Use of thrombin inhibitors in the preparation or screening of products for preventing and treating allergic diseases

Abstract

The present invention relates to the field of medicine, and in particular to the use of thrombin inhibitors in the preparation or screening of products for preventing and treating allergic diseases. The allergic disease is an allergic disease targeting thrombin. The allergic disease is a pulmonary allergic disease, which is selected from allergic asthma, allergic rhinitis, allergic lung infection or chronic sinusitis with nasal polyps. The present invention proposes the possibility of using thrombin as a drug target, and by inhibiting thrombin, thereby controlling IL-33 activity and ILC2 cell-mediated inflammatory reactions, provides a new theoretical basis and treatment strategy for the treatment of allergic diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the use of thrombin inhibitors in the preparation or screening of products for preventing and treating allergic diseases. Background Art

[0002] 1. Clinical characteristics and pathogenesis of allergic asthma

[0003] Allergic diseases can cause severe morbidity and even mortality, placing significant strain on healthcare systems. These diseases, characterized by a type II inflammatory response, include food allergies, asthma, allergic rhinitis, chronic sinusitis with nasal polyps, and atopic dermatitis. Allergic asthma is the most typical type II inflammatory disease of the lungs, with distinct clinical manifestations. Clinically, the primary manifestation of asthma is reversible airflow obstruction, which can generally be alleviated with bronchodilators or anti-inflammatory drugs. Many factors contribute to airflow obstruction, including airway hyperresponsiveness (AHR), a spasmodic response of bronchial smooth muscle to irritation, inflammation, edema, neovascularization, excessive mucus production, and airway remodeling. Airway remodeling refers to the cyclical damage-repair process that occurs during the progression of severe or long-standing chronic disease, leading to thickening of the airway basement membrane, damage to the airway lumen, and impaired airflow. Studies have suggested that this airway thickening is caused by chronic and persistent inflammation and further exacerbates chronic airway inflammation, goblet cell hyperplasia, airway smooth muscle hypertrophy, and edema. These pathological changes lead to persistent airflow obstruction, and in severe cases, the therapeutic effects of corticosteroid bronchodilators or anti-inflammatory drugs become very limited. After repeated stimulation of the respiratory tract by specific allergens (such as dust mites, pollen, fungi, etc.), allergic asthma patients produce an adaptive immune response mediated by Th2 cells (T Helper 2 cells, Th2), manifested by an increase in eosinophils, an increase in the levels of cytokines associated with type II immune responses (such as IL-4, IL-5, and IL-13), and an increase in serum IgE levels. Due to the heterogeneity of the disease, some patients exhibit more complex phenotypes, such as the absence of specific allergens, Th2 involvement, and IgE production, making the diagnosis and treatment of the disease more difficult.

[0004] During the pathogenesis of allergic asthma, in addition to bacteria, viruses, and inflammatory signals in the lung microenvironment that can damage and open the epithelial barrier, allowing allergens to penetrate, some allergens with their own protease activity can also directly damage epithelial cells. Upon entry, damaged epithelial cells release large amounts of the warning cytokines IL-33, IL-25, and TSLP, activating ILC2 cells and dendritic cells (DCs). Simultaneously, allergens that have entered the epithelial barrier can come into contact with and be taken up by dendritic cells (DCs). Mature DCs migrate to local lymph nodes and present processed allergen peptides to naive T cells via class II MHC molecules on their cell surfaces. These T cells, stimulated by polarizing factors such as IL-4 in the microenvironment, differentiate into Th2 cells and exert effector functions. Both Th2 and ILC2 cells secrete type II immune cytokines IL-4, IL-5, IL-9, and IL-13, mediating type II inflammatory responses and disease progression. IL-4 is involved in IgE production by B cells, which binds to FcεRI on mast cells (MCs) and sensitizes them. The subsequent release of mast cell-associated mediators, such as histamine, tryptase, prostaglandins, leukotrienes, and cytokines, can lead to goblet cell hyperplasia, smooth muscle contraction, and increased vascular permeability. IL-5 is primarily responsible for the recruitment and maturation of eosinophils, and the granular contents released by eosinophils can cause tissue damage and promote the occurrence of inflammation. IL-13 can regulate the proliferation of IgE-producing B cells, excessive secretion of mucus, airway hyperresponsiveness (AHR), and promote the opening of the epithelial barrier. In addition, immunoregulatory cytokines released by Treg cells, such as IL-10 and TGF-β, can inhibit type II immune responses and Th1 and Th17 immune responses. B regulatory cells (Breg) that produce IL-10 can also inhibit effector T cells. IL-9 secreted by Th9 cells can induce eosinophilic inflammation, mast cell growth, excessive mucus secretion, and airway hyperresponsiveness (AHR) ( Figure 1 ).

[0005] 2. Overview of IL-33 and Inflammation

[0006] Interleukin 1 (IL-1) is an important cytokine that regulates innate immunity and inflammation. In recent years, research on the IL-1 family has made rapid progress in terms of species and biological functions. In 2005, Schmitz et al. first discovered that IL-33 is a new member of the IL-1 family that can bind to the IL-1 receptor-related protein ST2 and induce the secretion of cytokines related to Th2 cells. Until now, many studies have found that IL-33 can not only induce type II immune responses of Th2 cells, but also stimulate ILC2, Treg cells, Th1 cells, CD8 +T cells and NK cells, etc., have enriched the important role of IL-33 in tissue and metabolic homeostasis, infection, inflammation, tumors and diseases related to the central nervous system. The IL-33 expressed in cells is actually a precursor form (full-length form, 270 amino acids), which contains the typical IL-1-like domain at the C-terminus. The substances secreted by neutrophils and mast cells in the body can process the precursor form of IL-33 released into the extracellular matrix after cell damage into a shorter mature form that retains the IL-1-like domain ( Figure 2 The mature form of IL-33 exhibits 10-30 times greater biological activity than the precursor form. Endogenous mature IL-33 can be detected in many diseases, such as acute liver injury and fungal infections, demonstrating that the mature form of IL-33 does exert biological functions in vivo. Currently, there are no definitive studies elucidating the relative importance of the full-length and mature forms of IL-33.

[0007] When lung epithelial cells are attacked by allergens or parasites, cell damage or necrosis releases full-length IL-33, which matures under the action of extracellular proteolytic enzymes and stimulates ST2 + Basophils, mast cells, ILC2s, and Th2 cells in the lung produce type II immune cytokines, chemokines, and eotaxin. While these cells mediate pathogen clearance during parasitic infections, chronic activation of this pathway in the persistent presence of allergens or allergens can lead to allergic pathology and tissue fibrosis. Therefore, while mediating inflammatory cell activation, IL-33 stimulates myeloid cells to secrete IL-2. These two synergistically promote Treg cell proliferation and balance type II immune responses. IL-33 also induces AREG expression in ILC2s, ILC2s, and Tregs, thereby supporting the proliferation and differentiation of lung stem cells. IL-33-activated ILC2s also secrete IL-13, promoting the production of repair-related M2 macrophages. However, in many pathological conditions, unresolved tissue damage leads to persistent IL-33 release, and acute inflammatory responses can progress to chronic inflammatory diseases, potentially contributing to the development of type II immune cytokine-driven pulmonary fibrosis.

[0008] 3. Overview of the Coagulation System and Inflammation

[0009] With the emergence of multicellular life, the immune system rapidly developed to protect against pathogen invasion. Subsequently, the coagulation system evolved from the early innate immune system. Ancient organisms such as horseshoe crabs can utilize a combination of coagulation and the immune system to plug wounds and capture pathogens with blood clots. However, in mammals, despite the link between coagulation and the immune system, previously identified mechanisms are generally indirect and slow-acting. When tissue barriers with rich vascular systems (such as the lungs and skin) are damaged, the coagulation system responds rapidly, activating a protease cascade through either intrinsic or extrinsic pathways, promoting rapid thrombin activation, fibrin deposition, and platelet activation, thereby mediating coagulation. The innate immune response is somewhat delayed, typically requiring the sensing of pathogen-associated molecular patterns to activate early cytokines such as interleukin-1 (IL-1), triggering inflammation and subsequent adaptive immunity. During this process, inflammation induces tissue factor to promote coagulation, while thrombin in the coagulation system cleaves and activates proteasome-activated receptors (PARs) on the surfaces of monocytes, lymphocytes, endothelial cells, and dendritic cells through proteolysis, mediating inflammation. This slower kinetic process may allow microorganisms to spread within the wound. Therefore, a faster and more direct connection between the mammalian coagulation system and the immune system would benefit host health. Summary of the Invention

[0010] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a use of a thrombin inhibitor in the preparation or screening of products for preventing and treating allergic diseases, so as to solve the problems in the prior art.

[0011] To achieve the above-mentioned and other related purposes, the present invention provides the use of a thrombin inhibitor in the preparation or screening of products for preventing and treating allergic diseases.

[0012] Preferably, the thrombin inhibitor is selected from one or more of direct thrombin inhibitors, indirect thrombin inhibitors, vitamin K antagonists or factor Xa inhibitors.

[0013] Preferably, the allergic disease is an allergic disease associated with IL-33 maturation. The allergic disease is selected from food allergies, pulmonary allergic diseases, or atopic dermatitis. The pulmonary allergic disease is selected from allergic asthma, allergic rhinitis, allergic lung infection, or chronic sinusitis with nasal polyps.

[0014] Preferably, the thrombin inhibitor inhibits thrombin, inhibits IL-33 maturation, and inhibits the ability of IL-33 to activate ILC2 cells, thereby inhibiting type II inflammatory response, thereby achieving the purpose of preventing and treating allergic diseases.

[0015] As described above, the use of the thrombin inhibitors of the present invention in the preparation or screening of products for the prevention and treatment of allergic diseases has the following beneficial effects: It reveals a novel mechanism by which thrombin regulates IL-33 maturation and promotes type II inflammatory responses mediated by ILC2 cells. This suggests the possibility of using thrombin as a drug target, thereby inhibiting thrombin to control IL-33 activity and ILC2 cell-mediated inflammatory responses, providing a new theoretical basis and therapeutic strategy for the clinical treatment of asthma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a schematic diagram of the type II immune response mechanism. Epithelial cells produce cytokines IL-33, IL-25, and TSLP that act on downstream ILC2 cells. The activated ILC2 cells rapidly produce type II cytokines IL-5 and IL-13, initiating a type II immune response. At the same time, they can present antigens to TH2 cells, promoting TH2 cell activation and promoting the occurrence and development of asthma.

[0017] Figure 2 Shown is a schematic diagram of the processing of full-length IL-33. The left figure shows that full-length IL-33 is stored in the cell nucleus as a warning factor and is released from cells that die after injury or necrosis. Biologically active full-length IL-33 can be cleaved at the N-terminus (arrow) to produce a shorter form. Compared with full-length IL-33, mature IL-33 after cleavage exhibits enhanced biological activity. Once the core IL-1 tetrahedral structure (pyramid) is altered, biological activity is lost. Due to the formation of disulfide bonds, extracellular oxidation inactivates IL-33's biological activity, thereby inhibiting the binding of IL-33 to its receptor. The right figure shows that in the case of apoptosis, full-length IL-33 is cleaved by apoptotic caspase-3 and caspase-7, inactivating it as a cytokine or warning factor.

[0018] Figure 3.1 It was shown that low molecular weight heparin inhibited the immune response of ILC2 in the papain model and the A. alternaria model.

[0019] A. Schematic diagram of papain-induced lung inflammation model and treatment methods.

[0020] B. Flow cytometric analysis of eosinophils (CD45 + CD11c - / lo SiglecF + ) Grouping and proportion.

[0021] C. Statistical analysis of the proportion and number of eosinophils in bronchoalveolar lavage fluid.

[0022] D. Statistical analysis of the number of macrophages and neutrophils in bronchoalveolar lavage fluid.

[0023] E. HE and PAS staining of lung tissue pathology sections (scale bar: 200 μm) and pathological scoring.

[0024] F.ELISA detection of IL-5 and IL-13 in bronchoalveolar lavage fluid, statistical analysis of the experimental group and the control group.

[0025] G. Flow cytometry was used to detect the number of ILC2 in lung monocytes.

[0026] The ratio of IL-5 and IL-13 in lung mononuclear ILC2 cells stimulated with HI.PMA, ionomycin and BFA for 4 hours was statistically analyzed.

[0027] J. Schematic diagram of the lung inflammation model induced by the fungus A. alternaria and its treatment methods.

[0028] K. Statistical analysis of the proportion and number of eosinophils in bronchoalveolar lavage fluid.

[0029] L. Statistical analysis of the number of macrophages and neutrophils in bronchoalveolar lavage fluid.

[0030] M. HE and PAS staining of lung tissue pathology sections (scale bar: 200 μm) and pathological scoring.

[0031] N.ELISA was used to detect IL-5 and IL-13 in bronchoalveolar lavage fluid, and statistical analysis was performed in the experimental and control groups.

[0032] O. Flow cytometry was used to detect the number of ILC2 in lung monocytes.

[0033] P. The ratio of IL-5 and IL-13 in lung mononuclear cell ILC2s stimulated with PMA, ionomycin and BFA for 4 hours and statistically analyzed.

[0034] P values ​​were calculated by Student's t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; n = 8; experiments were performed twice independently.

[0035] Figure 3.2 It shows that the inhibition of acute type II inflammatory response by low molecular weight heparin may depend on the IL-33 signaling pathway.

[0036] A. Schematic diagram of the lung inflammation model induced by recombinant cytokine IL-33 and its treatment method.

[0037] B. Statistical analysis of the proportion and number of eosinophils in bronchoalveolar lavage fluid.

[0038] C. Statistical analysis of the number of macrophages and neutrophils in bronchoalveolar lavage fluid.

[0039] D.ELISA was used to detect IL-5 and IL-13 in bronchoalveolar lavage fluid, and statistical analysis was performed between the experimental group and the control group.

[0040] E. HE and PAS staining of lung tissue pathology sections (scale bar: 200 μm) and pathological scoring.

[0041] F. Flow cytometry was used to detect the number of ILC2 in lung monocytes.

[0042] G. The ratio of IL-5 and IL-13 in lung mononuclear cell ILC2s stimulated with PMA, ionomycin and BFA for 4 hours and statistical analysis.

[0043] H.LMWH treatment did not affect the number of ILC2 cells.

[0044] I. Flow cytometry analysis showed that IL-5 + IL-13 + Proportion.

[0045] J. Secretion of IL-5 and IL-13 in ILC2 culture supernatant.

[0046] P values ​​were calculated by Student's t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; n = 8; experiments were performed twice independently.

[0047] Figure 3.3 Low molecular weight heparin was shown to inhibit the maturation and function of IL-33 in a fungal-mediated lung injury model, where:

[0048] A. ELISA was used to detect the IL-33 concentration in the bronchoalveolar lavage fluid of the LMWH-treated group and the control group 1 hour after A. alternata-induced lung injury.

[0049] B. qRT-PCR was used to detect the gene expression of IL-33 in the lungs 1 h after papain induction. The data were processed using the 2-△△Ct method with actin as a reference and statistically analyzed.

[0050] C. IL-33 protein in the freeze-dried bronchoalveolar lavage fluid after 1 hour of A. alternata induction was separated by SDS-PAGE, and the mature IL-33 levels in the control and LMWH-treated groups were detected by immunoblotting using endogenous mouse IL-33 antibody (R&D).

[0051] D. Statistical analysis of the mature proportion and mature / full-length ratio of IL-33 protein in the control group and LMWH-treated group in C. Proteins of different sizes were quantified using ImageJ software.

[0052] E. Primary ILC2 cells were cultured in BALF from mice induced with A. alternata for 1 h and treated with LMWH, ± mouse IL-33 neutralizing antibody (10 μg / ml). After 3 days of culture, the IL-5 and IL-13 concentrations in the culture supernatant were detected by ELISA and statistically analyzed.

[0053] F.ELISA was used to detect the concentration of thrombin-antithrombin complex in bronchoalveolar lavage fluid after 1 hour of lung injury induced by the fungus A. alternata.

[0054] G. IL-33 protein in the freeze-dried bronchoalveolar lavage fluid after 1 hour of A. alternata induction was separated by SDS-PAGE, and the mature IL-33 levels in the control and argatroban-treated groups were detected by immunoblotting using endogenous mouse IL-33 antibody (R&D).

[0055] H. Statistical analysis of the mature proportion and mature / full-length ratio of IL-33 protein in the control group and Argatroban-treated group in G. Proteins of different sizes were quantified using ImageJ software.

[0056] P values ​​were calculated by Student's t-test (A / B / E) or one-way ANOVA with Tukey's multiple comparisons test; *, P < 0.05; **, P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant; the experiment was performed twice independently.

[0057] Figure 3.4 Thrombin directly cleaves human full-length IL-33 by binding to specific sites, where:

[0058] AB. Western blots were used to examine the cleavage of full-length human IL-33 (C-terminally HA-tagged) expressed by thrombin in rabbit reticulocytes (RRL). The primary cleavage forms were ~20 kD and ~30 kD. The cells were treated with either DMSO or the direct thrombin inhibitor argatroban (50 μg / ml). Reaction conditions were: A. Thrombin activity gradient (0.01875 / 0.0375 / 0.075 / 0.15 / 0.3 U) in 15 μl of thrombin buffer, incubated at room temperature for 1 hour; B. Thrombin incubation time gradient (15 min / 30 min / 1 h / 2 h / 4 h), with 0.075 U of enzyme activity in 15 μl of thrombin buffer.

[0059] C. Western blot detection of WT, Arg48-His (R 48 H) and Arg106-His(R 106 H) Thrombin cleavage of three types of human full-length IL-33, thrombin activity 0.0375 / 0.075U incubated at room temperature for 1 hour. RRL-pro-IL-33 (or IL-33 FL ) was approximately 50 pg in each system, and protein quantification was performed using ELISA (total IL-33) and Western blot (HA).

[0060] DF. Detection of thrombin-cleaved RRL-IL-33 using an ILC2 in vitro culture system FL -WT, R 48 H and R 106 To investigate the biological activity of H, human IL-33 neutralizing monoclonal antibody was added during culture, and the levels of IL-5 and IL-13 produced by ILC2 activated by IL-33 enzymatic hydrolysis products were detected by ELISA.

[0061] G. Detection of different forms of RRL-hIL-33 (IL-33) in ILC2 in vitro culture system FL IL-33 49-270 IL-33 107-270 IL-33 112-270 ) biological activity, and ELISA was used to detect the levels of IL-5 and IL-13 produced by ILC2 after activation of different forms of IL-33. The culture conditions were 50 pg RRL-hIL-33 FL Primary ILC2s (selected from IL-33-induced mouse model, 5000 cells / well) were stimulated with enzymatic hydrolysate (thrombin 0.075U incubated at room temperature for 1 hour and then serum terminated) or different forms of RRL-hIL-33, and cultured with IL-2 and IL-7 for 18 hours.

[0062] H. Western blot detection of RRL-hIL-33 FL - WT was cleaved synergistically by fungi (0 / 50 / 100 μg / ml) and thrombin.

[0063] I. Detection of RRL-hIL-33 Synergistically Cleaved by Fungus and Thrombin in an ILC2 In Vitro Culture System FL -WT biological activity, and ELISA was used to detect the levels of IL-5 and IL-13 produced by ILC2 activation after IL-33 enzymatic hydrolysis products.

[0064] J. Western blot detection of RRL-hIL-33 FL -R 106 H was cleaved synergistically by fungi (0 / 50 / 100 μg / ml) and thrombin.

[0065] K.ILC2 in vitro culture system to detect RRL-hIL-33 cleaved by fungi and thrombin FL -R 106 The biological activity of H was detected by ELISA, and the levels of IL-5 and IL-13 produced by IL-33 enzymatic hydrolysis products after activating ILC2 were detected.

[0066] P values ​​were calculated by one-way ANOVA with Tukey's multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; experiments were performed independently three times.

[0067] Figure 3.5 Bivalirudin was shown to inhibit ILC2 responses and inflammatory responses in both the papain-induced model and the A. alternaria model, where:

[0068] A. Schematic diagram of papain-induced lung inflammation model and treatment methods.

[0069] B. Flow cytometric analysis of eosinophils (CD45 + CD11c - / lo SiglecF + ) Grouping and proportion.

[0070] C. Statistical analysis of the proportion and number of eosinophils in bronchoalveolar lavage fluid.

[0071] D. Statistical analysis of the number of macrophages and neutrophils in bronchoalveolar lavage fluid.

[0072] E. HE and PAS staining of lung tissue pathology sections (scale bar: 200 μm) and pathological scoring.

[0073] F.ELISA detection of IL-5 and IL-13 in bronchoalveolar lavage fluid, statistical analysis of the experimental group and the control group.

[0074] G. Flow cytometry analysis of ILC2 populations in pulmonary monocytes. Statistical analysis of the ratios of IL-5 and IL-13 in ILC2s from pulmonary monocytes stimulated with PMA, ionomycin, and BFA for 4 hours.

[0075] H. Schematic diagram of the lung inflammation model induced by the fungus A. alternaria and its treatment methods.

[0076] I. Statistical analysis of the proportion and number of eosinophils in bronchoalveolar lavage fluid.

[0077] J. Statistical analysis of the number of macrophages and neutrophils in bronchoalveolar lavage fluid.

[0078] K.ELISA was used to detect IL-5 and IL-13 in bronchoalveolar lavage fluid, and statistical analysis was performed in the experimental and control groups.

[0079] L. HE and PAS staining of lung tissue pathology sections (scale bar: 200 μm) and pathological scoring.

[0080] M. Flow cytometry was used to detect the number of ILC2 in lung monocytes.

[0081] N. The ratio of IL-5 and IL-13 in lung mononuclear cell ILC2s stimulated with PMA, ionomycin and BFA for 4 hours and statistical analysis.

[0082] P values ​​were calculated by Student's t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; n = 6-7; experiments were performed twice independently.

[0083] Figure 3.6 Bivalirudin was shown to suppress type II immune responses and inflammatory reactions in a dust mite-induced asthma model, where:

[0084] A. Starting from day 1, mice were sensitized with 30 μg of HDM by intrabronchial immunization for 3 consecutive days. Starting from day 14, mice were challenged with 6 μg of HDM antigen for 4 consecutive days. Bivalirudin (0.25 mg / kg) was administered simultaneously with HDM by intrabronchial immunization.

[0085] B. Pulmonary function test of mice lung resistance.

[0086] C. Flow cytometry was used to detect the proportion of eosinophils in bronchoalveolar lavage fluid and statistically analyze the results.

[0087] D.ELISA was used to detect the concentrations of IL-5 and IL-13 in bronchoalveolar lavage fluid.

[0088] E. Flow cytometry was used to detect the number of macrophages and neutrophils in bronchoalveolar lavage fluid.

[0089] F. HE and PAS staining of lung histopathological sections (scale bar, 200 μm).

[0090] G. Flow cytometry detection of IL-5 after 4 hours of PMA, ionomycin and BFA stimulation + and IL-5 + IL-13 + The proportion of ILC2 cells in the lungs.

[0091] H.ELISA was used to detect serum IgE and HDM-specific IgG1.

[0092] I. Primary Th2 cells (ST2) isolated from HDM model cultured with IL-33 after thrombin cleavage + The levels of IL-5 and IL-13 produced by ILC2 activated by IL-33 enzymatic hydrolysis products were detected by ELISA.

[0093] P values ​​were calculated by Student's t-test. *, P < 0.05; **, P < 0.01; *** P < 0.001; **** P < 0.0001. The experiment was repeated twice independently.

[0094] Figure 4 Shown is a hypothetical model of thrombin-mediated IL-33 maturation and II inflammatory responses. DETAILED DESCRIPTION

[0095] The inventors of the present invention have found through extensive research that:

[0096] 1. Low molecular weight heparin (LMWH), an indirect inhibitor of thrombin, can inhibit papain- or fungal-induced type II immune responses by inhibiting IL-33 maturation and activation.

[0097] 2. By analyzing potential thrombin protease cleavage sites, it was found that IL-33 can be directly cleaved by thrombin at specific amino acid sites (human 48R and 106R) to generate a mature form with higher biological activity.

[0098] 3. Bivalirudin, a direct inhibitor of thrombin, can also inhibit various type II mouse lung inflammation models, such as HDM and OVA-induced lung allergic asthma models.

[0099] Based on the above research results, the present invention first provides the use of thrombin inhibitors in the preparation or screening of products for preventing and treating allergic diseases.

[0100] In the present invention, the definition of "inhibitor" refers to an agent, treatment or intervention that can directly or indirectly lead to a decrease in thrombin expression, activity or function, including, for example, a decrease in expression, a decrease in activity, an inhibitory change in activity time and / or site, or another inhibitory control of activity. Other types of inhibition are also included.

[0101] The thrombin inhibitor is selected from one or more of direct thrombin inhibitors, indirect thrombin inhibitors, vitamin K antagonists or factor Xa inhibitors.

[0102] The direct thrombin inhibitor is selected from dabigatran etexilate, bivalirudin, argatroban, recombinant hirudin (lepirudin) and the like.

[0103] In a preferred embodiment, the direct thrombin inhibitor is bivalirudin, which can inhibit HDM- and OVA-induced pulmonary allergic asthma.

[0104] The indirect thrombin inhibitor is selected from unfractionated heparin or low molecular weight heparin (LMWH). Unfractionated heparin is a mixture with a molecular weight range of 3 to 30 kDa. The low molecular weight heparin has a molecular weight range of 1 to 10 kDa. Examples of low molecular weight heparins include enoxaparin, dalteparin, nadroparin, and bemiparin.

[0105] Low molecular weight heparin can inhibit type II inflammatory response by inhibiting IL-33 maturation and / or activation, thereby achieving the purpose of preventing and treating allergic diseases. In certain embodiments of the present invention, the inhibition of IL-33 maturation or activation refers to the inhibition of IL-33 cleavage. Specifically, the inhibition of IL-33 maturation or activation refers to the inhibition of IL-33 cleavage to produce a mature form. More specifically, the inhibition of IL-33 maturation or activation refers to the inhibition of IL-33 cleavage at the human 48R and / or 106R amino acid sites to produce a mature form. The mature form refers to the IL-33 precursor form produced and secreted by cells, which is processed into a biologically active form. The mature form has a 10-30-fold increase in biological activity compared to the precursor form. "Maturation" refers to the process by which the IL-33 precursor form is processed to form the mature form.

[0106] The vitamin K antagonist (VKA) is, for example, warfarin.

[0107] The factor Xa inhibitor is selected from rivaroxaban, apixaban, edoxaban, fondaparinux sodium, etc.

[0108] In the present invention, the term "prevention" includes preventive treatment that can lead to the desired pharmaceutical and / or physiological effects. Preferably, the effect is to medically block or delay the occurrence of the disease and / or reduce the risk of disease development or worsening.

[0109] As used herein, the term "treatment" includes curative or palliative treatment that results in the desired pharmaceutical and / or physiological effect. Preferably, the effect is a medical reduction in one or more symptoms of the disease or a complete elimination of the disease.

[0110] In the present invention, the allergic disease refers to an allergic disease associated with IL-33 maturation.

[0111] The allergic disease is selected from food allergy, pulmonary allergic disease or atopic dermatitis.

[0112] The allergic disease is characterized by type II inflammatory response as one of its main features.

[0113] In certain embodiments of the present invention, the pulmonary allergic disease is selected from allergic asthma, allergic rhinitis, allergic lung infection or chronic sinusitis with nasal polyps.

[0114] The present invention discovered a close link between thrombin and the type II inflammatory response mediated by ILC2 cells in allergic lung diseases such as asthma. Specifically, thrombin cleaves IL-33, thereby increasing IL-33 activity and promoting IL-33-mediated ILC2 cell activation and lung inflammatory responses. Thrombin inhibitors inhibit thrombin, thereby controlling IL-33 activity and ILC2 cell-mediated inflammatory responses to achieve the purpose of treating allergic diseases.

[0115] The allergic diseases described in the present invention are allergic diseases confirmed after medical diagnosis.

[0116] The product must include a thrombin inhibitor and use the thrombin inhibitor as an active ingredient for the aforementioned efficacy.

[0117] In the product, the active ingredient that performs its function may be only a thrombin inhibitor, or may contain other substances.

[0118] That is, the thrombin inhibitor is the only active ingredient or one of the active ingredients of the product.

[0119] The product can be a single-component substance or a multi-component substance.

[0120] The form of the product is not particularly limited and can be in the form of solid, liquid, gel, semi-fluid, aerosol or other substances.

[0121] The product is mainly targeted at mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0122] The products include but are not limited to medicines, health products, food, etc.

[0123] In certain embodiments of the present invention, the product is a drug, which further includes a pharmaceutically acceptable carrier or excipient.

[0124] "Pharmaceutically acceptable" means that the drugs do not produce adverse, allergic or other untoward reactions when properly administered to animals or humans.

[0125] "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the efficacy of the drug under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0126] The present invention also provides the use of a thrombin inhibitor in preparing or screening a product having any one or more of the following functions:

[0127] 1) Inhibit IL-33 maturation and / or activation;

[0128] 2) Inhibit the activation of ILC2 cells;

[0129] 3) Inhibit type II inflammatory response.

[0130] Inhibiting IL-33 maturation or activation refers to inhibiting IL-33 cleavage. Specifically, inhibiting IL-33 maturation or activation refers to inhibiting IL-33 cleavage to produce the mature form. More specifically, inhibiting IL-33 maturation or activation refers to inhibiting IL-33 cleavage at amino acid sites 48R and / or 106R of human origin, or amino acid sites 50R and 87R of mouse origin, to produce the mature form.

[0131] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0132] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0133] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0134] The experimental materials used in the embodiments of the present invention are as follows:

[0135] 1. Experimental Animals and Cells

[0136] The mice used in the experiments were of a C57BL / 6 genetic background. Wild-type mice were purchased from Shanghai Lingchang Co., Ltd. IL-33KO mice were generated by the Shanghai Southern Model Animal Center, and Rag1KO mice were bred in-house. All experimental mice were kept between 8 and 10 weeks old. Mice were housed and the model was induced at the SPF-grade animal facility of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All animal experiments were approved by the Animal Care Committee of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and performed in accordance with the National Institutes of Health (NIH) Guidelines for the Use of Laboratory Animals.

[0137] 293T cell lines were purchased from the Central Cell Bank.

[0138] 2. Allergens, enzymes and cytokines

[0139] The allergens used in the experiment include papain, fungal component Alternaria, dust mite extract HDM and ovalbumin OVA. Related enzymes include thrombin, collagenase and various tool enzymes for cloning construction such as restriction endonucleases. Cytokines and other proteins include human / mouse recombinant proteins mature-IL-33, IL-2 and IL-7 used in primary cell culture and animal model induction, as well as recombinant histones H2A, H2B and H3.

[0140] Papain was purchased from Sigma, and the fungal component Alternaria was purchased from Beijing Broad Biotechnology Co., Ltd. The two protease allergens were mainly used to induce ILC2 cell-mediated type II immune response and in vitro IL-33 cleavage experiments; dust mite extract HDM was purchased from GREER, and ovalbumin OVA was purchased from Sigma. The two allergens were mainly used to induce mouse asthma models; thrombin was purchased from MCE and used for in vitro IL-33 cleavage experiments; collagenase I for tissue digestion was purchased from Invitrogen, and commonly used restriction endonucleases were purchased from Takara; the cytokines used to culture primary cells include IL-2, IL-7, and IL-33, mouse IL-2 and IL-7 were purchased from Peprotech, mouse mature-IL-33 was purchased from Biolegend, human IL-2 and IL-7 were purchased from Peprotech, human mature-IL-33 was purchased from R&D, and recombinant histones H2A, H2B, and H3 were purchased from abcam.

[0141] 3. Antibodies

[0142] The antibodies used in the experiment are mainly divided into fluorescein-conjugated flow cytometry (IF) antibodies, non-fluorescein-conjugated immunoblotting (WB) antibodies, and cell culture neutralization (Neu) antibodies. The specific antibodies are as follows:

[0143] Table 1 Information of major antibodies

[0144]

[0145]

[0146] 4. Other main reagents

[0147] Reagents for mouse tissue processing and cell culture: RPMI 1640 medium and fetal calf serum (FCS) were purchased from ThermoFisher; double antibodies (penicillin-penicillin), glutamine, and β-mercaptoethanol were purchased from Gibco BRL; cell separation medium Percoll was purchased from GE Healthcare; cell stimulators PMA and ionomycin were purchased from Sigma-Aldrich; Golgi transport blocker was purchased from BD Pharmingen; 1xPBS was purchased from Gibco, and 20xPBS was purchased from Shanghai Biotechnology Co., Ltd.; Ficoll-Paque PLUS for human peripheral blood PBMC separation was purchased from GE Healthcare.

[0148] Mouse model induction-related reagents: anticoagulant low molecular weight heparin (LMWH, Enoxaparin) was purchased from Selleck; direct thrombin inhibitor Bivalirudin (TFA) was purchased from MCE; aluminum adjuvant was purchased from Thermo Fisher; anesthetic isoflurane was purchased from Reward.

[0149] Reagents related to the mRNA detection experiment: TRizol for cell lysis was purchased from Invitrogen; isopropanol, chloroform, anhydrous ethanol and glycogen for RNA extraction were purchased from Sigma; reverse transcription kit was purchased from Takara; SYBPGREEN was purchased from Toyobo.

[0150] Flow cytometry staining related reagents: Cell viability dye (Fixable Viability Stain-780 / 450) was purchased from BD Biosciences; cell apoptosis staining kit (Annexin V-FITC / PI Apoptosis Detection Kit) was purchased from Vazyme; nuclear transcription factor staining buffer (Foxp3 / Transcription Factor Staining Buffer) was purchased from ebioscience.

[0151] 5. Related kits

[0152] The mouse lineage-negative cell enrichment kit (EasySep Mouse ILC2 Enrichment kit) was purchased from Stem Cell Company and was used to enrich lung lineage-negative monocytes, thereby increasing the proportion of ILC2 cells and facilitating sorting.

[0153] Enzyme-linked immunosorbent assay (ELISA) detection kits included: mouse IL-4 / IL-5 / IL-13 / TSLP detection kits and human IL-5 / IL-13 detection kits were purchased from eBioscience, mouse IL-33 / IL-25 and human IL-33 detection kits were purchased from R&D, and mouse IgE / IgG1 / IgG2c detection reagents were purchased from BD.

[0154] Human and mouse TAT complex (thrombin-anti-thrombin complex) detection kits were purchased from Abcam.

[0155] 1. Implementation

[0156] 1. Induction of Mouse Inflammation Model

[0157] Papain-induced acute lung inflammation model: 8-10 week old female mice were selected and anesthetized with isoflurane via an anesthesia machine. After anesthesia, 40 μl of 1xPBS containing 4 μg of papain was instilled through the bronchus. The same treatment was given at a fixed time point every day for 5 consecutive days. On the 6th day, the mice were euthanized, and the bronchoalveolar lavage fluid (BALF) of the mice was isolated to detect the infiltration of inflammatory cells such as eosinophils and the production of cytokines IL-5 and IL-13. At the same time, lung tissue was isolated to detect the activation and proliferation of ILC2 cells. Lung tissue pathology sections were stained to observe inflammatory cell infiltration and goblet cell mucus secretion. In an experiment on the effects of the anticoagulant drug low molecular weight heparin (LMWH, Enoxaparin) and the direct thrombin inhibitor Bivalirudin (Bivalirudin TFA) on type II pulmonary inflammation, drug intervention was given through the bronchus at the same time as daily allergen immunization. The dose of low molecular weight heparin was 2.5 mg / kg and the dose of bivalirudin was 0.25 mg / kg.

[0158] Fungal-Induced Acute Lung Inflammation Model: To establish a fungal A. alternaria-induced airway inflammation model, mice were immunized intrabronchially for four consecutive days at a dose of 4 μg. Twenty-four hours after the final immunization, mice were sacrificed and samples collected for lung inflammation testing as described above. Similarly, intervention with the anticoagulants low molecular weight heparin (LMWH, enoxaparin) and bivalirudin (TFA) was administered intrabronchially concurrently with daily fungal immunizations, using the same doses as in the papain model.

[0159] IL-33-induced acute lung inflammation model: For IL-33-induced airway inflammation, recombinant mouse cytokine IL-33 (50 ng dissolved in 40 μl 1x PBS) was administered intrabronchially to mice on days 0, 1, and 2. Twenty-four hours after the final immunization, mice were sacrificed, and bronchoalveolar lavage fluid and lung tissue were collected for inflammatory assays. Low-molecular-weight heparin (LMWH) was administered intrabronchially daily with IL-33 at the same dose as in the papain model.

[0160] Dust mite HDM-induced allergic asthma model: After mice were anesthetized with isoflurane, 30 μg of dust mite HDM was administered intrabronchially on days 0, 1, and 2, respectively. Antigen challenge was carried out for 4 consecutive days starting on day 13, with each mouse being given 6 μg of dust mite HDM every day. The test was performed 24 hours after the last immunization, and venous blood was collected to detect serum immunoglobulins IgE, IgG1, and IgG2c. The alveolar lavage fluid was collected to detect the number of eosinophils, and the lung tissue pathology sections were stained. Single-cell suspensions of mediastinal lymph nodes (MeLN) and lung tissues were prepared, and ILC2 and Th2 cells were detected by flow cytometry. At the same time, the remaining cells were re-stimulated with HDM to detect the ability of T cells to secrete cytokines such as IL-4, IL-5, and IL-13.

[0161] Ovalbumin (OVA)-induced allergic asthma model: To construct an OVA-induced allergic asthma model, 20 μg of OVA and 1 mg of aluminum adjuvant were thoroughly mixed in 200 μl of 1xPBS per mouse. The mice were immunized by intraperitoneal injection on days 0 and 14, respectively. Starting from day 21, the mice were attacked with 10 mg / ml of OVA by spray for 5 consecutive days. The mice were tested 24 hours after the last immunization, and the test indicators were the same as those of HDM-induced allergic asthma.

[0162] 2. Mouse Lung Tissue Processing and Cell Suspension Preparation

[0163] To isolate primary cells from the lung tissue and mediastinal lymph nodes of 8-10 week old female mice on a C57 / BL6 background, the mice were first euthanized by CO2. The thoracic cavity was opened and the trachea was exposed. 500 μl and 800 μl of ice-bathed PBS were slowly instilled into the lungs twice using a 1 ml syringe equipped with an 18G needle. The supernatant of the first lavage and the total infiltrating cells of the two lavages were separated by centrifugation at 500 g for 3 min each. While puncturing the left atrial appendage, 10 ml of PBS was rapidly perfused from the right atrium with a syringe to drain the pulmonary venous blood of the mouse. The pulmonary mediastinal lymph nodes and lungs were removed and washed separately in 1xPBS in an ice bath. The pulmonary mediastinal lymph nodes can be directly placed in the wells of a 24-well plate with RPMI 1640 culture medium (2% FBS). The tissue is gently pressed with the piston head of a 1 ml syringe to fully release the cells, collect them into a suspension and count them; the lung tissue needs to be minced with scissors, collected with 3 ml of digestive solution (final concentration of 0.5 mg / ml collagenase I dissolved in RPMI-1640 culture medium containing 10% FCS and 1% double antibody), and then placed in a 37°C shaker for digestion for 30 minutes. The digested tissue was placed on a 70 μm filter membrane in a 6-well plate, and fresh culture medium was added. The tissue was ground with the piston of a 5 ml syringe and centrifuged at 500 g for 5 min. The cell clumps were collected and resuspended in 40% Percoll, a cell separation buffer, and gently added to the upper layer of 80% Percoll. The middle layer of pulmonary mononuclear cells was isolated and collected by density gradient centrifugation (600 g for 20 min, 5% increase and 0 decrease). The cells were washed with 10 ml of RPMI-1640 containing 2% FCS, centrifuged, and resuspended to obtain a pulmonary mononuclear cell suspension for subsequent detection.

[0164] 3. Flow Cytometry Staining

[0165] Cell surface staining method: Count the prepared alveolar lavage cells, mediastinal lymph node single cell suspension or lung mononuclear cell suspension using a cell counter, and take no more than 3×10 6The single-cell suspension of cells was placed in a 5 ml flow cytometry centrifuge tube (BD falcon). After centrifugation, the supernatant was removed and the cells were washed with 1xPBS. Then, FixableViability Dye was added at a dilution ratio of 1:1000 and stained on ice for 15 minutes to remove the interference of dead cells on flow cytometry detection. Then, 2 ml of cell surface staining buffer (1xPBS solution containing 2% FBS and 2 mM EDTA) was added for washing. The tube was centrifuged at 350 g at 4°C for 5 minutes. The supernatant was carefully removed and the cells were gently flicked to mix. The next step of cell surface marker staining (non-specific aggregation was blocked with Fc blocker at the same time) was carried out. The tube was stained on ice for about 30 minutes. The tube was washed with 2 ml of Stain Buffer and centrifuged at 350 g at 4°C for 5 minutes. The supernatant was carefully removed and the cells were gently flicked to mix. An appropriate amount of Stain Buffer was added to re-select the cells. After filtration, the cells can be tested on the machine. For ILC2 / Th2 detection in MeLN and lung monocytes, lineage markers (CD3, CD5, CD45R, CD11b, CD11c, NK1.1, Gr-1, TER119, FcεR1, and TCRγ / δ) were used to distinguish Lin. - cells, among which CD90.2 (Thy1.2), CD127, KLRG1 and ST2 (IL-33R) are highly expressed markers of ILC2 cells, while Lin + CD4 + ST2 + For Th2 cells.

[0166] Intracellular cytokine staining: Stimulate cells with 50 ng / ml PMA and 1 μg / ml ionomycin for 4-6 hours. Add 1 μg / ml of the Golgi transport inhibitor BFA for the last 2 hours. Collect treated cells, perform live / dead staining and cell surface staining, then wash with Stain Buffer. Fix and transfect cells using the eBioscience intracellular staining kit and incubate on ice for 30 minutes. Dilute 10x Perm buffer with ddH2O. After fixation, wash cells with 1x Perm buffer. Add intracellular cytokine antibodies to the fixed and transfected cells and stain on ice for 30 minutes. After staining, wash cells with 1x Perm buffer, reselect cells with Stain Buffer, and filter before analysis.

[0167] Nuclear transcription factor staining: For cell surface staining, use the eBioscience Foxp3 Staining Buffer Set permeabilization staining kit to stain for nuclear transcription factors. After the final cell surface staining step, wash the cells once with 1xPBS. Add 100μl of Foxp3 Solution A (prepared with Black Bottle Buffer at a 1:3 ratio of diluent) and fix and permeabilize the membrane at 4°C for 30 minutes. Then, add 2ml of 1xFoxp3 Solution B to wash the cells. Centrifuge at 500g for 5 minutes at 4°C, add the transcription factor antibody, and stain in the dark for 30 minutes on ice. After staining, wash the sample once with 2ml of 1xFoxp3 Solution B. Add the appropriate amount of Stain Buffer and analyze.

[0168] All flow cytometric data were read by BD LSR Fortessa or Beckman CytoFLEX3 flow cytometers and analyzed using FlowJo 10 software.

[0169] 4. Enrichment of Mouse Lineage-negative Cells and Flow Cytometry Isolation of ILC2 Cells

[0170] Lineage-negative cell enrichment method: ILC2 cells were isolated using the EasySep Mouse ILC2 Enrichment Kit. The specific steps are as follows: resuspend lung mononuclear cells in PBS containing 2% FBS to 1x10 8 cells / ml and transfer to a 5ml flow cytometry tube. Add 50μl of cocktail Ab to each tube, pipette evenly, and let stand at room temperature for 5 minutes. Vortex the beads for 30 seconds, add 75μl of beads to each tube, pipette evenly, and let stand at room temperature for 5 minutes. Add PBS containing 2% FBS to each tube, make up to 2.5ml, pipette evenly, and insert the sorting magnet. Let stand at room temperature for 3 minutes. Pour the supernatant into a 15ml centrifuge tube. Repeat once to collect the cells.

[0171] Flow cytometry: After enrichment with the EasySep Mouse ILC2 Enrichment kit, wash the cells with 1xPBS and then add Fixable Viability Dye to stain the cells for viability and remove the interference of dead cells. Then, perform antibody surface staining. The main antibodies are lineage cocktail antibody, CD90.2, ST2 and CD45.2. After staining, resuspend the cells in RPMI 1640 medium containing 1% FBS and 1mM EDTA, filter through a 0.45μm filter and then sort the cells for CD45.2. + Lin- CD90.2 + ST2 + The entire staining and sorting process of ILC2 cells was performed aseptically. Cell sorting instruments mainly used were BD bioscience's Aria II and Aria Sorp flow cytometry sorters.

[0172] 5. Culture of Primary Mouse ILC2 and Th2 Cells

[0173] ILC2 cell culture: For mice with IL-33-induced lung inflammation, lung tissue mononuclear cells were first isolated and CD45 mononuclear cells with a purity of more than 95% were sorted according to the ILC2 enrichment and sorting method. + Lin - CD90.2 + ST2 + ILC2 cells were resuspended in RPMI-1640 medium containing 10% FCS and 1% double-antibody, and 3000-5000 cells were plated per well of a 96-well U-shaped plate. Cytokines 100U / ml hIL-2 and 20ng / ml mIL-7 were added. When studying the effects of drugs such as low molecular weight heparin Enoxaparin and Bivalirudin TFA on ILC2 cells, the activation factor 0.5ng / ml mature IL-33 should be added at the same time. After culturing for 3-5 days, the cells and supernatant were collected for flow cytometry and ELISA analysis, respectively.

[0174] Th2 cell culture: For HDM-induced allergic asthma model mice, the lung tissue mononuclear cells were first isolated and then surface stained to select CD45 Th2 cells with a purity of more than 95%. + CD4 + ST2 + Th2 cells were resuspended in RPMI-1640 medium containing 10% FCS, 1% double antibody, 1% glutamine and 1 / 25000 β-mercaptoethanol. 5000 cells were plated per well of a 96-well U-shaped plate. Cytokines 100U / ml hIL-2, 20ng / ml mIL-7 and corresponding experimentally treated IL-33 were added and cultured for 3-5 days. The cells and supernatant were collected for flow cytometry and ELISA analysis, respectively.

[0175] 6. Plasmid Construction and Protein Expression

[0176] The plasmid construction process used in the experiment was as follows: First, full-length cDNAs sequences with HA fusion tag sequences at the 3' end of the nucleic acid sequence were synthesized, namely human-IL-33 (NM_033439) and mouse-IL-33 (NM_133775). Based on the full-length sequences, truncations or single-point amino acid mutations were PCR amplified to generate different mutant forms required for the study, including human-IL-33 FL -WT / R 48 H / R 106 H / IL-33 49-270 / IL-33 107-270 and mouse-IL-33 FL -WT / R 50 H / R 50 Q / R 51 H / R 87 H / IL-33 51-266 / IL-33 87-266 , Each amplified fragment was constructed into the pcDNA3.1 (Invitrogen) vector.

[0177] The protein expression of wild-type (WT) and mutant (MUT) IL-33 was performed using the rabbit reticulocyte lysate system RRL (TNT-T7 kit from Promega), and the protein products were quantified by immunoblotting (WB) combined with IL-33 ELISA kit detection.

[0178] 7. Enzyme cleavage reaction and activity detection of IL-33

[0179] Enzymatic digestion system: 50 / 100 pg (0.5 / 1 μl) of full-length IL-33 protein in vitro translated by the RRL system was mixed with 0.01875-0.3 U of thrombin in a 15 μl reaction solution (20 mM Tris pH 8.4, 150 mM NaCl, 2.5 mM CaCl2) and incubated at room temperature for 1 hour. The thrombin inhibitor argatroban (50 μg / ml) was added or not. The products were then used for Western blotting and primary cell viability assays. For other allergens with protease activity, papain (0.625-1.25 μg / ml) and fungal enzymes (25-100 μg / ml) were used to digest RRL-IL-33 in 15 μl of PBS at 37°C for 5 minutes.

[0180] IL-33 activity detection: Use the sorted primary cells ILC2 and Th2 as effector cells. After adding IL-2 and IL-7 (Th2 setting with or without anti-CD3 and anti-CD28 to activate TCR signaling) as cell proliferation maintenance factors and culturing for 1-3 days, add RRL-IL-33 treated with different forms and methods and culture for 18 hours. Then, detect the cytokines IL-5 and IL-13 in the supernatant (IL-4 is also detected for Th2) as a basis for judging the strength of IL-33 activity.

[0181] 8. Enzyme-linked immunosorbent assay (ELISA) for cytokine detection

[0182] To detect the severity of lung inflammation in the mouse model and the activation of ILC2 cells cultured in vitro, we used IL-4, IL-5, IL-13, IL-33, IL-25, and TSLP ELISA detection kits from eBioscience or R&D. We first coated 96-well plates (Nunc) with our own coating buffer (antibody coating buffer: NaHCO3 0.738g, Na2CO3 0.397g, add water to 250ml, pH = 9.5) overnight (4°C). The next day, we blocked the plates with 1% BSA in 1xPBS for 1 hour. At the same time, we diluted the collected samples to the appropriate concentration and completed the dilution of the standards. The plates were then washed three times with 1xPBS containing Tween-20. The samples and standards were added to the corresponding wells of the 96-well plates. After incubation at room temperature for 2 hours, the plates were washed three times with 1xPBS containing Tween, and horseradish peroxidase (HRP)-labeled secondary antibodies were added. Antibody), continue incubation at room temperature for 1-2 hours, wash the plate three times with 1xPBS containing Tween-20, add HRP, incubate at room temperature for 40 minutes, wash the plate five times with 1xPBS containing Tween-20, add the color development solution in the kit, and start the color reaction. After timely termination with 2M concentrated sulfuric acid, read the OD value at 450nm using a microplate reader.

[0183] 9. Pathological tissue section staining

[0184] Lung tissue from the mouse lung inflammation model was fixed in 4% formaldehyde overnight. After 48 hours, it could be embedded and sliced.

[0185] First, fix the tissue sample: gently remove the lung tissue from the lung inflammation model mouse to avoid artificial tissue damage, wash it in PBS, and then place it in a 4ml EP tube containing 3ml formaldehyde. Use a cotton ball to plug the lung tissue into the formaldehyde and fix it overnight. After 24 hours, wait until the lung tissue sinks to the bottom of the EP tube and proceed to the next embedding operation.

[0186] Paraffin embedding and sectioning were then performed in a fume hood. Fixed lung tissue samples were placed in different concentrations of ethanol (50%, 70%, 80%, 95%, and 100%) for 1 hour each, followed by gradient dehydration. The samples were then placed in a 1:1 mixture of ethanol and xylene for 30 minutes. After removal, the samples were placed in xylene for 5-10 minutes (depending on the clarity of the tissue) to clear the tissue. Once the tissue was clear, it was removed and immersed in paraffin at 60°C overnight. The tissue was then sectioned the next day using a Leica microtome. The sections were 5 μm thick, dried at 42°C, and stored at room temperature.

[0187] Finally, HE staining was performed: xylene for 15 minutes twice, xylene: anhydrous ethanol = 1:1 for 2 minutes, 100% ethanol for 5 minutes twice, 80% ethanol for 5 minutes, distilled water for 5 minutes, hematoxylin staining for 5 minutes, washing away the hematoxylin with running water for 5 minutes, 1% hydrochloric acid ethanol for 1-3 seconds, washing with water for 10-30 seconds, washing with distilled water for 1-2 seconds, and staining with 0.5% eosin solution for 1-3 minutes.

[0188] PAS staining was performed at the same time: paraffin sections were dewaxed, rinsed with distilled water, washed with 70% ethanol, immersed in periodic acid alcohol solution for 10 minutes (17-20°C), washed with 70% ethanol, added to reducing solution for 1 minute (17-20°C), washed with 70% ethanol, added to colorless fuchsin solution for 1-1.5 hours (placed in a 37°C incubator), rinsed with running water for 10 minutes, counterstained with hematoxylin counterstain solution for 3-5 minutes, and then differentiated with 1% hydrochloric acid alcohol, rinsed with running water, dehydrated, transparent, and sealed.

[0189] The final tissue sections were scanned using a Zesis 60 instrument at 20× magnification, and the images were analyzed using Zesis software.

[0190] 10. RNA Extraction and Reverse Transcription

[0191] RNA extraction: Phenol / chloroform extraction was used. First, cells were collected into RNase-free 1.5ml EP tubes, centrifuged, supernatant removed, appropriate amount of TRizol was added and vortexed to fully lyse the cells. Lysed cells can be directly used for the next step of extraction or frozen in a -80℃ refrigerator and extracted together after all samples were collected. 200μl chloroform was added to the sample and vortexed to mix thoroughly. After mixing, the sample was allowed to stand on ice for 10 minutes and centrifuged at 4℃ 13000rpm for 30 minutes. After the centrifugation, the solution was separated into 3 layers: the supernatant layer (containing RNA), the middle layer (containing protein) and the organic layer. RNase Gently aspirate the supernatant aqueous phase with a free pipette tip (be careful not to aspirate the middle layer), add an equal volume of ice-cold isopropanol solution and 5μl glycogen, gently invert and mix, and place in -20℃ for precipitation for more than 1 hour; wash the precipitated RNA once with 75% ethanol (prepared with DEPC water), remove the supernatant, air-dry, add appropriate amount of DEPC water, place in a 4℃ refrigerator to dissolve, then measure the concentration and store in a -80℃ refrigerator.

[0192] Reverse transcription: TAKARA's PrimeScript RT Master Mix Perfect Real Time Kit (Cat#RR036A) was used. The reaction system was as follows:

[0193] Table 2.2 Reverse transcription reagent preparation

[0194]

[0195] Mix the mixture containing RNA and centrifuge briefly to allow the reaction solution to settle to the bottom of the PCR tube. Invert the reaction using a PCR instrument. Procedure:

[0196] Table 2.3 Reverse transcription procedures

[0197]

[0198] After PCR, 20 μl of ddH2O was added to each tube to dilute the cDNA and stored at -20°C.

[0199] 11. Real-time PCR

[0200] The SRBR PCR Master Mix (ABI) system was used to perform real-time fluorescence quantitative detection of the target molecule mRNA level on the cDNA obtained by reverse transcription. Three replicate wells were set up for each sample when detecting the target gene. The HPRT gene was used as an internal reference for experiments and analysis. Real-time fluorescence quantitative PCR used the SYBR Green method. The reagents were purchased from TaKaRa and the testing instrument was a QuanStudio6 Flex 384-well real-time fluorescence quantitative PCR instrument (Life Technologies). The data were analyzed using QuantStudio™ Real-Time PCR Software using 2 -△△Ct The reaction system is as follows:

[0201] Table 2.4 Real-time PCR reagent preparation

[0202]

[0203] 12. Data analysis and statistical processing

[0204] All experimental results were independently repeated at least three times, or with at least six independent samples per experiment. Significant differences were analyzed using two-tailed Student's t-test or one-way ANOVA. All statistical analyses were performed using Graphpad Prism software. Experimental results are presented as mean ± standard error. A p-value < 0.05 was considered significant. P < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) were considered significant.

[0205] 2. Example Results and Conclusions

[0206] 1. Low molecular weight heparin can inhibit type II inflammation mediated by ILC2 cells

[0207] This example uses models commonly used in the study of acute type II inflammation mediated by ILC2 cells—the mouse papain model and the fungal A. alternaria model. Compared to conventional chronic allergic asthma models, this acute injury model induces more direct damage and minimizes the involvement of Th2 cells, DCs, and mast cells in allergic reactions, making it more suitable for investigating the early regulation of IL-33 and ILC2 cell immune responses.

[0208] After the mice were modeled, the cells in the bronchoalveolar lavage fluid were stained by flow cytometry on the 6th day to detect the eosinophils (CD45 + CD11c - / lo SiglecF + )( Figure 3.1B), statistical analysis showed that LMWH could significantly inhibit the proportion and number of eosinophils in BALF ( Figure 3.1 C) but did not affect the number of macrophages and neutrophils ( Figure 3.1 D).

[0209] At the same time, the same lung area was cut and fixed with PFA, paraffin sections were made and stained with HE and PAS. Analysis of the HE sections showed that the LMWH-treated group had less inflammatory cell infiltration in the lungs than the control group ( Figure 3.1 E), analysis of PAS sections revealed that LMWH significantly inhibited the ability of goblet cells to secrete mucus ( Figure 3.1 E).

[0210] ELISA detection showed that the concentrations of IL-5 and IL-13 in BALF were significantly decreased after Enoxaparin treatment ( Figure 3.1 F).

[0211] Since ILC2 is the main source of IL-5 and IL-13 in the lungs, we isolated monocytes from the lungs and performed surface flow cytometry to detect ILC2 cells. We found that the number of ILC2 cells in lung monocytes was significantly reduced after Enoxaparin treatment ( Figure 3.1 G).

[0212] At the same time, in order to detect whether the immune response of ILC2 cells, that is, the ability to produce IL-5 and IL-13, was affected, the pulmonary monocytes were stimulated with stimulators PMA and ionomycin for 4-6 hours, and the Golgi transport inhibitor BFA was added for the last 2 hours. The cells were then collected and the expression of intracellular cytokines in ILC2 was detected by flow cytometry. It was found that compared with the control group, IL-13 in lung ILC2 was increased after Enoxaparin treatment. + Cells and IL-5 + IL-13 + The proportion of cells decreased significantly, and IL-5 + The proportion of cells also decreased to a certain extent ( Figure 3.1 HI).

[0213] The above experiments showed that LMWH inhibited the immune response and inflammatory response of ILC2 cells in the papain-induced mouse type II acute inflammation model.

[0214] Similarly, consistent conclusions were obtained in the fungus A. alternaria model ( Figure 3.1 JP).

[0215] 2. The inhibitory effect of low molecular weight heparin on type II inflammatory response depends on the IL-33 signaling pathway upstream of ILC2

[0216] In order to further verify whether the ability of LMWH to inhibit ILC2 response and type II inflammatory response depends on the IL-33 signaling pathway upstream of ILC2, the present invention directly uses the mouse recombinant protein IL-33114-266 to induce a mouse type II inflammatory model to observe whether the ILC2 cell response directly activated by mature active IL-33 can be inhibited by LMWH. WT mice were immunized with recombinant IL-33 bronchiolitis for 3 consecutive days and LMWH intervention was performed ( Figure 3.2 A), ILC2 response and lung inflammation were detected on day 4. It was found that in the type II inflammation model directly induced by mature IL-33, LMWH intervention had an effect on the number and proportion of eosinophils in BALF ( Figure 3.2 B) IL-5 / IL-13 concentrations in BALF ( Figure 3.2 D) and pulmonary inflammatory cell infiltration and mucus secretion ( Figure 3.2 E) had no significant effect. The number of ILC2 cells in the lungs ( Figure 3.2 F) and the IL-5 / IL-13 ratio in ILC2 ( Figure 3.2 G), and there was no obvious change. Therefore, the present invention believes that the inhibition of LMWH on type II acute lung inflammation may depend on the IL-33 signaling pathway, and the phenomenon that LMWH has no effect on the biological function of mature IL-33 in vivo can also be inferred that it may regulate the maturation of full-length IL-33 and other mechanisms to affect the IL-33 signal. Based on the previous mouse model experiments, it is speculated that the inhibitory function of LMWH on ILC2 may be indirectly through affecting IL-33. In order to further verify this point, the present invention conducted an in vitro culture experiment of ILC2 cells, that is, primary ILC2 cells from the lungs of mice in an IL-33 induced model (continuous immunization for 3 days, sorting on the 7th day) were sorted by flow cytometry, IL-2, IL-7 and IL-33 were added in vitro for culture, and Enoxaparin was used for treatment. The supernatant IL-5 and IL-13 concentrations were detected by ELISA for 3-5 days, and the cytokine expression of ILC2 was detected by flow cytometry. The results showed that LMWH treatment did not affect the number of ILC2 cells ( Figure 3.2 H), Flow cytometry analysis showed that IL-5 + IL-13 + Proportion( Figure 3.2 The secretion of IL-5 and IL-13 in the culture supernatant of ILC1 and ILC2 ( Figure 3.2 J) No significant change. The above experiments indicate that LMWH may indeed mediate ILC2 responses and lung inflammation by regulating IL-33 signaling.

[0217] 3. Low molecular weight heparin inhibits the maturation of endogenous IL-33 in mice during acute type II lung inflammation

[0218] To investigate whether LMWH inhibits the biological activity of endogenous IL-33 by affecting IL-33 maturation, a fungal-mediated lung injury model with mild protease activity was used. First, ELISA assays showed that the total IL-33 released into BALF 1 hour after fungal induction was not affected by LMWH ( Figure 3.3 A), qRT-PCR detection of IL-33 mRNA levels in lung homogenate also showed no significant changes ( Figure 3.3 B) To detect the maturation of endogenous IL-33, bronchoalveolar lavage fluid was lyophilized after fungal induction for 1 hour (and protease inhibitors were added to inhibit IL-33 degradation). Protein lysates were then prepared and separated by SDS-PAGE. Western blot analysis was then performed using a mouse endogenous IL-33 antibody to detect the distribution of bands of different sizes during the IL-33 maturation process. The results showed that compared with the control group, LMWH treatment significantly increased the amount of full-length IL-33 (pro-IL-33) at approximately 30 kD, while a corresponding decrease was observed in mature IL-33 (cleaved IL-33) at approximately 20 kD. Figure 3.3 C). Statistical analysis showed that the maturation of IL-33 was significantly inhibited by LMWH, that is, the ratio of activity to full length and the ratio of activity to total protein were significantly decreased ( Figure 3.3 D). At the same time, endogenous IL-33 activity detection found that the ability of BALF in the LMWH-treated group to activate ILC2 to produce IL-5 and IL-13 was significantly lower than that in the control group. This difference was eliminated after the addition of IL-33 neutralizing antibodies ( Figure 3.3 E). The results of the fungal and papain-induced lung injury models were consistent, that is, in protease-induced lung injury, LMWH did not inhibit the biological activity of endogenous IL-33 by affecting the expression or release of IL-33. The above functional and biochemical experimental results are consistent, indicating that LMWH does inhibit the biological activity of endogenous IL-33 by affecting the maturation of IL-33. In order to prove that the function of LMWH is related to thrombin, the thrombin-antithrombin complex (i.e., thrombin activity) in the fungal-induced lung injury and inflammation models was detected. The experiment found that thrombin activity was significantly increased after fungal induction ( Figure 3.3 F). At the same time, the thrombin activity in the alveolar lavage fluid after LMWH treatment was significantly lower than that in the control group ( Figure 3.3 F) In addition, the direct thrombin inhibitor argatroban can also inhibit the maturation of endogenous IL-33 ( Figure 3.3 The above experiments indicate that thrombin activity is positively correlated with the maturation of endogenous IL-33. Anticoagulants such as LMWH may inhibit IL-33 maturation and ILC2 activation-mediated type II acute lung inflammation by inhibiting thrombin activity.

[0219] 4. Thrombin can directly cleave and activate IL-33

[0220] The previous experiment of intervening in mouse lung injury models with anticoagulant drugs proved that endogenous thrombin may be related to the maturation and activity of IL-33. In order to further prove the mechanism by which thrombin regulates the maturation of IL-33, this example conducted an in vitro enzyme digestion experiment. First, a pcDNA3.1-human-pro-IL-33-3'HA plasmid was constructed, which can express the full-length human IL-33 protein with an HA fusion tag at the C-terminus. Since full-length IL-33 is a nuclear protein that is difficult to express and purify, rabbit reticulocyte lysate (RRL) was used for in vitro translation, and then incubated with different doses of thrombin for different times, and RRL-h-pro-IL-33 (also known as RRL-hIL-33) was detected by western blot. FL ) is cleaved by thrombin. Experiments have found that thrombin can cleave human full-length IL-33 into two main forms, a mature form of about 20kD and an intermediate form of about 30kD, and the increase in the mature form and the decrease in the intermediate form are thrombin dose-dependent ( Figure 3.4 A) and thrombin hydrolysis time dependence ( Figure 3.4 B). The related cleavage is completely inhibited by the direct thrombin inhibitor argatroban ( Figure 3.4 AB), indicating that thrombin can indeed directly cleave full-length IL-33.

[0221] Next, in order to explore the thrombin recognition and cleavage sites, the present invention predicted and analyzed the protein sequence of human full-length IL-33 based on the size of the cleavage band and the sequence specificity of the thrombin cleavage site, and speculated that the two cleavage sites may be R48 and R106. In order to verify the enzyme cleavage sites, the pcDNA3.1-human-pro-IL-33-3'HA plasmid was mutated so that the R at these two sites on the IL-33 protein expressed by RRL was mutated to H. Western blot detection of RRL-hIL-33 FL -WT / R 48 H / R 106 The results of thrombin cleavage of H showed that R48H lacked a 30kD intermediate form compared to WT after thrombin cleavage, while R106H lacked a 20kD mature form compared to WT ( Figure 3.4 C) The above experiments demonstrate that thrombin can directly cleave human full-length IL-33 by binding to specific sites.

[0222] In order to verify the biological functions of the two forms of IL-33 after thrombin cleavage, the present invention conducted a mouse primary ILC2 cell culture experiment. First, ELISA was used to detect the activation of IL-5 and IL-13 downstream of the ST2 signaling of ILC2 cells after full-length IL-33 was cleaved by thrombin. Compared with the control group without thrombin incubation, RRL-hIL-33 FL -WT can significantly activate the ability of ILC2 to secrete IL-5 and IL-13 after being processed by thrombin, and this enhancement is basically inhibited after the addition of IL-33 neutralizing antibodies ( Figure 3.4 D). Is 107-270 the main active form? The present invention also investigates R 48 H and R 106 The same experiment was conducted on two mutant proteins, and it was found that only the mutation at R106 significantly inhibited the activation of downstream ST2 signaling by IL-33 after thrombin cleavage ( Figure 3.4 F), while the thrombin cleavage product of the R48 mutation can still significantly activate ILC2 ( Figure 3.4 E) Simultaneously construct an intermediate form of IL-33 expressing thrombin cleavage 49-270 and mature form of IL-33 107-270 The plasmid was used to culture primary ILC2 with proteins expressed by the RRL system, and ELISA was used to detect the IL-5 and IL-13 produced by ILC2 activation. It was found that these two forms were similar to full-length IL-33. FL and the shortest active form of IL-33 114-270 In contrast, the activity of IL-33 protein increases with the decrease of amino acid sequence, and IL-33 107-270 than IL-33 49-270 Have stronger biological functions ( Figure 3.4 G). In order to explore whether thrombin and proteases mediating type II inflammatory response can synergistically regulate the process of IL-33 maturation and activation, the effects of the coexistence of exogenous protease A. alternata (cleavage site Q103) and thrombin (cleavage sites R48 and R106) on the cleavage of IL-33 and the ability to activate ILC2 cells in vitro were simulated in vitro. A. alternata and thrombin were combined at different concentrations to incubate RRL in vitro translated human IL-33 FL Western blots revealed that thrombin and A. alternata can synergize with each other to produce more mature IL-33, and the amount of mature IL-33 increases with increasing concentrations of the two proteases ( Figure 3.4 H). Compared with A. alternata alone, the combination of A. alternata and thrombin more strongly stimulated ILC2 to secrete type II cytokines IL-5 and IL-13 ( Figure 3.4I). However, when combined with IL-33 FL -R 106 When incubated with H, A. alternata still promoted thrombin to produce more mature IL-33, but conversely, the ability of thrombin to synergistically enhance the production of mature IL-33 and activate ILC2 by A. alternata disappeared ( Figure 3.4 These data suggest that the synergistic effect of thrombin on A. alternata depends on the R106 site cleavage, and A. alternata should promote the production of the thrombin intermediate IL-33 through its own Q102 site. 49-270 Such coordinated regulation is likely to play an important role in the maturation and activation of endogenous IL-33 in type II inflammatory responses.

[0223] In summary, the present invention demonstrates that thrombin specifically cleaves and activates IL-33 at a specific site, revealing the mechanism of action between thrombin and IL-33-mediated type II immune response.

[0224] 5. Bivalirudin, a direct thrombin inhibitor, can inhibit type II acute lung inflammation in mice

[0225] Previous in vitro experiments have demonstrated that thrombin can mediate the maturation and activation of IL-33, so it is speculated that anticoagulants that are direct inhibitors of thrombin may also have anti-inflammatory effects. By comparing existing drugs, the 20-peptide analog of hirudin, Bivalirudin (TFA), is preferred. It can specifically and reversibly bind to the catalytic and anionic binding sites of thrombin, directly inhibiting thrombin activity. Compared with the indirect thrombin inhibitor low molecular weight heparin that is widely used and studied in clinical practice, the mechanism of action of bivalirudin is simpler and suitable for verifying the function of endogenous thrombin in type II acute lung inflammation. First, in order to explore the effect of bivalirudin on ILC2-mediated acute type II inflammatory response, a papain-induced lung inflammation model was constructed. Bivalirudin (0.25 mg / kg) and papain were administered intrabronchially at the same time ( Figure 3.5 A). Lung ILC2 response and type II inflammation were detected on day 6. Compared with the control group, inflammation-related indicators such as the number and proportion of eosinophils in BALF in the bivalirudin intervention group were significantly higher ( Figure 3.5 BC), pulmonary inflammatory cell infiltration and goblet cell hyperplasia ( Figure 3.5 E) and cytokines IL-5 and IL-13 in bronchoalveolar lavage fluid decreased significantly ( Figure 3.5 F) were significantly reduced. At the same time, bivalirudin could significantly inhibit the number of lung ILC2 cells and the ratio of IL-5 and IL-13 in ILC2 in the papain-induced model ( Figure 3.5G). This experiment shows that bivalirudin can effectively inhibit the ILC2 response and type II inflammatory response in the papain-induced model.

[0226] Then we explored the intervention effect of bivalirudin on the mouse fungus (A. alternaria) model, an acute type II lung inflammation model that is closer to the clinical model, and obtained consistent results of inhibiting inflammation ( Figure 3.5 HN).

[0227] In summary, the present invention found that the direct thrombin inhibitor bivalirudin can effectively inhibit the ILC2 response and type II inflammatory response in the protease allergen-induced lung inflammation model.

[0228] 6. Bivalirudin has a therapeutic effect on the mouse allergic asthma model

[0229] A mouse allergic asthma model induced by dust mites (HDM) was constructed, which is closer to clinical asthma because dust mites themselves have certain protease activity and can mediate epithelial damage. Model construction process: mice were sensitized with the allergen HDM by intrabronchial immunization for 3 consecutive days starting on day 1, followed by HDM antigen challenge for 4 consecutive days starting on day 14. Bivalirudin (0.25 mg / kg) was administered simultaneously with HDM by intrabronchial immunization. Finally, mice were sacrificed on day 18, and the type II immune response and inflammatory response in peripheral blood and lungs were detected ( Figure 3.6 A).

[0230] Pulmonary function tests showed that compared with the control group, mice in the bivalirudin group showed lower pulmonary resistance ( Figure 3.6 B). Bivalirudin also significantly inhibited inflammation-related indicators such as the number and proportion of eosinophils in BALF ( Figure 3.6 C) and IL-5 and IL-13 concentrations in bronchial lavage fluid ( Figure 3.6 D), pulmonary inflammatory cell infiltration and goblet cell hyperplasia ( Figure 3.6 F) was significantly reduced. At the same time, IL-5 + and IL-5 + IL-13 + The cell ratio was also significantly inhibited ( Figure 3.6 G). ELISA test showed that compared with the control group, the serum total IgE and HDM-specific IgG1 ( Figure 3.6 H) In addition, because the HDM model is weaker than the OVA model, the production of IL-4, IL-5, and IL-13 by HDM-restimulated lung monocytes did not change significantly (not shown), indicating that the Th2 response was not affected by LMWH.

[0231] In summary, bivalirudin can inhibit the type II immune response and inflammatory response dominated by ILC2 in the mouse allergic asthma model, and may have good prospects for the intervention and treatment of allergic asthma. In the process of type II inflammatory response such as asthma, mature IL-33 can activate a variety of immune cells, such as ILC2, Th2, Treg, mast cells and DC, by interacting with cell surface ST2. The previous experiments mainly used primary ILC2 as IL-33 detection cells. In order to verify the regulation of IL-33 by thrombin in other cells at the same time, the primary Th2 in the HDM allergic asthma model was also sorted, and it was found that IL-33 cleaved by thrombin can also more strongly activate primary Th2 cells to produce IL-4, IL-5 and IL-13 ( Figure 3.6 I), further supplemented the role of thrombin-mediated IL-33 maturation in asthma.

[0232] Due to the complexity of the body's microenvironment, the specific regulatory mechanism of the coagulation system and type II inflammation is still unclear. In order to study the relationship between thrombin and IL-33 maturation and IL-33-mediated type II inflammatory response, the present invention carried out a series of in vivo and in vitro experiments in mouse animal models. The results showed that low molecular weight heparin (LMWH) can effectively inhibit the ILC2 response and type II inflammatory response in the mouse acute pneumonia model induced by papain and fungi, but the similar inhibitory effect disappeared in the ILC2-mediated inflammation model directly induced by IL-33. Further studies found that the effect of LMWH does not depend on resident T / B cells, nor does it directly inhibit the function of ILC2 cells themselves. It is speculated that it is likely to act by affecting IL-33 signals. To explore the effect of LMWH on endogenous IL-33, LMWH was used to intervene in protease-induced lung injury to detect the expression, release, and activity of endogenous IL-33. A series of experiments showed that LMWH did not affect the expression and release of IL-33, but significantly inhibited the ability of IL-33 in the lungs to activate ILC2 cells. Further detection of different forms of endogenous IL-33 in bronchoalveolar lavage fluid found that LMWH indeed inhibited the maturation of IL-33 and was associated with thrombin activity. By analyzing potential thrombin protease cleavage sites, in vitro experiments found that full-length IL-33 can be directly cleaved and activated by thrombin at specific amino acid sites (human 48R and 106R, mouse 50R and 87R), generating a mature form with more effective biological activity and an intermediate form that may have histone-related biological functions. To further verify the theory that thrombin can promote IL-33 maturation and response, the present invention used bivalirudin, a direct inhibitor of thrombin, and found that it can indeed effectively inhibit the acute pneumonia model induced by papain and fungi in mice. Intrabronchial administration of bivalirudin in HDM- and OVA-induced allergic asthma models revealed promising clinical applications for the treatment of asthma. Related studies have revealed a novel mechanism by which thrombin regulates IL-33 maturation and type II inflammatory responses in the lungs, providing a new theoretical basis and therapeutic strategy for the clinical treatment of asthma targeting thrombin.

[0233] Regarding basic research, the present invention reports for the first time that thrombin can directly promote the maturation and activation of IL-33 through specific sites, and explores the mechanism of synergistic action between thrombin and fungal proteases that can mediate IL-33 maturation. In addition, the study of the biological activity of IL-33 after histone enhancement by thrombin cleavage is likely to provide a new and important regulatory mechanism for the function of IL-33. In summary, the relevant research further improves the in vivo model of the interaction between the thrombin system and the immune system in acute lung inflammation and allergic asthma mediated by protease allergens (such as Figure 4 ).

[0234] With regard to clinical applications, this invention serves as a valuable bridge between past and future work, further revealing the mechanism of action of low-molecular-weight heparin, an anticoagulant drug with clinical asthma treatment potential, and providing a new theoretical basis for its efficacy. Finally, supported by previous theoretical research, this invention targets the thrombin-IL-33-inflammatory pathway and discovers that bivalirudin, an anticoagulant drug that directly inhibits thrombin, has a significant therapeutic effect on allergic asthma in mice. This finding suggests that bivalirudin, a promising new drug for asthma intervention and treatment, could offer a novel strategy for refractory complex asthma.

[0235] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of bivalirudin in the preparation of a medicament for treating an allergic disease, wherein the allergic disease is type II acute pulmonary inflammation or allergic asthma.

2. The use according to claim 1, characterized in that The thrombin inhibitor inhibits thrombin, inhibits IL-33 maturation, and inhibits the ability of IL-33 to activate ILC2 cells, thereby inhibiting type II inflammatory response and achieving the purpose of treating allergic diseases.

3. The use according to claim 2, characterized in that The inhibition of IL-33 maturation is to inhibit the cleavage of human IL-33 into a mature form at amino acid sites 48R and / or 106R, or to inhibit the cleavage of mouse IL-33 into a mature form at amino acid sites 50R and / or 87R.

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