Use of ceramide molecule in treatment of asthma

Abstract

Use of a ceramide molecule in the treatment of asthma.

Description

Application of ceramide molecules in the treatment of asthma Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of ceramide molecules in the treatment of asthma. Background Technology

[0002] Asthma can be further classified into different phenotypes based on the proportion of induced sputum cells. The subtype with ≥61% neutrophils and <3% eosinophils is called neutrophilic asthma. Neutrophilic asthma is characterized by airway neutrophilic inflammation driven by non-type 2 inflammatory factors such as T helper cell 17 (Th17) and its cytokine interleukin-17 (IL-17), and exhibits resistance to inhaled corticosteroids. Patients with neutrophilic asthma often present with refractory asthma and may even develop severe asthma. Currently, various biological targeted agents have been developed for personalized treatment of asthma; however, 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 therapies such as bronchial thermoplasty.

[0003] With in-depth research into the pathogenesis of asthma, it has been found that IL-17 is associated with inflammatory cells in asthma, especially neutrophils and eosinophils, as well as airway remodeling, thus participating in the occurrence and development of asthma. Targeting Th17 cells holds promise as a potential target for regulating asthma, especially neutrophilic asthma. Significant sphingolipid metabolism disorders exist in neutrophilic asthma, and ceramides, as important bioactive sphingolipids, play a crucial role in immune regulation. Currently, drugs for neutrophilic asthma are mainly inhaled corticosteroids and bronchodilators, lacking corresponding targeted drugs. Although Th17-related cytokines act as central effector factors in airway inflammatory storms and airway remodeling, substantial progress is still needed in the treatment of IL-17A monoclonal antibodies. Their therapeutic efficacy, drug resistance issues, and high treatment costs remain obstacles to their widespread application. Studies have found that sphingolipids such as ceramides can effectively regulate the function of immune cells such as T cells. Therefore, this invention focuses on the therapeutic effects of ceramides in asthma. As a metabolic byproduct inherent in the human body, exploring its mechanisms and applying it to the treatment of neutrophilic asthma through complementary therapies will help reduce drug side effects and improve the efficacy of precision treatment.

[0004] Cer24:1 ceramide is a naturally occurring long-chain monounsaturated ceramide with the structural formula N-tetracosylsphingosine. It contains a fatty acid side chain with 24 carbon atoms and a double bond, and a sphingosine moiety with 18 carbon atoms and a double bond. Its molecular formula is C42H81NO3, and its molecular weight is 648.097. Cer24:1 ceramide possesses various physicochemical properties, with 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 widely distributed in mammalian cells, playing a crucial role in maintaining cell membrane structure, signal transduction, and metabolic regulation, and is also an important bioactive molecule. As a ceramide with a specific chain length, Cer24:1 is commercially available. Technical issues

[0005] The purpose of this invention is to provide the use of ceramide molecules in the preparation of medicaments for the treatment and / or prevention of asthma.

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

[0007] Among them, asthma is neutrophilic asthma.

[0008] Applications of this invention include:

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

[0010] Application of substances that supplement ultra-long chain ceramide Cer24:1 and / or increase Smpd1 expression or enzyme activity in the preparation of products to relieve neutrophilic asthma.

[0011] Application of substances that supplement ultra-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 applications, the improvement of neutrophilic asthma can manifest as reducing respiratory neutrophil recruitment, decreasing Th17-related inflammatory factors, airway remodeling, and alleviating airway hyperresponsiveness.

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

[0014] Another object of the present invention is to provide CD4 + Application of T cell EP2 protein in the preparation of products for relieving neutrophilic asthma; the application is achieved through CD4... +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 Th17 cell differentiation inhibitors; the application is through CD4 + 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 manifest as reduced neutrophil recruitment in the respiratory tract, decreased Th17-related inflammatory factors, airway remodeling, and relief of airway hyperresponsiveness.

[0017] Another object of the present invention is to provide a pharmaceutical composition containing ceramide molecules.

[0018] The pharmaceutical compositions of the present invention can be prepared into any pharmaceutically acceptable dosage form.

[0019] Another object of the present invention is to provide a liposome containing a ceramide molecule Cer24:1 and a pharmaceutically acceptable carrier.

[0020] The liposomes of the present invention can be prepared according to existing technologies.

[0021] Another object of the present invention is to provide the use of liposomes in the preparation of medicaments for the treatment and / or prevention of asthma.

[0022] Among them, asthma is neutrophilic asthma.

[0023] The pharmaceutical uses of this invention were obtained after extensive experimental research:

[0024] This invention first uses targeted sphingolipid metabolomics to detect plasma, exhaled condensate, and induced sputum supernatant from patients and healthy controls. The detection revealed that among various differential metabolites, the reduction of ultra-long chain ceramide Cer24:1 was the most significant. Correlation analysis showed that ultra-long chain ceramide was significantly negatively correlated with Th17. C57 mice were sensitized with a combination of HDM (house dust mites) and LPS (lipopolysaccharide), and a neutrophilic asthma model was constructed through continuous HDM stimulation. Flow cytometry analysis revealed a significant increase in Th17 cells in lung tissue and a marked increase in Th17-related cytokines in bronchoalveolar lavage fluid and plasma. Furthermore, the neutrophilic asthma model mice also exhibited a widespread decrease in Cer24:1 levels in lung tissue and BALF (bronchoalveolar lavage fluid) supernatant. Detection of ceramide metabolic enzymes revealed reduced expression of several enzymes regulating Cer24:1 synthesis, including ASM (acidic sphingomyelin, encoded by the Smpd1 gene, whose main function is to promote the hydrolysis of sphingomyelin to ceramides, especially ultra-long-chain ceramides). By further constructing Smpd1KO mice and establishing a neutrophilic asthma model, we found that KO mice exhibited reduced Cer24:1 content, as well as increased airway neutrophil ratio, airway resistance, and Th17-related inflammation levels.

[0025] To investigate the role of the ultra-long-chain ceramide Cer24:1 in neutrophilic asthma, mice were first intraperitoneally injected with Cer24:1. Airway resistance was assessed using a FlexiVent pulmonary function testing instrument. The proportion of Th17 cells and the level of neutrophil inflammation were detected by flow cytometry, ELISA, specific pathological staining, and immunohistochemistry of lung tissue. The degree of lung fibrosis was statistically analyzed using Masson staining and immunohistochemistry. The results showed that the proportion of Th17 cells in the lung tissue of mice was significantly reduced after injection of Cer24:1, and the Masson staining score of the lung tissue was significantly alleviated. Further in vitro experiments confirmed that Cer24:1 inhibits Th17 cell differentiation in vitro. Transcriptomic and Western blot analyses revealed that Cer24:1 inhibits Th17 cell differentiation by downregulating the JAK2-STAT3 signaling pathway.

[0026] Mechanistically, the long-chain ceramide Cer24:1 inhibits Th17 differentiation by binding to the T cell surface protein EP2. Molecularly, it inhibits the downstream JAK2-STAT3 pathway by binding to EP2, thereby suppressing the expression of RORγt transcription factor and leading to naïve CD4. +Impaired T cell differentiation into Th17 cells. Administration of prostaglandin E2 (PGE2), a specific ligand of the EP2 receptor protein, effectively reversed the inhibitory effect of the ultra-long-chain ceramide C24:1 on Th17 differentiation. In animal models, we found that PGE2 infusion also reversed the protective effect of Cer24:1 on HDM / LPS-induced mice. This invention has significant application value; the above results mean that ultra-long-chain ceramide compounds such as Cer24:1 can be used as novel monotherapy for neutrophilic asthma or as a component of pharmaceutical drugs, and can be used in different dosage forms and routes of administration to treat neutrophilic asthma.

[0027] The beneficial effects of this invention are that, in vitro, it has been demonstrated that the ultralong-chain ceramide C24:1 can inhibit Th17 cell differentiation and the secretion of the IL-17A inflammatory factor. In vivo experiments have confirmed its effective inhibition of airway inflammation in neutrophilic asthma and its relief of airway remodeling, revealing its potential for treating neutrophilic asthma. As a natural sphingolipid small molecule compound, it is easy to synthesize, and as an inherent lipid molecule contained in the body, the side effects of supplementation therapy are relatively small, thus possessing significant pharmaceutical value. Attached Figure Description

[0028] Figure 1: Metabolomics analysis revealed a widespread reduction in the ultralong-chain ceramide Cer24:1 in the exhaled condensate (EBC) of patients with neutrophilic asthma. (AC): EBCs were provided by 60 asthma patients and 16 healthy controls. Volcano plots depict differences in glycerophospholipids and sphingomyelin in EBCs between different groups: asthma vs. control (A), neutrophilic asthma vs. control (B), and neutrophilic asthma vs. non-neutrophilic asthma (C). The color of the dots represents the trend of metabolites between different groups (P < 0.05 and log2FC > 0.8 were considered significant differences between the two groups). We found that Cer24:1 was significantly reduced in all three comparison combinations.

[0029] Figure 2: Significant reduction of Cer24:1 in neutrophilic asthma patients: In this clinical cohort, EBCs were provided by 60 asthma patients and 16 healthy controls, plasma by 59 asthma patients and 16 healthy controls, and induced sputum by 54 asthma patients and 23 healthy controls. The figure shows the relative abundance of ceramides of different chain lengths between neutrophilic asthma (red) and healthy controls (blue). The levels of ultralong-chain ceramides, represented by Cer24:1, were significantly reduced in neutrophilic asthma patients in EBC (A), plasma (B), and sputum supernatant (C). Data are expressed as mean ± SEM. Statistical analysis was performed using the two-tailed Student's test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0030] Figure 3: Diagnostic value of Cer24:1 in neutrophilic asthma: (AC) ROC curves of Cer24:1 in EBC (A), plasma (B), and induced sputum supernatant (C) were used to differentiate neutrophilic asthma patients from healthy controls. The results showed that the area under the curve was greater than 0.7 for all samples. (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 some beneficial effects.

[0031] Figure 4: Neutrophilic asthma induced by HDM combined with LPS in mice: (A) Schematic diagram of experimental design: Mice were sensitized and challenged with house dust mites (HDM), while control mice received an equal volume of PBS intranasally. (B) Airway resistance in each group of mice was measured using a pulmonary function instrument to characterize airway hyperresponsiveness. Rrs represents respiratory resistance. The results showed that the airway resistance in the model mice was significantly higher than that in the control group. (C) Bar chart showing the percentage of neutrophils, eosinophils, macrophages, and lymphocytes in BALF cell smears (n=3 mice / group). The results showed that the proportion of neutrophils in the BALF of the model group mice was significantly increased. Data are expressed as mean ± SEM. Statistical analysis was performed using the two-tailed Student's test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Figure 5: Inflammatory infiltration, airway remodeling, and hypermucus secretion in mouse lung tissue induced by HDM combined with LPS: Immunohistochemical staining images and pathological score histograms (GI, PR) of representative H&E staining (A, D), PAS staining (B, E), Masson trichrome staining (C, F), and α-SMA (J, M), MPO (K, N), and Ly6G (L, O) in mouse lung tissue sections, with a scale bar of 50 μm. The results showed that the model mice exhibited exacerbated airway neutrophilic inflammation, hypermucus secretion, and airway remodeling at the pathological level (n=4 mice / group). Data are expressed as mean ± SEM. Statistical analysis was performed using the two-tailed Student's test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0033] Figure 6: Hydromycin combined with LPS-induced neutrophilic asthma dominated by Th17 inflammation in mice: (AC) Bar chart analysis of plasma and BALF concentrations of IL-17A (A), IL-17F (B), and IL-22 (C) showed a significant increase in the concentrations of Th17-related cytokines in both plasma and BALF supernatant. (D) Flow cytometry statistical analysis of the proportions of Th1, Th2, Th17, and Treg cells in mouse lung tissue (n=6 mice / group) showed a significant increase in the proportion of Th17 cells in the lung tissue of the model mice. Data are expressed as mean ± SEM. Statistical analysis was performed using the two-tailed Student's test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0034] Figure 7: Widespread reduction of ultralong-chain ceramides in HDM / LPS-induced neutrophilic asthmatic mice: (AB) Cluster heatmap of sphingolipid content in BALF and lung tissue of PBS-induced control mice (red) and HDM / LPS-induced mice (green), showing that most sphingolipids and ceramide concentrations were significantly reduced in the model group mice. (CD) Bar chart of relative abundance of ceramides of different chain lengths in BALF and lung tissue of PBS-induced control mice (blue), HDM / LPS-induced neutrophilic asthmatic mice (red), and HDM-induced eosinophilic asthmatic mice (yellow), showing that the ceramide concentration in BALF and lung tissue of neutrophilic asthmatic mice was lower than that in both the control group and the eosinophilic asthmatic mice. 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.

[0035] Figure 8: Decreased ceramide metabolism in lung tissue of neutrophil-asthmatic mice: (A) Introduction to the metabolic pathways of ceramide. This figure illustrates the metabolic pathways of 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 aminohydrolase; SPHK, sphingosine kinase; sphingomyelin synthase; sphingomyelin diesterase; CERK, ceramide kinase). (B) Analysis of RNA-seq data from mouse lungs induced by PBS and HDM / LPS revealed a downregulation of the de novo sphingolipid synthesis pathway in HDM / LPS-induced mice (n=6 mice / group), and decreased transcription of multiple sphingolipid metabolic enzyme genes, represented by Smpd1.

[0036] Figure 9: Decreased expression of multiple sphingolipid metabolism enzymes in lung tissue of neutrophil-asthmatic mice. (AM) RT-qPCR analysis of ceramide synthase-related genes in the lungs of HDM / LPS-induced mice compared to the control group. In HDM / LPS-induced mice, the transcription of three sphingolipid metabolism enzymes, Sptlc2, Cers2, and Smpd1, was downregulated (n=4-5 mice / group). (N) The protein expression of Sptlc2, Cers2, and Smpd1 in the lung tissues of both HDM / LPS-induced and control mice was also significantly decreased. Statistical analysis was performed using a two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001,

[0037] Figure 10: Exacerbation of Th17 inflammation in Smpd1KO neutrophilic asthmatic mice: (AB) Airway resistance in all groups under acetylcholine stimulation and at baseline showed that Smpd1KO-induced mice had significantly higher airway resistance than wild-type mice. (CE) Concentrations of Th17-related cytokines such as IL-17A, IL-17F, and IL-22 in plasma and BALF showed a significant increase in IL-22 concentration in the plasma of Smpd1KO-induced mice. (FG) Flow cytometry statistical analysis of the proportions of Th1, Th2, Th17, and Treg cells in the lungs (F) and spleen (G) (n=6 mice / group). (ZombieAqua...) - CD3 + CD8 - CD4 + T-cell gating analysis revealed the proportion of all Th subgroups. Results showed an increased proportion of Th17 cells in the lung tissue of Smpd1KO mice. Data are expressed as mean ± SEM. Statistical analysis was performed using the two-tailed Student's test and one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0038] Figure 11: Increased inflammatory infiltration in lung tissue of Smpd1KO neutrophilic asthmatic mice: (AH) lung tissue sections showing representative H&E, PAS and MPO, Ly6G immunohistochemical staining (AP) maps and pathological score histograms (QT). The results indicate increased inflammatory infiltration in the lung tissue of Smpd1KO-induced mice and increased neutrophil recruitment in the lung tissue (scale bar = 50 μm). 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.

[0039] Figure 12: Increased neutrophil recruitment in the BALF of Smpd1KO neutrophil-asthmatic mice: (A, B) Flow cytometry analysis of the 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 are based on CD45. + The proportion of neutrophils in cells. Results showed increased recruitment of neutrophils in the BALF of Smpd1KO model mice. 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.

[0040] Figure 13: Cer24:1 injection alleviates neutrophilic asthma inflammation: (A) Schematic diagram of experimental design: Cer24:1 ceramide (10 mg / kg body weight) was injected once daily during the modeling period. (B) Airway resistance in mice under acetylcholine stimulation. Cer24:1 did not significantly improve airway resistance in modeled mice. (CD) Bar chart of IL-17A and IL-17F concentrations in plasma and BALF. The results showed that the Cer24:1 intervention group significantly reduced the plasma IL-17A concentration. (E) Single-cell sequencing annotation results of lung tissue. (F) Single-cell sequencing showed that the proportion of Th17 in the lung tissue of Cer24:1-treated mice was lower than that in the modeled group. 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.

[0041] Figure 14: Flow cytometry analysis and statistical evaluation of Th1, Th2, Th17, and Treg in lung tissue of Cer24:1-injected mice with reduced neutrophilic asthma: (A, B) (A) Lung tissue (A), spleen (B) (n=6 mice / group). ZombieAqua was used. - CD3 +CD8 - CD4 + T-cell gating analysis of T-cell subset proportions. Results showed that Cer24:1 intervention suppressed the proportion of Th17 cells in lung tissue but did not affect the proportion of Th17 cells in the spleen. 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.

[0042] Figure 15: Cer24:1 injection alleviates lung inflammation and mucus hypersecretion in neutrophilic asthmatic mice: (AP) representative H&E and PAS staining, MPO and Ly6G immunohistochemical staining (AL), and histogram of pathological scores (MP) in lung tissue sections, scale bar 50 μm. 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 ANOVA; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0043] Figure 16: Cer24:1 injection alleviates airway remodeling in neutrophilic asthmatic mice: (AL) Histograms of representative Masson trichrome staining, α-SMA and Fibronectin immunohistochemical staining, and pathological scores in lung tissue sections, scale bar 50 μm. Results showed that Cer24:1 alleviated airway remodeling in neutrophilic asthmatic mice (n=4 mice / group). (MP) Bar chart analysis of plasma and BALF airway remodeling-related factors MMP-9 and TIMP-1 concentrations. Results showed that plasma and BALF supernatant MMP-9 and BALF TIMP-1 concentrations were significantly reduced after Cer24:1 intervention. 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.

[0044] Figure 17: Neutrophil recruitment in the BALF of Cer24:1 injection-inhibited neutrophilic asthmatic mice: (A, B) Flow cytometry analysis and statistical evaluation of neutrophils, eosinophils, alveolar macrophages (AM), and interstitial macrophages (IM) in BALF (A, n=3 mice / group) and lungs (B, n=6 mice / group). The proportion of all myeloid cells was gated by CD45. +Cellular analysis. Results showed that Cer24:1 intervention significantly reduced the proportion of neutrophils in BALF. 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.

[0045] Figure 18: Cer24:1 inhibits Th17 differentiation in vitro: (AF) Flow cytometry analysis of control solvent or Cer24:1-treated mouse naïve CD4 + Statistical analysis of the intracellular staining proportions of non-pathogenic Th17 (A), pathogenic Th17 (B, C), Treg, Th1, and Th2 cells in T cells. Results showed that Cer24:1 treatment significantly inhibited the differentiation of both pathogenic and non-pathogenic Th17 cells, but did not affect the in vitro differentiation of Th1, Th2, and Treg cells. 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.

[0046] Figure 19: Cer24:1 inhibits Th17 cell differentiation in vitro, but does not affect Th17 cell apoptosis or proliferation: (AB) Representative immunoblotting analysis of RORγt in pathogenic Th17 cells treated with control solvent or Cer24:1 showed that Cer24:1 treatment inhibited RORγt protein expression. (C, D) RT-qPCR analysis of Il17a and Il17f mRNA expression after 3 days of treatment with control solvent or Cer24:1 (n=4) showed that Cer24:1 treatment inhibited gene transcription of Il17a and Il17f. (E) ELISA showed that the concentration of IL-17A in cell culture supernatant decreased with increasing Cer24:1 concentration. (F) Statistical analysis of the percentage of pathogenic Th17 cells treated with control solvent or amitriptyline (Smpd1 inhibitor) for 3 days (n=4) showed that inhibiting Smpd1 activity effectively promoted Th17 cell differentiation. (G) Ceramides of different chain lengths (C16, C20, 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 expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0047] Figure 20: Cer24:1 inhibits the differentiation of pathogenic Th17 cells by downregulating the JAK2-STAT3 signaling pathway: (A) Transcriptome analysis of Th17 cells treated with Cer24:1 and control solvents showed that most differentially expressed genes in pathogenic Th17 cells cultured with control solvent and 10 μM Cer24:1 were downregulated. (B) GSEA enrichment analysis based on transcriptome showed that the JAK-STAT pathway was significantly downregulated in the Cer24:1 intervention group. (C) KEGG enrichment of differentially phosphorylated signaling pathway proteins based on phosphorylated protein microarray detection. The JAK-STAT pathway is highlighted in red. The results showed that the JAK-STAT signaling pathway was significantly downregulated after Cer24:1 intervention.

[0048] Figure 21: Cer24:1 intervention inhibits STAT3 pathway phosphorylation in pathogenic Th17 cells: (AC) Representative immunoblotting analysis of phosphorylated JAK2 and STAT3 in control or Cer24:1-treated Th17 cells. Results show that Cer24:1 intervention inhibits STAT3 pathway activation. (D, E) Representative immunoblotting analysis of phosphorylated JAK2 and STAT3 in control, Cer24:1, or Cer24:1 combined with Colivelin (a STAT3 activator)-treated Th17 cells shows that Colivelin effectively reverses the decrease in STAT3 phosphorylation levels induced by Cer24:1. (F) Flow cytometry analysis of pathogenic Th17 cells treated with Cer24:1 or Cer24:1 combined with Colivelin shows that Colivelin effectively promotes Th17 differentiation. (G) ELISA shows that Colivelin effectively promotes an increase in IL-17A concentration in cell supernatant. Data are expressed as mean ± SEM. Statistical analysis was performed using the two-tailed Student's test and one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0049] Figure 22: Cer24:1 specifically binds to EP2, inhibiting the activation of the STAT3 signaling pathway in Th17 cells: (A) Single-cell sequencing data revealed CD4 + (B) Flow cytometry analysis of the percentage of Th17 cells treated with Cer24:1 or Cer24:1 in combination with PGE2 (a specific ligand of EP2) showed that PGE2 significantly promoted 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 the interacting proteins were separated by streptavidin agarose gel extraction, followed by Western blotting analysis using anti-EP2 and anti-EP4 antibodies. (E) Molecular docking prediction of the EP2-Cer24:1 binding site. 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.

[0050] Figure 23: Activation of the EP2 receptor significantly promotes STAT3 pathway activation and Th17 differentiation: (AC) Representative immunoblotting analysis of phosphorylated JAK2-STAT3 pathway proteins and RORγt in Th17 cells treated with control solvent, Cer24:1, or Cer24:1 combined with PGE2 showed that PGE2 (EP2 agonist) treatment significantly promoted RORγt and STAT3 phosphorylation activation. (D) AH6809 (EP2 inhibitor) treatment significantly reduced pathogenic Th17 cell differentiation. (E) HDM / LPS induced a significant increase in plasma PGE2 concentration in mice (n=6 mice / group). 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.

[0051] Figure 24: PGE2 significantly reversed the ameliorative effect of Cer24:1 on HDM / LPS-induced mice: (A) Schematic diagram of 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 cytometry analysis and statistical evaluation of Th1, Th2, Th17, and Treg cells in lung tissue (n=6 mice / 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 ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0052] Figure 25: PGE2 significantly reversed and aggravated the anti-inflammatory effect of Cer24:1 on HDM / LPS-induced lung tissue in mice: (AT) lung sections with representative H&E, PAS, MPO, and Ly6G immunohistochemical staining (AP), and pathological score histogram (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 expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0053] Figure 26: PGE2 promotes HDM / LPS-induced neutrophil recruitment in mouse BALF: (AB) Flow cytometry analysis and statistical evaluation of neutrophils in 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 BALF, but not in tissue. (C, D) Representative immunoblotting analysis of phosphorylated JAK2-STAT3 pathway proteins in lung tissue of control, Cer24:1 intervention, or Cer24:1 combined with PGE2 treatment mice 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 Implementation

[0054] The present invention will be further illustrated by the following examples.

[0055] The product and its preparation method according to the present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials can be obtained from publicly available commercial sources.

[0056] Example 1: Confirmation of reduced levels of ultralong chain ceramides in neutrophilic asthma patients.

[0057] (1) Clinical studies included asthma patients:

[0058] ① Cross-sectional study:

[0059] This study consecutively enrolled asthma patients visiting the Department of Respiratory and Critical Care Medicine at Peking University Third Hospital, and also collected data from healthy non-asthmatic patients matched for gender, age, and BMI. Asthma patients were categorized based on sputum cytology counts into four types: 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%). The main exclusion criteria were: acute asthma exacerbation, comorbid chronic obstructive pulmonary disease, bronchiectasis, pneumonia, obstructive sleep apnea-hypopnea syndrome, malignant tumors, various acute and chronic respiratory failures, and severe cardiovascular diseases.

[0060] Demographic information of the subjects was collected and recorded, such as gender, age, BMI, age of onset, lung function, complete blood count, induced sputum cell differential count, lung CT results, asthma control test (ACT) score, allergen testing, etc.

[0061] ② Exhaled breath condensate (EBC) collection and specimen processing

[0062] EBCs were collected using a TURBO-DECCS exhaled condensate collector (Medivac PARMA, Italy). During EBC collection, participants wore a nasal clip and took deep breaths for 10 minutes. EBC samples were immediately stored at -80°C until analysis.

[0063] ③Induced sputum collection, specimen processing, and cell counting and classification:

[0064] Sputum samples were collected from subjects after nebulized inhalation of 3% hypertonic saline for 20–30 min. Sputum plugs were removed from saliva, mixed with 4 volumes of 0.1% dithiothreitol, vortexed, and incubated for 30 min. After filtration, the sputum supernatant and cells were separated by centrifugation. Cell smears were stained with Wright-Giemsa, and 200 inflammatory cells were counted and classified under high magnification. The remaining cells were resuspended in flow cytometry staining buffer.

[0065] ④ Peripheral blood sample collection and processing:

[0066] Collect 2-4 mL of EDTA-anticoagulated blood from patients or healthy controls. Immediately centrifuge the collected blood at 3000 rpm for 10 minutes to obtain the supernatant plasma.

[0067] ⑤ Targeted metabolomics detection of phospholipid content in plasma, induced sputum supernatant, and other samples:

[0068] Lipids were extracted from human exhaled breath condensate using a chloroform / methanol extraction method, and qualitative and quantitative analyses of lipid molecules were performed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Targeted analysis was used to detect phospholipid molecules such as sphingolipids and glycerophospholipids (e.g., Cer, SM, PC, PE, PI, etc.). Further differential analysis of exhaled breath condensate lipids among different groups (asthmatic patients vs. controls, neutrophilic asthmatic patients vs. controls, and neutrophilic asthmatic patients vs. neutrophilic asthmatic patients) identified the differentially expressed phospholipid molecule Cer24:1 (Figure 1), which was found to be significantly reduced in all three comparison combinations.

[0069] Further analysis of plasma and induced sputum supernatant samples verified the differences in ceramide molecules of different chain lengths between neutrophilic asthma and healthy individuals, revealing that ultra-long chain ceramides, represented by Cer24:1, were significantly reduced in neutrophilic asthma patients (Figure 2).

[0070] Next, we analyzed the ROC curves of Cer24:1 in EBC, plasma, and induced sputum supernatant. The results showed that the area under the curve was greater than 0.7, indicating that Cer24:1 has diagnostic value in differentiating neutrophilic asthma from the control population (Figure 3A-C). Furthermore, we explored the correlation between plasma Cer24:1 levels and peripheral blood Th17 and the proportion of neutrophils in induced sputum using Pearson correlation analysis (Figure 3D, 3E).

[0071] (2) A mouse model of neutrophilic asthma was induced using HDM and LPS:

[0072] ① House dust mite HDM + lipopolysaccharide LPS combined to induce neutrophilic asthma model (Figure 4A):

[0073] Experimental group: C57BL / 6 mice aged 6 to 8 weeks were selected. For the first three days, they were sensitized by nasal drops of 25 μg HDM + 10 μg LPS. Starting from day 7, they were challenged by nasal drops of 25 μg HDM for 9 consecutive days. The mice were harvested 24 hours after the last challenge.

[0074] Control group: C57BL / 6 mice aged 6 to 8 weeks were selected. For the first three days, mice were given an equal volume of PBS via intranasal instillation. Starting from day 7, mice were given an equal volume of PBS via intranasal instillation for 9 consecutive days. Mice were harvested 24 hours after the last instillation.

[0075] ② Assay for airway hyperresponsiveness in neutrophilic asthmatic mice:

[0076] Using the FinePointe™ systemic pulmonary function testing instrument, different concentrations of methacholine (0, 6.25, 12.5, 25, 50 mg / mL) were applied to model mice for gradient stimulation, and changes in airway resistance were recorded. Airway responsiveness was calculated based on the maximum airway response (Figure 4B). The results showed that the airway resistance of the model mice was significantly higher than that of the control mice.

[0077] ③ Neutrophil inflammation and Th17 detection in lung tissue of neutrophil-asthmatic mice:

[0078] Bronchoalveolar lavage fluid was collected from two groups of mice, smeared, and dried. The proportions of neutrophils, eosinophils, macrophages, and lymphocytes in the BALF cells of mice were analyzed using Swiss Giemsa staining. The staining results showed that the proportion of neutrophils in the BALF of the model group mice was significantly increased (Figure 4C).

[0079] Left lung lobe was harvested for lung tissue pathological staining, such as hematoxylin-eosin (HE) staining (0-4 semi-quantitative score) to detect airway inflammatory cell infiltration; periodic acid-Schiff (PAS) staining for airway goblet cell proliferation (0-4 semi-quantitative score); peri-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); peri-airway smooth muscle proliferation (α-SMA immunohistochemical staining, image analysis to calculate the peri-airway smooth muscle area / basement membrane thickness), etc. The results showed that the lung tissue of the model mice pathologically exhibited characteristics of increased airway neutrophil inflammation, hypersecretion of mucus, and airway remodeling (Figure 5).

[0080] The levels of cytokines such as IL-17A, IL-17F, and IL-22 in mouse serum and bronchoalveolar lavage fluid were detected using a multifactor flow cytometry kit. Significant increases in the concentrations of Th17-related cytokines in plasma and BALF supernatant were found (Figures 6A-C). Lung tissues from two groups of mice were collected, minced, enzymatically digested, ground, filtered, and lysed with erythrocytes to prepare single-cell suspensions. T cells were stimulated to secrete cytokines such as IL-17A, IL-4, and IFN-γ using PMA and iomycin. After incubation at 37°C for 4-6 hours, the proportions of various T cell subsets, including Th1 / 2 / 17 / Treg, in the tissues were detected using flow cytometry staining with cytokine internal standards. Flow cytometry analysis showed a significant increase in the proportion of Th17 cells in the lung tissues of the model mice (Figure 6D).

[0081] ④ Targeted metabolomics detection of phospholipid content in mouse tissues

[0082] Lipid molecules were extracted from mouse plasma, bronchoalveolar lavage fluid supernatant, and lung tissue homogenate using a chloroform / methanol extraction method. Qualitative and quantitative analysis of lipid molecules was performed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Targeted analysis was used to detect phospholipid molecules such as sphingolipids and glycerophospholipids (e.g., Cer, SM, PC, PE, PI). Furthermore, differential analysis of ceramides was performed among different groups (HDM+LPS-induced neutrophilic asthmatic mice, HDM-induced eosinophilic mice, and healthy control mice). The results showed that the concentration of ceramides in BALF and lung tissue of neutrophilic asthmatic mice was lower than that in both the control and eosinophilic asthmatic mice (Figure 7).

[0083] Example 2 confirms that ultra-long chain ceramides have a protective effect against neutrophilic asthma.

[0084] (1) It was confirmed that ceramide synthesis was reduced in the lung tissue of neutrophil-asthmatic mice:

[0085] The metabolic pathways of ceramide synthesis include the sphingomyelinase pathway and the de novo synthesis pathway (Figure 8A). Transcriptome sequencing analysis was used to investigate altered pathways in the lung tissue of neutrophil-asthmatic mice. The study found that the de novo sphingolipid synthesis pathway was downregulated in the model mice, and that the transcriptional levels of several sphingolipid metabolic enzyme genes, represented by Smpd1, were reduced (Figure 8B). RT-qPCR and Western blot analysis were used to verify the expression of multiple sphingolipid metabolic enzymes in the lung tissue of model and control mice, revealing a significant downregulation of the expression of three sphingolipid metabolic enzymes: Sptlc2, Cers2, and Smpd1 (Figure 9).

[0086] (2) Construction and intervention of Smpd1 gene knockout mouse model:

[0087] Smpd1 gene knockout mice (C57BL / 6J background) were further constructed. Smpd1 gene synthesis, via acid sphingomyelinase, regulates the synthesis of ultra-long-chain ceramides. In this mouse model, the deletion of the Smpd1 gene leads to a deficiency in the synthesis of C22-C24 ultra-long-chain ceramides, facilitating the investigation of the role of ultra-long-chain ceramides in neutrophilic asthma. Wild-type mice with the same background were used as a control group. A neutrophilic asthma mouse model was established using the same method of HDM+LPS combined exposure, and Smpd1 levels were measured. - / - Asthmatic mice and Smpd1 + / +Differences in airway resistance, lung tissue inflammation levels, neutrophil ratio, and Th17 inflammation in asthmatic mice were investigated using the methods described above. Airway resistance in Smpd1 KO mice was significantly higher than in wild-type mice under acetylcholine stimulation and at baseline (Fig. 10A, 10B). Furthermore, plasma IL-22 concentration was significantly increased in KO mice (Fig. 10C-E), and the proportion of Th17 cells in lung tissue was significantly higher in KO mice than in wild-type mice (Fig. 10F, 10G), while no significant difference was observed in the proportion of Th17 cells in the spleen.

[0088] Further evaluation of the pathological staining of lung tissue from Smpd1 knockout mice revealed increased inflammatory infiltration in the lung tissue of Smpd1 KO mice and increased neutrophil recruitment in the lung tissue, without affecting airway mucus hypersecretion (Figure 11). Mouse lung tissue was collected, minced, enzymatically digested, ground, filtered, and lysed with erythrocytes to prepare a single-cell suspension. Flow cytometry staining with cell surface antibodies (CD45, CD11b, CD11c, SiglecF, Ly6G) was used to detect the proportions of macrophages, neutrophils, and eosinophils in the lung tissue single-cell suspension and BALF cells. Increased neutrophil recruitment in BALF was found in Smpd1 KO mice (Figure 12).

[0089] (3) Cer24:1 metabolic supplementation experiment:

[0090] A mouse model of neutrophilic asthma was constructed. Ceramide C24:1 standard was dissolved in anhydrous ethanol. Mice in the intervention group were intraperitoneally injected daily with 10 mg / kg of Cer24:1 solution, while the control group received an equal volume of saline (Figure 13A). The improvement in airway resistance, lung inflammation levels, and airway remodeling in the model mice was assessed, with specific indicators as described previously. Although Cer24:1 intervention did not affect airway resistance (Figure 13B), it significantly reduced plasma IL-17A concentration (Figures 13C, 13D). Further single-cell sequencing analysis of lung tissue from healthy control mice, the model group, and the Cer24:1 intervention group (Figure 13E) revealed a decreasing trend in the proportion of Th17 cells in the lung tissue of the intervention group compared to the model group (Figure 13F). Flow cytometry analysis 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 cells in lung tissue but did not affect the proportion of Th17 cells in the spleen (Figure 14).

[0091] Further pathological evaluation of the effects of Cer24:1 injection on lung inflammation, mucus hypersecretion, and airway remodeling in neutrophilic asthmatic mice revealed that Cer24:1 intervention significantly reduced inflammatory infiltration and neutrophil recruitment in lung tissue and alleviated pulmonary mucus hypersecretion (Figure 15). Cer24:1 also showed some alleviating effect on airway remodeling in neutrophilic asthmatic mice. In addition to Masson staining and Fibronectin immunohistochemistry, ELISA detection of epithelial remodeling-related cytokines showed a significant decrease in plasma and BALF supernatant MMP-9 and BALF TIMP-1 concentrations after Cer24:1 intervention (Figure 16). Flow cytometry analysis of the proportion of granulocytes and macrophages in lung tissue and BALF showed that Cer24:1 intervention significantly reduced the proportion of neutrophils in BALF (Figure 17).

[0092] Example 3: Ceramide Cer24:1 participates in the pathogenesis of neutrophilic asthma through Th17 cells.

[0093] (1) naïve CD4 + In vitro T cell differentiation induction experiment:

[0094] ①naïve CD4 + T-cell acquisition:

[0095] Single-cell suspensions were obtained from the spleen and lymph nodes of C57BL / 6J mice, and naïve CD4 cells were used to... + Naïve CD4 cells were isolated using a T-cell magnetic bead sorting kit. + T cells were cultured using T cell culture medium (RPMI-1640, containing 10% FBS, 1% L-glutamine, etc.) for CD4 cells. + T cells were activated using anti-CD3 (5 μg / mL) and anti-CD28 (5 μg / mL) monoclonal antibodies.

[0096] ②Induction of differentiation of Th1, Th2, Th17, and iTreg cells:

[0097] Th1-induced differentiation conditions: addition of IL-12 (20 ng / mL) and IL-2 (10 ng / mL);

[0098] Th2-induced differentiation conditions: addition of IL-4 (10 ng / mL) and IL-2 (10 ng / mL);

[0099] Pathogenic Th17 induction differentiation conditions: addition of IL-6 (20 ng / mL), TGF-β (3 ng / mL), IL-23 (10 ng / mL) and IL-1β (10 ng / mL);

[0100] Non-pathogenic Th17 induction differentiation conditions: addition of IL-6 (20 ng / mL) and TGF-β (3 ng / mL);

[0101] iTreg induction differentiation conditions: addition of TGF-β (8 ng / mL).

[0102] ③ Under the above differentiation conditions, different concentrations of Cer24:1 (0, 5 μM, 10 μM, 20 μM) were added for metabolic intervention, and T cell proliferation and differentiation were monitored during induction. After 72 hours of culture, cell culture supernatant was collected to detect cytokine levels, and cells were collected for cell function testing.

[0103] Flow cytometry was used to detect cell polarization efficiency: Flow cytometry antibodies such as CD4, IL-17A, IL-4, IFN-γ, and Foxp3 were used as markers, and intracellular factor staining was used to analyze the proportions of Th1, Th2, Th17, and iTreg cells (Figure 18). Western blot analysis was used to analyze the expression levels of Th17 cell-specific proteins (such as RORγt) (Figure 19A, 19B). RT-qPCR analysis was used to analyze the expression levels of Th17 cell-specific genes (such as Il-17a and Il-17f) (Figure 19C, 19D). ELISA was used to detect the level of IL-17A in the culture supernatant (Figure 19E). The results showed that Cer24:1 inhibited Th17 differentiation in vitro.

[0104] Adding different concentrations of amitriptyline (Amt), an inhibitor of acid sphingomyelinase (Smpd1), to the culture medium significantly increased the in vitro differentiation rate of Th17 cells (Figure 19F). However, intervention with ceramides of other chain lengths such as C16, C20, and C22 did not significantly change the in vitro differentiation rate of Th17 cells (Figure 19G).

[0105] Apoptosis rate was analyzed using Annexin V / PI staining (Fig. 19H), and T cell proliferation was detected using CFSE dye (Fig. 19I). The results showed that Cer24:1 intervention did not affect Th17 cell proliferation or apoptosis.

[0106] Example 4: Ceramide Cer24:1 regulates Th17 differentiation through the EP2-STAT3 axis

[0107] (1) Screening and verification of the signaling pathways affected by Cer24:1:

[0108] Total RNA was extracted from Th17 cells after intervention in the Cer24:1 and solvent control groups, and RNA sequencing was performed. The expression levels of genes related to Th17 differentiation were compared among the groups (Figure 20A), and enrichment analysis was conducted based on differentially expressed genes to screen for significantly downregulated JAK-STAT pathways (Figure 20B). Simultaneously, total protein was extracted from both groups of cells, and phosphorylated protein microarray analysis was performed. Based on differentially expressed protein enrichment analysis, it was found that phosphorylated proteins of the JAK-STAT pathway were also significantly downregulated in the Cer24:1 group (Figure 20C).

[0109] Proteins were extracted from Th17 cells after Cer24:1 intervention-induced differentiation. 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 STAT3 pathway protein phosphorylation (Figure 21A-C).

[0110] Based on Cer24:1 intervention to induce Th17 differentiation, a STAT3 agonist (Colivelin) was used for reversal experiments. 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 study verified whether Cer24:1 regulates Th17 cell differentiation through the JAK2 / STAT3 pathway. The results showed that Colivelin, as an effective STAT3 agonist, significantly promoted Th17 differentiation (Figures 21D-F).

[0111] (2) Cer24:1 receptor prediction and validation

[0112] ① Receptor agonist function recovery experiment:

[0113] Screening for CD4 using single-cell sequencing data + The G protein-coupled receptor (GPCR) gene is highly expressed on T cells (Figure 22A). Further functional recovery experiments were conducted by adding corresponding G protein-coupled receptor agonists or ligands to the Cer24:1 intervention for Th17 induced differentiation. It was found that adding EP2 receptor agonists (such as PGE2) to the recovery experiment could effectively promote Th17 cell differentiation (Figure 22B).

[0114] ② Surface plasmon resonance (SPR) detection:

[0115] Using SPR technology to detect Cer24:1 and CD4 + The binding affinity of the G protein-coupled receptor (EP2) that may bind on the surface of T cells was analyzed, and it was found that Cer24:1 interacts with EP2, with a dissociation constant of 417.5 μM (Figure 22C).

[0116] ③ Co-immunoprecipitation assay (Co-IP):

[0117] Immunoprecipitation was used to verify whether the EP2 receptor interacts with Cer24:1. CD4+ was precipitated using a biotin-coupled anticeramide antibody. + The ceramide-protein complex in T cells was further examined to confirm the binding of EP2 protein to Cer24:1 (Figure 22D).

[0118] ④ Molecular dynamics and molecular simulation docking:

[0119] The chemical structure of Cer24:1 and the molecular structure of the receptor protein to be detected were obtained, and computer molecular simulation docking experiments were performed using AutoDock Vina v1.2.5 software (Figure 22E).

[0120] (3) In vitro reversion experiment of EP2 receptor agonist:

[0121] Based on Cer24:1 intervention to induce Th17 differentiation, an EP2 receptor agonist (such as PGE2) was added, and total protein was extracted from cells in each group. Western blot analysis was used to verify 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. The results showed that the EP2 agonist effectively promoted STAT3 pathway activation (Figure 23A-C). However, the addition of an EP2 inhibitor (AH6809) significantly inhibited Th17 differentiation (Figure 23D).

[0122] The levels of PGE2 in the plasma of model mice and control mice were detected by ELISA. It was found that the plasma PGE2 concentration of model mice was significantly increased (Figure 23E), which was consistent with the trend of inflammation levels found in neutrophil asthma model mice.

[0123] (4) Effects of EP2 receptor agonists in neutrophilic asthmatic mice:

[0124] Using an established mouse model of neutrophilic asthma, EP2 receptor agonist PGE2 was injected into Cer24:1-treated mice and control mice (Figure 24A). Lung tissue inflammation levels and airway remodeling were measured in each group using the same methods described above. This aimed to clarify whether ultra-long-chain ceramides affect Th17 cell differentiation by binding to EP2 receptors, thereby exerting a protective effect against neutrophilic asthma. Results showed that PGE2 treatment not only increased plasma IL-22 concentration (Figures 24B, 24C) but also increased the proportion of Th17 cells in lung tissue (Figure 24D). Pathologically, PGE2 significantly reversed and impaired the anti-inflammatory effect of Cer24:1 on HDM / LPS-induced lung tissue in mice, promoting lung tissue inflammation levels and the proportion of neutrophils in lung tissue (Figure 25). Flow cytometry analysis confirmed that PGE2 treatment promoted neutrophil recruitment in BALF (Figures 26A, 26B). Detection of STAT3 pathway proteins in lung tissue revealed that, compared to Cer24:1 intervention, PGE2 treatment also promoted STAT3 phosphorylation in the lung tissue of model mice (Figures 26D, 26E).

[0125] The above description is merely a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the described embodiments. Various changes, modifications, substitutions, combinations, and simplifications can be made without departing from the principle of the present invention, and all such substitutions should be considered equivalent replacements and should also be considered within the scope of protection of the present invention.

Claims

1. The use of ceramide molecules in the preparation of drugs for the treatment and / or prevention of asthma.

2. Use according to claim 1, characterized in that, in, The ceramide molecule has a Cer24:1 ratio.

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

4. Use according to claim 1, characterized in that, Ceramide molecules have the effect of inhibiting Th17 cell differentiation; inhibiting the secretion of IL-17A inflammatory factor; inhibiting airway inflammation in neutrophil asthma; and alleviating airway remodeling.

5. The use according to claim 1, characterized in that, The use of ceramide Cer24:1 and / or substances that increase Smpd1 expression or enzyme activity in the preparation of drugs for treating neutrophilic asthma.

6. Use according to claim 1, characterized in that, Application of ceramide Cer24:1 and / or substances that increase Smpd1 expression or enzyme activity in the preparation of drugs that inhibit Th17 cell differentiation.

7. CD4 + Use of T cell EP2 protein in the preparation of a medicament for alleviating neutrophilic asthma.

8. Use of pharmaceutical compositions containing the ceramide molecule Cer24:1 in the preparation of medicaments for the treatment and / or prevention of asthma.

9. The use of liposomes containing the ceramide molecule Cer24:1 in the preparation of drugs for the treatment and / or prevention of asthma.

10. The use of liposomes containing the ceramide molecule Cer24:1 in the preparation of drugs for the treatment and / or prevention of neutrophilic asthma.

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