Application of ceramide molecule in treating asthma

Abstract

The invention belongs to the field of biological medicine, and relates to application of ceramide molecules in treating asthma. Experiments prove that the traditional Chinese medicine composition has the effects of effectively inhibiting neutrophil asthma airway inflammation and relieving airway remodeling.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of ceramide molecules in treating asthma. Background Art

[0002] Asthma can be further divided into different phenotypes based on the proportion of cells in the patient's induced sputum. The subtype with sputum neutrophils ≥61% and eosinophils <3% is called neutrophilic asthma. Neutrophilic asthma is characterized by airway neutrophilic inflammation driven by non-type 2 inflammatory factors such as helper T cells-17 (T helper cell 17, Th17) and its cytokine interleukin-17 (interleukin-17, IL-17), and is resistant to inhaled corticosteroids. Patients with neutrophilic asthma often present with refractory asthma and even develop severe asthma. Currently, a variety of biological targeted agents have emerged for personalized treatment of asthma, but there are no approved biological agents for non-type 2 asthma such as neutrophilic asthma. Therefore, the treatment of this type of asthma still relies on conventional drug control and possible alternative treatments such as bronchial thermoplasty.

[0003] In-depth research into the pathogenesis of asthma has revealed that IL-17 is associated with asthma inflammatory cells, particularly neutrophils and eosinophils, and airway remodeling, thereby contributing to the development and progression of asthma. Targeting Th17 cells is expected to become a potential target for regulating asthma, particularly neutrophilic asthma. Neutrophilic asthma is associated with significant sphingolipid metabolism disorders, and ceramide, as an important bioactive sphingolipid, plays a crucial role in immune regulation. Currently, the main treatments for neutrophilic asthma are inhaled corticosteroids and bronchodilators, and there is a lack of corresponding targeted drugs. Although Th17-related cytokines act as central effectors in airway inflammation and airway remodeling, substantial progress is still needed in the treatment of IL-17A monoclonal antibodies. Factors such as their therapeutic efficacy, drug resistance, and high treatment costs remain obstacles to their widespread use. Research has found that sphingolipids such as ceramide can effectively regulate the functions of immune cells such as T cells. Therefore, the present invention focuses on the therapeutic role of ceramide in asthma. As an intrinsic metabolite of the human body, exploring its mechanism and applying it to the treatment of neutrophilic asthma through complementary therapies will help reduce drug side effects and improve the efficacy of targeted treatments.

[0004] Cer24:1 ceramide is a naturally occurring long-chain monounsaturated ceramide with the structural formula N-tetracosylsphingosine. It consists of a fatty acid side chain containing 24 carbon atoms with a double bond and an 18-carbon sphingosine moiety with a double bond. Its molecular formula is C42H81NO3, and its molecular weight is 648.097. Cer24:1 ceramide exhibits various physicochemical properties, including a density of 0.9±0.1 g / cm3, a boiling point of 749.6±60.0°C, and a flash point of 407.1±32.9°C. Cer24:1 is ubiquitous in mammalian cells, playing a key role in maintaining cell membrane structure, signal transduction, and metabolic regulation, and is an important bioactive molecule. Cer24:1, a ceramide of a specific chain length, is commercially available. Summary of the Invention

[0005] The object of the present invention is to provide the use of ceramide molecules in the preparation of drugs for treating and / or preventing asthma.

[0006] Among them, the ceramide molecule is Cer24:1.

[0007] Among them, asthma is neutrophilic asthma.

[0008] Applications of the present invention include:

[0009] Ceramide molecules have the effect of inhibiting Th17 cell differentiation and the secretion of IL-17A inflammatory factors.

[0010] Use of substances that supplement very long chain ceramide Cer24:1 and / or increase Smpd1 expression or enzyme activity in the preparation of products for alleviating neutrophil asthma.

[0011] Use of substances that supplement very long chain ceramide Cer24:1 and / or increase Smpd1 expression or enzyme activity in the preparation of products that inhibit Th17 cell differentiation.

[0012] In the above application, the improvement of neutrophilic asthma can be manifested as reducing the recruitment of neutrophils in the respiratory tract, reducing Th17-related inflammatory factors, airway remodeling, and alleviating airway hyperresponsiveness.

[0013] In any of the above applications, the substance that increases the expression level of Smpd1 may be a substance that overexpresses Smpd1 in the body and / or a Smpd1-specific agonist.

[0014] Another object of the present invention is to provide CD4 + Application of T cell EP2 protein in preparing products for alleviating neutrophil asthma; said application is through the +This is achieved by inhibiting EP2 expression and / or EP2 function inhibitors in T cells.

[0015] Another object of the present invention is to provide CD4 + Application of T cell EP2 protein in the preparation of inhibitory Th17 cell differentiation; the application is through the + This is achieved by inhibiting EP2 expression and / or EP2 function inhibitors in T cells.

[0016] In the above applications, the treatment or improvement of neutrophilic asthma can be manifested as reduced recruitment of neutrophils in the respiratory tract, reduction of Th17-related inflammatory factors, airway remodeling, and relief of airway hyperresponsiveness.

[0017] The drug of the present invention can be prepared into any pharmaceutically acceptable dosage form.

[0018] The pharmaceutical uses of the present invention are obtained after extensive experimental research:

[0019] The present invention first uses targeted sphingolipid metabolomics to detect the plasma, exhaled breath condensate and induced sputum supernatant of patients and healthy controls. The detection found that among the multiple differential metabolites, very long-chain ceramide Cer24:1 was the most significantly reduced. Correlation analysis found that very long-chain ceramide was significantly negatively correlated with Th17. By co-sensitizing C57 mice with HDM (House dust mites) and LPS (Lipopolysaccharide), and establishing a neutrophilic asthma model through continuous HDM stimulation, flow cytometry analysis revealed a significant increase in Th17 cells in lung tissue, and a significant increase in Th17-related cytokines in bronchoalveolar lavage fluid and plasma. It was also found that neutrophilic asthma model mice also showed a widespread decrease in Cer24:1 content in lung tissue and BALF (Bronchoalveolar Lavage Fluid) supernatant. Through the detection of ceramide metabolic enzymes, we found that the expression of various enzymes regulating Cer24:1 synthesis, such as ASM (ASM, also known as acidic sphingomyelin, is encoded and synthesized by the Smpd1 gene, and its main function is to promote the hydrolysis of sphingomyelin to ceramide, especially very long-chain ceramide), was reduced. By further constructing Smpd1KO mice and performing a neutrophil asthma model, we found that KO mice showed reduced Cer24:1 content and also had increased airway neutrophil ratio, airway resistance, and Th17-related inflammation levels.

[0020] To investigate the role of very-long-chain ceramide Cer24:1 in neutrophilic asthma, mice were first intraperitoneally injected with very-long-chain ceramide Cer24:1. Airway resistance was assessed using the FlexiVent pulmonary function tester. Th17 cell proportions and neutrophil inflammation levels were measured by flow cytometry, ELISA, special pathological staining, and immunohistochemistry. The degree of lung fibrosis was assessed by Masson staining and immunohistochemistry. The results showed that the Th17 proportion in the lungs of mice was significantly reduced after injection of very-long-chain ceramide Cer24:1, as was the Masson staining score. Further in vitro experiments confirmed that Cer24:1 inhibited Th17 cell differentiation in vitro. Transcriptome and Western blot analyses revealed that Cer24:1 inhibited Th17 cell differentiation by downregulating the JAK2-STAT3 signaling pathway.

[0021] Mechanistically, very long chain ceramide Cer24:1 inhibits Th17 differentiation by binding to T cell surface protein EP2. In terms of molecular mechanism, it inhibits the downstream JAK2-STAT3 pathway by binding to EP2 protein, thereby inhibiting the expression of RORγt transcription factor, leading to CD4 + T cell differentiation into Th17 is impaired. The inhibitory effect of very long chain ceramide C24:1 on Th17 differentiation can be effectively reversed by administering prostaglandin E2 (PGE2), a specific ligand of EP2 receptor protein. In animal models, we found that infusion of PGE2 can also reverse the protective effect of Cer24:1 on HDM / LPS-induced mice. The present invention has important application value. The above results mean that very long chain ceramide compounds such as Cer24:1 can become a new single drug for the treatment of neutrophilic asthma or as a component of a drug, and can be used as different dosage forms and administration methods to treat neutrophilic asthma.

[0022] The beneficial effects of the present invention lie in its in vitro demonstration of the ability of very long-chain ceramide C24:1 to inhibit Th17 cell differentiation and the secretion of the inflammatory factor IL-17A. In vivo experiments have confirmed its effectiveness in inhibiting airway inflammation and alleviating airway remodeling in neutrophilic asthma, revealing its potential for treating neutrophilic asthma. As a natural small molecule sphingolipid compound, it is easy to synthesize, and as an intrinsic lipid molecule in the body, it has relatively few side effects associated with supplemental therapy, thus possessing significant pharmaceutical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Metabolomic analysis reveals widespread reductions in the very-long-chain ceramide Cer24:1 in exhaled breath condensate (EBC) from patients with neutrophilic asthma. (AC) Sixty patients with asthma and 16 healthy controls provided EBC. Volcano plots depict differences in glycerophospholipids and sphingomyelins in EBC between asthma and controls (A), neutrophilic asthma and controls (B), and neutrophilic asthma and non-neutrophilic asthma (C). The color of the dots represents the trend of the metabolite between the groups (P < 0.05 and log2FC > 0.8 were considered significant between the two groups). Cer24:1 was found to be a significantly reduced sphingolipid in all three comparison combinations.

[0024] Figure 2 :Cer24:1 is significantly reduced in patients with neutrophilic asthma: In this clinical cohort, 60 asthma patients and 16 healthy controls provided EBC, 59 asthma patients and 16 healthy controls provided plasma, and 54 asthma patients and 23 healthy controls provided induced sputum. The figure shows the relative abundance of ceramides of different chain lengths between neutrophilic asthma (red) and healthy controls (blue). In EBC (A), plasma (B) and sputum supernatant (C), the levels of very long chain ceramides represented by Cer24:1 were significantly reduced in patients with neutrophilic asthma. Data are expressed as mean ± SEM. Statistical analysis was performed using a two-tailed Student's t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0025] Figure 3 Diagnostic value of Cer24:1 in neutrophilic asthma: (AC) Receiver operating characteristic (ROC) curves for Cer24:1 in EBC (A), plasma (B), and induced sputum supernatant (C) for distinguishing patients with neutrophilic asthma from healthy controls, showing areas under the curves greater than 0.7. (DE) Plasma Cer24:1 levels were significantly negatively correlated with the proportion of Th17 cells in peripheral blood and the proportion of granulocytes in sputum, suggesting that Cer24:1 may have beneficial effects.

[0026] Figure 4HDM combined with LPS induces neutrophilic asthma in mice, characterized by neutrophilic inflammation. (A) Schematic diagram of the experimental design: Mice were sensitized and challenged with house dust mites (HDM), while control mice received an equal volume of PBS intranasally. (B) Airway hyperresponsiveness to acetylcholine was measured using a pulmonary function test. Rrs represents respiratory resistance. Results showed that airway resistance in model mice was significantly higher than in control mice. (C) Bar graph of the percentages of neutrophils, eosinophils, macrophages, and lymphocytes in BALF smears (n = 3 / group) shows a significant increase in the proportion of neutrophils in the BALF of model mice. Data are expressed as mean ± SEM. Statistical analysis was performed using a two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0027] Figure 5 HDM combined with LPS induces inflammatory infiltration, airway remodeling, and mucus hypersecretion in mouse lung tissue. Representative H&E staining (A, D), PAS staining (B, E), Masson's trichrome staining (C, F), and immunohistochemical staining for α-SMA (J, M), MPO (K, N), and Ly6G (L, O) in mouse lung tissue sections, along with pathological scoring histograms (G, R). Scale bar, 50 μm. Results show that the model mice exhibited pathological features of enhanced neutrophilic inflammation, mucus hypersecretion, and airway remodeling (n = 4 / group). Data are expressed as mean ± SEM. Statistical analysis was performed using a two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0028] Figure 6 HDM combined with LPS induces Th17-dominated neutrophilic asthma in mice. (A-C) Bar graphs of IL-17A (A), IL-17F (B), and IL-22 (C) concentrations in plasma and BALF demonstrate significant increases in Th17-related cytokine concentrations in both plasma and BALF supernatants. (D) Flow cytometric analysis of the proportions of Th1, Th2, Th17, and Treg cells in mouse lung tissue (n = 6 / group) demonstrates a significant increase in the proportion of Th17 cells in the lung tissue of modeled mice. Data are expressed as mean ± SEM. Statistical analysis was performed using a two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Figure 7Extensive reduction of very-long-chain ceramides in HDM / LPS-induced neutrophilic asthma mice: (A-B) Clustered heatmaps of sphingolipid content in the BALF and lung tissues of PBS-induced control mice (red) and HDM / LPS-induced mice (green), showing that the concentrations of most sphingomyelins and ceramides were significantly reduced in these models. (B-C) Histograms of the relative abundance of different chain-length ceramides in the BALF and lung tissues of PBS-induced control mice (blue), HDM / LPS-induced neutrophilic asthma mice (red), and HDM-induced eosinophilic asthma mice (yellow) show that ceramide concentrations in the BALF and lung tissues of neutrophilic asthma mice were lower than those in both control mice and eosinophilic asthma mice. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0029] Figure 8 Reduced ceramide metabolism in lung tissue of neutrophil-induced asthmatic mice: (A) Introduction to the ceramide metabolic pathway. This figure illustrates the metabolic pathways for ceramide synthesis, including the sphingomyelinase pathway and the de novo synthesis pathway. (Abbreviations: SPTLC, serine palmitoyltransferase; KDSR, 3-ketodihydrosphingosine reductase; CERS, ceramide synthase; DEGS, denatured spermatocyte homolog; N-acylsphingosine amidohydrolase; SPHK, sphingosine kinase; sphingomyelin synthase; sphingomyelin diesterase; CERK, ceramide kinase). (B) Analysis of RNA-seq data from the lungs of PBS- and HDM / LPS-induced mice revealed downregulation of the de novo sphingolipid synthesis pathway in HDM / LPS-induced mice (n = 6 / group), and decreased transcription of genes encoding multiple sphingolipid metabolism enzymes, including Smpd1.

[0030] Figure 9 :The expression of multiple sphingolipid metabolism enzymes in the lung tissues of neutrophil asthma mice was reduced. (AM) RT-qPCR analysis of ceramide synthase-related genes in the lungs of HDM / LPS-induced model mice compared with the control group. In HDM / LPS-induced mice, gene transcription of three sphingolipid metabolism enzymes, Sptlc2, Cers2 and Smpd1, was downregulated (n=4-5 / group). (N) The expression of Sptlc2, Cer2 and Smpd1 proteins in the lung tissues of HDM / LPS-induced mice and control mice was also significantly reduced. Statistical analysis was performed using a two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001,

[0031] Figure 10:Th17 inflammation is aggravated in Smpd1KO neutrophil asthma mice: (AB) The airway resistance of each group of mice under acetylcholine stimulation and at baseline showed that the airway resistance of Smpd1KO model mice was significantly higher than that of wild-type model mice. (CE) The concentrations of Th17-related cytokines such as IL-17A, IL-17F, and IL-22 in plasma and BALF showed that the IL-22 concentration in the plasma of Smpd1KO model mice was significantly increased. (FG) Flow cytometric statistical analysis of the proportions of Th1, Th2, Th17, and Treg in the lung (F) and spleen (G) (n=6 / group). By ZombieAqua - CD3 + CD8 - CD4 + Gated T cell analysis revealed an increase in the proportion of all Th subsets in the lung tissue of Smpd1KO mice. Data are presented as mean ± SEM. Statistical analysis was performed using a two-tailed Student's t-test and one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0032] Figure 11 Smpd1KO neutrophilic asthma mice show exacerbated inflammatory infiltration in the lungs. (A-H) Representative H&E, PAS, MPO, and Ly6G immunohistochemical staining images (AP) and pathological scoring histograms (QT) are shown in lung tissue sections. Results show exacerbated inflammatory infiltration and increased neutrophil recruitment in the lungs of Smpd1KO mice. Scale bar = 50 μm. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Figure 12 : Increased neutrophil recruitment in BALF of Smpd1KO neutrophilic asthma mice: (A, B) Flow cytometric analysis of the cell proportions of neutrophils, eosinophils, alveolar macrophages (AM), and interstitial macrophages (IM) in BALF (A, n=3 mice / group) and lung tissue (B, n=6 mice / group). The proportions of all myeloid cells were based on CD45 + The results showed increased neutrophil recruitment in the BALF of Smpd1KO mice. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0033] Figure 13: Cer24:1 injection alleviates neutrophilic asthma inflammation: (A) Schematic diagram of experimental design: Ceramide Cer24:1 (dose of 10 mg / kg body weight) was injected once daily during the modeling period. (B) Airway resistance of each group of mice under acetylcholine stimulation. Cer24:1 did not significantly improve airway resistance in model mice. (CD) Bar graphs of IL-17A and IL-17F concentrations in plasma and BALF showed that the Cer24:1 intervention group significantly reduced plasma IL-17A concentrations. (E) Annotation results of single-cell sequencing of lung tissue. (F) Single-cell sequencing showed that the proportion of Th17 in lung tissue of Cer24:1-treated mice was lower than that in lung tissue of model mice. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0034] Figure 14 :Cer24:1 injection reduces the proportion of Th17 in lung tissue of neutrophil asthmatic mice: (A, B) Flow cytometric analysis and statistical evaluation of Th1, Th2, Th17, and Treg in lung tissue (A) and spleen (B) (n=6 / group). - CD3 + CD8 - CD4 + T cell gating was used to analyze T cell subset proportions. Results showed that Cer24:1 treatment suppressed Th17 cell proportions in lung tissue but did not affect Th17 cell proportions in the spleen. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0035] Figure 15 Cer24:1 injection alleviates lung inflammation and mucus hypersecretion in mice with neutrophilic asthma: (AP) Representative H&E and PAS staining of lung tissue sections, as well as immunohistochemical staining for MPO and Ly6G (AL) and pathological score histograms (MP). Scale bar, 50 μm. The results showed that Cer24:1 intervention significantly reduced lung inflammatory infiltration and neutrophil recruitment and alleviated lung mucus hypersecretion. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0036] Figure 16Cer24:1 injection alleviates airway remodeling in mice with neutrophilic asthma. (AL) Representative lung tissue sections showing Masson's trichrome staining, α-SMA and Fibronectin immunohistochemical staining, and pathological scoring histograms. Scale bar, 50 μm. Results show that Cer24:1 alleviates airway remodeling in mice with neutrophilic asthma (n = 4 / group). (MP) Histogram analysis of plasma and BALF airway remodeling-related factors, MMP-9 and TIMP-1. Results show that Cer24:1 treatment significantly decreased plasma and BALF supernatant MMP-9 levels, as well as BALF TIMP-1 levels. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0037] Figure 17 : Cer24:1 injection inhibits neutrophil recruitment in BALF of neutrophilic asthmatic mice: (A, B) Flow cytometric analysis and statistical evaluation of neutrophils, eosinophils, alveolar macrophages (AM), and interstitial macrophages (IM) in BALF (A, n=3 mice / group) and lung (B, n=6 mice / group). The proportion of all myeloid cells was gated on CD45 + Cells. The results showed that Cer24:1 treatment significantly reduced the proportion of neutrophils in BALF. Data are presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0038] Figure 18 :Cer24:1 inhibits Th17 differentiation in vitro: (AF) Flow cytometric analysis of control vehicle or Cer24:1-treated mice CD4 + Statistical analysis of the intracellular staining ratios of nonpathogenic Th17 (A), pathogenic Th17 (B, C), Treg, Th1, and Th2 T cells. Results showed that Cer24:1 treatment significantly inhibited the differentiation of pathogenic and nonpathogenic Th17 cells but did not affect the in vitro differentiation of Th1, Th2, or Treg cells. Data are presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0039] Figure 19Cer24:1 inhibits Th17 differentiation in vitro but does not affect Th17 cell apoptosis or proliferation. (A-B) Representative immunoblot analysis of RORγt in pathogenic Th17 cells treated with either vehicle or Cer24:1 demonstrates that Cer24:1 treatment inhibits RORγt protein expression. (C, D) RT-qPCR analysis of Il17a and Il17f mRNA expression in cells treated with either vehicle or Cer24:1 for 3 days (n=4) demonstrates that Cer24:1 treatment inhibits Il17a and Il17f gene transcription. (E) ELISA analysis demonstrates that IL-17A concentrations in cell culture supernatants decrease with increasing Cer24:1 concentrations. (F) Statistical analysis of the percentage of pathogenic Th17 cells treated with either vehicle or amitriptyline (Smpd1 inhibitor) for 3 days (n=4) demonstrates that inhibition of Smpd1 activity effectively promotes Th17 cell differentiation. (G) Ceramides of different chain lengths (C16, C20, and C22) had no significant effect on Th17 differentiation. (H, I) Treatment of pathogenic Th17 cells with the control solvent or Cer24:1 for 3 days did not affect apoptosis (H) or proliferation (I). Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Figure 20 Cer24:1 inhibits pathogenic Th17 cell differentiation by downregulating the JAK2-STAT3 signaling pathway. (A) Transcriptome analysis of Th17 cells treated with Cer24:1 and a control solvent showed that the majority of differentially expressed genes between pathogenic Th17 cells cultured with the control solvent and 10 μM Cer24:1 were downregulated. (B) Transcriptome-based GSEA pathway enrichment analysis showed that the JAK-STAT pathway was significantly downregulated in the Cer24:1-treated group. (C) KEGG enrichment pathways of differentially phosphorylated signaling pathway proteins detected by phosphoprotein microarray. The JAK-STAT pathway is highlighted in red. Results show that the JAK-STAT signaling pathway is significantly downregulated after Cer24:1 treatment.

[0040] Figure 21Cer24:1 Intervention Inhibits STAT3 Pathway Phosphorylation in Pathogenic Th17 Cells: (A-C) Representative immunoblot analysis of phosphorylated JAK2 and STAT3 in control or Cer24:1-treated Th17 cells. Results demonstrate that Cer24:1 intervention inhibits STAT3 pathway activation. (D, E) Representative immunoblot analysis of phosphorylated JAK2 and STAT3 in Th17 cells treated with control, Cer24:1, or Cer24:1 combined with Colivelin (a STAT3 activator) demonstrates that Colivelin effectively reverses the Cer24:1-induced decrease in STAT3 phosphorylation. (F) Flow cytometric analysis of pathogenic Th17 cells treated with Cer24:1 or Cer24:1 combined with Colivelin demonstrates that Colivelin effectively promotes Th17 differentiation. (G) ELISA analysis demonstrates that Colivelin effectively increases IL-17A concentrations in cell supernatants. Data are presented as mean ± SEM. Statistical analysis was performed using a two-tailed Student's t-test and one-way analysis of variance. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0041] Figure 22 :Cer24:1 specifically binds to EP2 and inhibits the activation of STAT3 signaling pathway in Th17 cells: (A) Single-cell sequencing data revealed that CD4 + The top 20 G protein-coupled receptor (GPCR) family genes expressed on T cells. (B) Flow cytometric analysis of the percentage of Th17 cells treated with Cer24:1 or Cer24:1 combined with PGE2 (a specific ligand for EP2) showed that PGE2 could significantly promote Th17 cell differentiation. (C) SPR analysis showed that Cer24:1 interacted with EP2 with a dissociation constant of 417.7 μM. (D) Biotin-ceramide pull-down assay showed that CD4 + Cer24:1 and EP2 interact in T cells. Cell lysates were incubated with biotin-ceramide or biotin, and interacting proteins were isolated by streptavidin-agarose pulldown, followed by immunoblotting analysis using anti-EP2 and anti-EP4 antibodies. (E) Molecular docking prediction of the binding site between EP2 and Cer24:1. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0042] Figure 23Activation of the EP2 receptor significantly promotes STAT3 pathway activation and Th17 differentiation. (A-C) Representative immunoblot analysis of phosphorylated JAK2-STAT3 pathway proteins and RORγt in Th17 cells treated with control vehicle, Cer24:1, or Cer24:1 combined with PGE2 shows that PGE2 (EP2 agonist) treatment significantly promotes RORγt and STAT3 phosphorylation. (D) Treatment with AH6809 (EP2 inhibitor) significantly reduces pathogenic Th17 cell differentiation. (E) HDM / LPS-induced significant increase in plasma PGE2 concentrations in mice (n = 6 / group). Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0043] Figure 24 : PGE2 significantly reversed the improvement effect of Cer24:1 on HDM / LPS-induced mice: (A) Schematic diagram of the experimental design. (B, C) Concentrations of IL-17A and IL-17F in plasma and BALF. The results showed that PGE2 treatment increased the concentration of IL-22 in plasma. (D) Flow cytometric analysis and statistical evaluation of Th1, Th2, Th17, and Treg cells in lung tissue (n=6 / group). The results showed that PGE2 treatment increased the proportion of Th17 cells in lung tissue. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0044] Figure 25 PGE2 significantly reversed and aggravated the anti-inflammatory effects of Cer24:1 on HDM / LPS-induced lung tissue in mice: (AT) Representative H&E, PAS, MPO, and Ly6G immunohistochemical staining (AP), and pathological score histograms (QT). Scale bar = 50 μm (n = 3 mice / group). Results showed that PGE2 treatment reversed and promoted inflammatory infiltration and neutrophil recruitment in lung tissue. Data are presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0045] Figure 26PGE2 promotes neutrophil recruitment into the BALF of HDM / LPS-induced mice: (A-B) Flow cytometric analysis and statistical evaluation of neutrophils in the BALF (A, n = 3-4 mice / group) and lung tissue (B, n = 6 mice / group). Results showed that PGE2 treatment increased the proportion of neutrophils in the BALF but not in the lung tissue. (C, D) Representative immunoblot analysis of phosphorylated JAK2-STAT3 pathway proteins in lung tissue of mice treated with the control group, Cer24:1, or Cer24:1 combined with PGE2 showed that PGE2 treatment promoted STAT3 phosphorylation. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0046] The present invention is further illustrated by the following examples.

[0047] The products and preparation methods of the present invention are further described below with reference to specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from publicly available commercial sources unless otherwise specified.

[0048] Example 1: Confirmation of reduced levels of very long chain ceramides in patients with neutrophilic asthma (1) Clinical studies involving asthma patients:

[0049] ① Cross-sectional study:

[0050] Asthma patients attending the Department of Respiratory and Critical Care Medicine at Peking University Third Hospital were consecutively enrolled. Healthy non-asthmatic patients matched for gender, age, and BMI were also included. Based on sputum cytology, asthma patients were categorized into eosinophilic asthma (sputum neutrophils <61%, eosinophils ≥3%), neutrophilic asthma (sputum neutrophils ≥61%, eosinophils <3%), mixed granulocytic asthma (sputum neutrophils ≥61%, eosinophils ≥3%), and oligogranulocytic asthma (sputum neutrophils <61%, eosinophils <3%). Exclusion criteria included acute asthma exacerbation, concomitant chronic obstructive pulmonary disease, bronchiectasis, pneumonia, obstructive sleep apnea-hypopnea syndrome, malignant tumors, various acute and chronic respiratory failures, and severe cardiovascular disease.

[0051] The demographic information of the subjects was collected and recorded, such as gender, age, BMI, age of onset, lung function, blood routine, induced sputum cell differential count, lung CT results, asthma control test (ACT) score, allergen determination, etc.

[0052] ②Exhaled breath condensate (EBC) collection and specimen processing

[0053] EBC was collected using a TURBO-DECCS exhaled breath condensate collector (Medivac PARMA, Italy). Participants wore a nose clip and breathed deeply for 10 minutes. EBC samples were immediately stored at −80°C until analysis.

[0054] ③Induced sputum collection and specimen processing, cell counting and classification:

[0055] Sputum samples were collected from subjects after 20–30 minutes of nebulized inhalation of 3% hypertonic saline. Sputum plugs were collected from the saliva and mixed with four volumes of 0.1% dithiothreitol, incubated with shaking for 30 minutes, filtered, and centrifuged to separate the sputum supernatant and cells. Cell smears were stained with Wright-Giemsa, and 200 inflammatory cells were counted and classified under a high-power microscope. The remaining cells were resuspended in flow cytometry staining buffer.

[0056] ④Peripheral blood sample acquisition and processing:

[0057] 2-4 mL of EDTA-anticoagulated blood was collected from patients or healthy controls and immediately centrifuged at 3000 rpm for 10 minutes to obtain the upper plasma layer.

[0058] ⑤Targeted metabolomics detection of phospholipid content in plasma, induced sputum supernatant and other samples:

[0059] Chloroform / methanol extraction was used to extract lipids from human exhaled breath condensate, and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used for qualitative and quantitative analysis of lipid molecules. Targeted analysis was used to detect phospholipid molecules such as sphingolipids and glycerophospholipids (such as Cer, SM, PC, PE, PI, etc.), and further differential analysis of exhaled breath condensate lipids between different groups (asthma patients and controls, neutrophil asthma patients and controls, neutrophil asthma patients and neutrophil asthma patients) was performed to screen out the differential phospholipid molecules Cer24:1 ( Figure 1 ) and found that it was significantly reduced in all three comparison combinations.

[0060] Further testing of plasma and induced sputum supernatant samples verified the differences in ceramide molecules of different chain lengths between neutrophil asthma and healthy subjects, and found that ultra-long chain ceramides represented by Cer24:1 were significantly reduced in neutrophil asthma patients ( Figure 2 ).

[0061] Next, we analyzed the ROC curves of Cer24:1 in EBC, plasma, and induced sputum supernatants. The results showed that the areas under the curves were all greater than 0.7, indicating that Cer24:1 has a diagnostic value in distinguishing neutrophilic asthma from controls ( Figure 3 AC), and the correlation between plasma Cer24:1 levels and the proportion of peripheral blood Th17 and induced sputum neutrophils was also explored by Pearson correlation analysis ( Figure 3 D, 3E). (2) Using HDM and LPS to induce neutrophil asthma mouse model:

[0062] ① Neutrophil asthma model induced by HDM+LPS Figure 4 A):

[0063] Experimental group: 6- to 8-week-old C57BL / 6 mice were selected and sensitized with 25μg HDM + 10μg LPS by intranasal drops every day for the first three days. Starting from the 7th day, 25μg HDM intranasal drops were administered every day for 9 consecutive days. The mice were collected 24 hours after the last challenge.

[0064] Control group: Six- to eight-week-old C57BL / 6 mice were selected and administered an equal volume of PBS intranasally for the first three days. Starting on the seventh day, the same volume of PBS was administered intranasally for nine consecutive days. Samples were collected 24 hours after the last intranasal instillation.

[0065] ② Determination of airway hyperresponsiveness in neutrophil asthma mice:

[0066] FinePointe system pulmonary function tester TM ), by applying different concentrations of methacholine (0, 6.25, 12.5, 25, 50 mg / mL) to the model mice for gradient stimulation, the changes in airway resistance were recorded, and the airway responsiveness ( Figure 4 B) The results showed that the airway resistance of the model mice was significantly higher than that of the control mice.

[0067] ③ Neutrophil inflammation and Th17 detection in lung tissue of neutrophilic asthma mice:

[0068] The alveolar lavage fluid of the two groups of mice was collected and smeared and dried. The proportion of neutrophils, eosinophils, macrophages and lymphocytes in the BALF cells of the mice was analyzed by Swiss Giemsa staining. The staining results showed that the proportion of neutrophils in the BALF of the modeling group mice was significantly increased ( Figure 4 C).

[0069] The left lung lobe was taken for lung tissue pathological staining, such as hematoxylin-eosin (HE) staining (0-4 semi-quantitative score) to detect airway inflammatory cell infiltration; airway goblet cell hyperplasia periodic acid Schiff (PAS) staining (0-4 semi-quantitative score); airway collagen deposition (Masson's staining, 0-4 semi-quantitative score); airway neutrophil infiltration (MPO, Ly6G immunohistochemical staining, image analysis to calculate the positive staining ratio); airway smooth muscle hyperplasia (α-SMA immunohistochemical staining, image analysis to calculate the airway smooth muscle area / basement membrane thickness), etc. The results showed that the lung tissue of the modeled mice showed the characteristics of aggravated airway neutrophil inflammation, mucus hypersecretion and airway remodeling at the pathological level ( Figure 5 ).

[0070] The levels of IL-17A, IL-17F, IL-22 and other cytokines in mouse serum and bronchoalveolar lavage fluid were detected using a multifactor flow cytometry kit. It was found that the concentrations of Th17-related cytokines in plasma and BALF supernatant increased significantly ( Figure 6 AC). Lung tissues from both groups of mice were collected, minced, enzymatically digested, ground, filtered, and red blood cell lysed to prepare single-cell suspensions. T cells were stimulated with PMA and ionomycin to secrete cytokines such as IL-17A, IL-4, and IFN-γ. After incubation at 37°C for 4-6 hours, the proportions of various T cell subsets, such as Th1 / 2 / 17 / Treg, in the above tissues were detected by flow cytometry using cytokine internal standards. The results of flow cytometry analysis showed that the proportion of Th17 in the lung tissues of the modeling mice was significantly increased ( Figure 6 D).

[0071] ④ Targeted metabolomics detection of phospholipid content in mouse tissues

[0072] Chloroform / methanol extraction was used to extract lipid molecules from mouse plasma, bronchoalveolar lavage fluid supernatant, lung tissue homogenate and other samples. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used for qualitative and quantitative analysis of lipid molecules. Targeted analysis was used to detect phospholipid molecules such as sphingolipids and glycerophospholipids (such as Cer, SM, PC, PE, PI, etc.), and further analysis of ceramide differences between different groups (HDM+LPS-induced neutrophil asthma mice, HDM-induced eosinophil mice, and healthy control mice) was performed. The results showed that the ceramide concentrations in BALF and lung tissues of neutrophil asthma mice were not only lower than those in the control group mice, but also lower than those in eosinophil asthma mice ( Figure 7 ).

[0073] Example 2: Confirmation of the protective effect of very long chain ceramide on neutrophil asthma

[0074] (1) Confirmed that ceramide synthesis is reduced in the lung tissue of neutrophil asthma mice:

[0075] The metabolic pathways for ceramide synthesis include the sphingomyelinase pathway and the de novo synthesis pathway ( Figure 8 A). Transcriptome sequencing analysis was used to investigate the altered pathways in the lung tissues of neutrophil asthma mice. It was found that the de novo sphingolipid synthesis pathway was downregulated in the model mice, and the transcription levels of multiple sphingolipid metabolism enzyme genes, represented by Smpd1, were reduced ( Figure 8 B). RT-qPCR and Western blot analysis were used to verify the expression of multiple sphingolipid metabolism enzymes in the lung tissues of model mice and control mice, and it was found that the expression of three sphingolipid metabolism enzymes, Sptlc2, Cers2, and Smpd1, was significantly downregulated ( Figure 9 ).

[0076] (2) Construction and intervention of Smpd1 gene knockout mouse model:

[0077] Smpd1 gene knockout mice (C57BL / 6J background) were further constructed. The acid sphingomyelinase synthesized by the Smpd1 gene regulates the synthesis of very long chain ceramides. In this mouse model, the loss of the Smpd1 gene will lead to defects in the synthesis of very long chain ceramides of C22-C24, which is convenient for studying the role of very long chain ceramides in neutrophil asthma. In addition, wild-type mice with the same background were selected as the control group. According to the above method, HDM+LPS combined exposure was also performed to establish a neutrophil asthma mouse model to detect Smpd1. - / - Asthmatic mice and Smpd1 + / + The differences in airway resistance, lung tissue inflammation level, neutrophil ratio, and Th17 inflammation in asthmatic mice were determined using the same methods as described above. The airway resistance of each group of mice under acetylcholine stimulation and at baseline showed that the airway resistance of Smpd1KO model mice was significantly higher than that of wild-type model mice ( Figure 10 A, 10B), and the plasma IL-22 concentration in KO model mice was significantly increased ( Figure 10 CE), and the proportion of Th17 in lung tissue was significantly higher than that in wild-type model mice ( Figure 10 F, 10G), but no significant difference was found in the proportion of Th17 in the spleen.

[0078] Further evaluation of the pathological staining of the lung tissue of Smpd1 knockout mice revealed that the inflammatory infiltration of the lung tissue of Smpd1KO model mice was aggravated, and the recruitment of neutrophils in the lung tissue was increased, without affecting the high secretion of airway mucus ( Figure 11Mouse lung tissue was collected, minced, enzymatically digested, ground, filtered, and red blood cells were lysed to prepare single-cell suspensions. Cell surface antibodies (CD45, CD11b, CD11c, SiglecF, Ly6G) were used for flow cytometry to detect the proportion of macrophages, neutrophils, and eosinophils in the single-cell suspensions of lung tissue and BALF cells. It was found that the recruitment of neutrophils in the BALF of Smpd1KO model mice was increased ( Figure 12 ).

[0079] (3) Cer24:1 metabolic supplementation experiment:

[0080] A neutrophil asthma mouse model was established. Ceramide C24:1 standard was dissolved in anhydrous ethanol. The intervention group mice were intraperitoneally injected with 10 mg / kg of Cer24:1 solution per day, and the control group was injected with an equal amount of normal saline ( Figure 13 A). Detect whether the airway resistance, lung tissue inflammation level and airway remodeling of the model mice have improved. The specific detection indicators are the same as described above. Although Cer24:1 intervention does not affect airway resistance ( Figure 13 B), but significantly reduced the plasma IL-17A concentration ( Figure 13 C, 13D). Single-cell sequencing analysis was further performed on the lung tissues of healthy control mice, model mice, and Cer24:1 intervention mice ( Figure 13 E), and found that the proportion of Th17 in the lung tissue of the intervention group mice was lower than that of the model group mice ( Figure 13 F). Flow cytometry was then used to further verify the proportion of T cell subsets in the lung tissue of the model mice. The results showed that Cer24:1 intervention inhibited the proportion of Th17 in lung tissue, but did not affect the proportion of Th17 in the spleen ( Figure 14 ).

[0081] Further pathological evaluation and analysis of the effects of Cer24:1 injection on lung tissue inflammation, mucus hypersecretion, and airway remodeling in neutrophilic asthma mice revealed that Cer24:1 intervention significantly reduced lung tissue inflammatory infiltration and neutrophil recruitment, and alleviated lung tissue mucus hypersecretion ( Figure 15 Cer24:1 also has a certain alleviating effect on airway remodeling in neutrophil asthma mice. In addition to Masson staining and Fibronectin immunohistochemistry, ELISA detection of epithelial remodeling-related cytokines found that the concentrations of MMP-9 in plasma and BALF supernatant and TIMP-1 in BALF were significantly reduced after Cer24:1 intervention ( Figure 16 Flow cytometry was used to further analyze the proportion of granulocytes and macrophages in lung tissue and BALF. The results showed that Cer24:1 intervention significantly reduced the proportion of neutrophils in BALF ( Figure 17 ).

[0082] Example 3: Ceramide Cer24:1 participates in the pathogenesis of neutrophilic asthma via Th17 cells (1) CD4 + T cell differentiation in vitro experiment:

[0083] ① CD4 + T cell acquisition:

[0084] Single cell suspensions were obtained from the spleen and lymph nodes of C57BL / 6J mice using CD4 + T cell magnetic bead separation kit isolated CD4 + T cells. CD4 T cells were cultured using T cell culture medium (RPMI-1640, containing 10% FBS, 1% L-glutamine, etc.). + T cells were activated using anti-CD3 (5 μg / mL) and anti-CD28 (5 μg / mL) monoclonal antibodies.

[0085] ②Induction and differentiation of Th1, Th2, Th17, and iTreg cells:

[0086] Th1 differentiation induction conditions: addition of IL-12 (20 ng / mL) and IL-2 (10 ng / mL);

[0087] Th2 differentiation induction conditions: addition of IL-4 (10 ng / mL) and IL-2 (10 ng / mL);

[0088] Pathogenic Th17 differentiation induction conditions: addition of IL-6 (20 ng / mL), TGF-β (3 ng / mL), IL-23 (10 ng / mL), and IL-1β (10 ng / mL);

[0089] Non-pathogenic Th17 differentiation induction conditions: addition of IL-6 (20 ng / mL) and TGF-β (3 ng / mL);

[0090] iTreg differentiation induction conditions: addition of TGF-β (8 ng / mL).

[0091] ③ Under the above differentiation conditions, different concentrations of Cer24:1 (0, 5μM, 10μM, 20μM) were added for metabolic supplementation intervention, and the proliferation and differentiation of T cells were monitored during the induction period. After 72 hours of culture, the cell culture supernatant was collected to detect cytokine content, and cells were collected for cell function testing:

[0092] Flow cytometry was used to detect cell polarization efficiency: CD4, IL-17A, IL-4, IFN-γ, Foxp3 and other flow cytometry antibody markers were used to analyze the proportion of Th1, Th2, Th17 and iTreg cells by intracellular factor staining ( Figure 18 ); Western blot analysis of the expression levels of Th17 cell-specific proteins (such as RORγt) ( Figure 19 A, 19B); RT-qPCR analysis of the expression levels of Th17 cell-specific genes (such as Il-17a, Il-17f, etc.) ( Figure 19 C, 19D); ELISA was used to detect the level of IL-17A in the culture supernatant ( Figure 19 E); The results showed that Cer24:1 inhibited Th17 differentiation in vitro.

[0093] After adding different concentrations of amitriptyline (Amt), an inhibitor of acid sphingomyelinase (Smpd1), to the culture medium, the proportion of Th17 differentiation in vitro was significantly increased ( Figure 19 F). However, after intervention with ceramides of other chain lengths, such as C16, C20, and C22, no significant changes were observed in the Th17 differentiation ratio in vitro ( Figure 19 G).

[0094] The apoptosis rate was analyzed by Annexin V / PI staining. Figure 19 H), CFSE dye was used to detect T cell proliferation ( Figure 19 I), the results showed that Cer24:1 intervention had no effect on the proliferation and apoptosis of Th17 cells.

[0095] Example 4: Ceramide Cer24:1 regulates Th17 differentiation via the EP2-STAT3 axis

[0096] (1) Screening and verification of the signaling pathways affected by Cer24:1:

[0097] The Th17 cells after intervention with Cer24:1 and solvent control groups were collected for total RNA extraction and RNA sequencing to compare the expression levels of genes related to Th17 differentiation among the groups ( Figure 20 A), and enrichment analysis was performed based on differentially expressed genes to screen for significantly downregulated JAK-STAT pathways ( Figure 20 B). Total protein was extracted from both groups of cells and analyzed by phosphorylation protein chip. Based on the differential phosphorylated protein enrichment analysis, it was found that the phosphorylated proteins of the JAK-STAT pathway were also significantly downregulated in the Cer24:1 group ( Figure 20 C).

[0098] Proteins of Th17 cells induced by Cer24:1 were extracted and the expression levels of JAK2 / STAT3 signaling pathway related proteins (such as p-STAT3, STAT3, p-JAK2, JAK2, etc.) were verified by Western blot. The results showed that Cer24:1 intervention inhibited the phosphorylation of STAT3 pathway proteins ( Figure 21 AC).

[0099] Based on the intervention of Cer24:1 on Th17 differentiation, a response experiment was conducted using a STAT3 agonist (Colivelin). The proportion of Th17 cells was detected by flow cytometry, and the expression of IL-17A in the cell culture supernatant was detected by ELISA. This was done to verify whether Cer24:1 regulates Th17 cell differentiation through the JAK2 / STAT3 pathway. The results showed that Colivelin, as an effective STAT3 agonist, can significantly promote Th17 differentiation ( Figure 21 DF).

[0100] (2) Prediction and validation of Cer24:1 receptor

[0101] ① Receptor agonist function recovery experiment:

[0102] Screening for CD4 + G protein-coupled receptor (GPCR) genes that are highly expressed on T cells ( Figure 22 A), further adding corresponding G protein-coupled receptor agonists or ligands to the Cer24:1 intervention Th17 differentiation induced functional recovery experiments, found that adding EP2 receptor agonists (such as PGE2) to the recovery experiment can effectively promote Th17 cell differentiation ( Figure 22 B).

[0103] ②Surface Plasmon Resonance (SPR) Detection:

[0104] Detection of Cer24:1 and CD4 using SPR technology + The binding affinity of Cer24:1 to the G protein-coupled receptor (EP2) that may bind to the surface of T cells was analyzed. The results showed that Cer24:1 interacted with EP2 with a dissociation constant of 417.5 μM ( Figure 22 C).

[0105] ③Co-immunoprecipitation experiment (Co-IP):

[0106] The binding of EP2 receptor to Cer24:1 was verified by immunoprecipitation. CD4 was precipitated using biotin-conjugated anti-ceramide antibody. + Ceramide-protein complex in T cells, further detection confirmed the binding of EP2 protein to Cer24:1 ( Figure 22 D).

[0107] ④Molecular dynamics and molecular simulation docking:

[0108] The chemical structure of Cer24:1 and the molecular structure of the receptor protein to be tested were obtained, and the computer molecular simulation docking experiment was performed using AutoDock Vinav1.2.5 software ( Figure 22 E).

[0109] (3) EP2 receptor agonist in vitro response experiment:

[0110] On the basis of Cer24:1 intervention for Th17 differentiation induction, EP2 receptor agonists (such as PGE2) were added, and the cells in each group were collected for total protein extraction. The expression levels of JAK2 / STAT3 signaling pathway related proteins (such as p-STAT3, STAT3, p-JAK2, JAK2, etc.) and RORγt proteins in each group were verified by Western blot. The results showed that EP2 agonists can effectively promote the activation of STAT3 pathway ( Figure 23 AC). However, the addition of EP2 inhibitor (AH6809) in vitro significantly inhibited the differentiation level of Th17 ( Figure 23 D).

[0111] The PGE2 levels in the plasma of model mice and control mice were detected by ELISA, and it was found that the plasma PGE2 concentration of model mice was significantly increased ( Figure 23 E), consistent with the trend of inflammation levels found in neutrophil-induced asthma model mice.

[0112] (4) Effects of EP2 receptor agonists in neutrophil-induced asthma mice:

[0113] Using the established neutrophil asthma mouse model, the EP2 receptor agonist PGE2 ( Figure 24 A), the level of lung inflammation and airway remodeling in each group of mice were detected. The specific detection methods were the same as described above, and then it was determined whether very long chain ceramide affects Th17 cell differentiation by binding to EP2 receptors, thereby exerting a protective effect on neutrophil asthma. The results showed that PGE2 treatment not only increased the plasma IL-22 concentration ( Figure 24 B, 24C), and also increased the proportion of Th17 cells in lung tissue ( Figure 24 D). From a pathological perspective, PGE2 significantly reversed and impaired the anti-inflammatory effect of Cer24:1 on HDM / LPS-induced lung tissue in mice, and promoted the level of lung inflammation and the proportion of neutrophils in lung tissue ( Figure 25Flow cytometry analysis showed that PGE2 treatment promoted the recruitment of neutrophils in BALF ( Figure 26 A, 26B). Detection of STAT3 pathway proteins in lung tissue revealed that, compared with Cer24:1 intervention, PGE2 treatment also promoted the phosphorylation of STAT3 in the lung tissue of model mice ( Figure 26 C, 26D).

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the embodiments described. Without departing from the principles of the present invention, a number of changes, modifications, substitutions, combinations, and simplifications may be made, all of which are equivalent replacement methods and should also be considered as the scope of protection of the present invention.

Claims

1. Use of ceramide molecules in the preparation of drugs for treating and / or preventing asthma.

2. The use according to claim 1, characterized in that in, The ceramide molecule is Cer24:

1.

3. The use according to claim 1, characterized in that in, Asthma is neutrophilic asthma.

4. The use according to claim 1, characterized in that Ceramide molecules have the effect of inhibiting Th17 cell differentiation.

5. The use according to claim 1, characterized in that Ceramide molecules have the effect of inhibiting the secretion of IL-17A inflammatory factor.

6. The use according to claim 1, characterized in that Ceramide molecules have the effect of inhibiting neutrophil-induced airway inflammation in asthma.

7. The use according to claim 1, characterized in that Ceramide molecules have the effect of alleviating airway remodeling.

8. The use according to claim 1, characterized in that Use of ceramide Cer24:1 and / or a substance that increases Smpd1 expression or enzyme activity in the preparation of a drug for treating neutrophilic asthma.

9. The use according to claim 1, characterized in that Use of ceramide Cer24:1 and / or a substance that increases Smpd1 expression or enzyme activity in the preparation of a drug for inhibiting Th17 cell differentiation. 10.CD4 + Application of T cell EP2 protein in the preparation of drugs for alleviating neutrophilic asthma.

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