shows that the pulmonary ventilation function of asthmatic mice significantly deteriorated under O3 exposure conditions; FH shows that O3 exposure significantly worsened airway hyperresponsiveness in asthmatic mice; I and J show that the serum total IgE and OVA-specific IgE levels of asthmatic mice were significantly increased, indicating that the asthma model was successfully established.

[0049] like Figure 2 As shown, A and B are representative images and statistical results of H&E staining, showing that ozone exposure further aggravated the infiltration of inflammatory cells in the lung tissue of asthmatic mice; C and D are representative images and statistical results of Congo red staining, showing that ozone exposure further aggravated the infiltration of eosinophils in the lung tissue of asthmatic mice; E and F are representative images and statistical results of AB-PAS staining, showing significant proliferation of goblet cells in the airway epithelium of asthmatic mice.

[0050] like Figure 3 As shown, ozone exposure significantly increased the proportion of ILC2 in CD45 in the lung tissue of asthmatic mice. +Cell proportions; B and C show that ozone exposure further exacerbates the imbalance of Th1 and Th2 in the lung tissue of asthmatic mice; D and E show that ozone exposure further exacerbates the imbalance of Th17 and regulatory T cells (Treg) in the lung tissue of asthmatic mice.

[0051] like Figure 4 As shown, ozone exposure significantly increased the IL-33 content in the lung tissue of asthmatic mice and control mice.

[0052] like Figure 5 As shown, A illustrates the experimental protocol for establishing the asthma model, ozone exposure, and IL-33 antibody intervention; B shows the ozone concentration in the FA chamber and O3 exposure chamber during the exposure period; CE shows that the pulmonary ventilation dysfunction induced by O3 exposure in asthmatic mice became insignificant after IL-33 antibody intervention; FH shows that IL-33 antibody intervention significantly alleviated the deterioration of airway hyperresponsiveness in asthmatic mice induced by O3 exposure.

[0053] like Figure 6 As shown, A and B are representative images and statistical results of H&E staining, showing that IL-33 antibody intervention significantly reduced the infiltration of inflammatory cells in the lung tissue of asthmatic mice induced by O3 exposure; C and D are representative images and statistical results of Congo red staining, showing that IL-33 antibody intervention significantly reduced the infiltration of eosinophils in the lung tissue of asthmatic mice induced by O3 exposure.

[0054] like Figure 7 As shown, IL-33 antibody intervention significantly reduced the proportion of ILC2 in CD45 in the lung tissue of O3-exposed asthmatic mice. + Increased cell proportion; B and C show that IL-33 antibody intervention significantly restored the imbalance of Th1 and Th2 in the lung tissue of ozone-exposed asthmatic mice; D and E show that IL-33 antibody intervention significantly restored the imbalance of Th17 and Treg in the lung tissue of ozone-exposed asthmatic mice.

[0055] In summary, IL-33 antibody intervention significantly alleviated ozone exposure-induced pulmonary dysfunction, inflammatory cell infiltration in lung tissue, lung tissue damage, and imbalance in the ratio of ILC2, Th2, and Th17 cells in lung tissue in asthmatic mice.

[0056] This application describes in detail the implementation methods, technical solutions, and beneficial effects of the present invention. The above description is merely an example of implementation of the present invention and does not limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, or other changes made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of interleukin-33 antibody in the preparation of drugs to alleviate asthma exacerbations.

2. The use according to claim 1, characterized in that, The aforementioned asthma exacerbation is an asthma exacerbation caused by ozone exposure.

3. The use according to claim 1 or 2, characterized in that, The interleukin-33 antibody inhibits the biological activity of IL-33, alleviates ozone-induced asthma-related pulmonary dysfunction, lung tissue inflammation, and lung tissue damage, and the intervention can rebalance ozone-induced pulmonary immune imbalance.

4. The use according to claim 1 or 2, characterized in that, The IL-33 antibody is prepared by immunizing animals or through genetic engineering techniques.

5. The use according to claim 1 or 2, characterized in that, The IL-33 antibody was prepared as a drug to alleviate asthma exacerbations via intraperitoneal injection.

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