Use of icariside II in the preparation of a drug for improving macrophage-related airway pathological phenotypes

Abstract

The application relates to the field of biological medicine, and discloses the use of icariin II in the preparation of a medicine for improving a macrophage-related airway pathological phenotype. The use of ICAII in the preparation of the medicine for improving the macrophage-related airway pathological phenotype can realize multiple beneficial effects such as improving lung function, reducing inflammatory infiltration, reducing collagen deposition, reducing mucus secretion, and relieving the pathological phenotype related to macrophages. The application effectively solves the technical deficiency that the prior art lacks targeted intervention on the chronic asthma pathological process involving immune cells, and has outstanding technical advantages and application value.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically the use of the traditional Chinese medicine small molecule compound icariin II in the preparation of a drug for improving macrophage-related airway pathological phenotypes. Background Technology

[0002] Chronic asthma is a chronic respiratory disease characterized by persistent airway inflammation, airway hyperresponsiveness, and airway structural changes. It is characterized by high incidence, long course, and frequent relapses. Current clinical treatment mainly relies on inhaled corticosteroids, long-acting β2-receptor agonists, anticholinergics, leukotriene modulators, and some targeted biologics. These drugs have some effect in controlling acute symptoms and reducing asthma attacks, but they primarily target bronchospasm or the regulation of inflammatory mediators, and are insufficient to comprehensively improve the gradually accumulating airway tissue damage and structural changes caused by chronic inflammation during the course of the disease.

[0003] Especially during the long-term progression of chronic asthma, significant immune cell infiltration occurs in airway tissues, including the repeated aggregation of various inflammatory cells, such as macrophages, which easily leads to persistent inflammatory responses, epithelial damage, abnormal mucus secretion, and tissue fibrosis. However, most existing control medications do not specifically regulate the chronic inflammatory phenotype involving immune cells, resulting in some patients experiencing recurrent attacks, significant airway remodeling progression, and poor treatment response even after standardized use of existing medications. This suggests that in the treatment of chronic asthma, there is still a lack of effective and precise interventions for the pathological processes related to macrophage infiltration and related tissue damage phenotypes.

[0004] With the development of natural drug research, active ingredients derived from traditional Chinese medicine have attracted attention due to their well-defined structures, clear sources, and potentially high safety. Epimedium, a commonly used traditional medicinal herb, contains various flavonoid active substances in its extracts, some of which have been reported to have potential anti-inflammatory or tissue-damaging effects. However, existing publicly available research mainly focuses on total flavonoids or icariin from Epimedium, with extremely limited information on the specific uses of icariin II (ICAII) in chronic respiratory diseases. A search of existing literature and patent databases has not yielded any publicly available technical solutions using ICAII as the main active ingredient to improve the pathological phenotype of chronic asthma associated with immune cell infiltration and airway structural damage.

[0005] In summary, current treatments remain significantly insufficient in controlling macrophage-mediated chronic inflammation and the resulting airway structural changes, and a natural monomeric compound capable of improving these pathological features is still lacking. In particular, there is currently no publicly available information on the use of ICAII in improving chronic asthma and its related pathological manifestations. Therefore, it is necessary to develop a novel drug formulation based on ICAII to address the shortcomings of existing technologies in the treatment of immune cell-mediated chronic asthma and expand the application scope of natural active ingredients in the long-term management of asthma. Summary of the Invention

[0006] The purpose of this invention is to provide a material option that can be used as an effective ingredient in the preparation of drugs to improve chronic asthma, given that existing treatments cannot effectively cover the pathological features involving immune cells and there is a lack of targeted natural compounds for improving chronic asthma. This would fill the technological gap in long-term disease regulation, especially in the intervention of pathological phenotypes related to macrophage infiltration.

[0007] To achieve the above-mentioned objectives, this application adopts the following technical solution:

[0008] In one aspect, this application provides the use of icariin II in the preparation of a medicament for improving macrophage-related airway pathological phenotypes.

[0009] Furthermore, the drug is used to improve pulmonary dysfunction and airway inflammation caused by chronic asthma.

[0010] Furthermore, the drug is used to improve airway remodeling caused by chronic asthma.

[0011] Furthermore, the drug is used to reduce the infiltration of M2 macrophages in a chronic asthma model.

[0012] Furthermore, the drug is used to downregulate the expression of M2 macrophage markers, including ARG1 and / or CD206.

[0013] Furthermore, the drug is used to inhibit macrophage polarization towards the M2 type.

[0014] Secondly, this application provides a pharmaceutical composition for improving macrophage-related airway pathological phenotypes, comprising a therapeutically effective amount of icariin II, and a pharmaceutically acceptable carrier or excipient.

[0015] Compared to existing technologies, the use of ICAII in the preparation of drugs that improve macrophage-related airway pathological phenotypes, as described in this application, effectively overcomes the technical deficiencies of existing technologies in the long-term management of chronic asthma, such as insufficient inflammation control, unsatisfactory improvement in airway remodeling, and lack of targeted intervention in pathological processes involving abnormal immune cells. Because this application selects ICAII as the active ingredient and has systematically validated it in animal models, this technical solution can produce the following significant and reproducible beneficial effects: First, the technical solution of this application can improve the abnormal lung function in animal models of chronic asthma. After ICAII treatment, airway resistance decreased, lung compliance increased, and stimulus-induced airway responsiveness weakened, indicating that the present invention can effectively alleviate the common airflow limitation and airway hyperresponsiveness problems in chronic asthma.

[0016] Secondly, this invention can significantly reduce airway inflammation associated with immune cell infiltration. Histological observations in animal models showed that ICAII treatment significantly reduced peri-airway inflammatory cell infiltration, including a reduction in the inflammatory infiltration area associated with M2 macrophages and a decrease in the degree of inflammation, indicating that ICAII has a good ameliorative effect on chronic inflammatory phenotypes involving abnormal macrophage activation.

[0017] Furthermore, this invention can improve airway tissue structure damage in chronic asthma. Masson staining showed that ICAII can reduce collagen fiber deposition and decrease airway wall thickening, thereby improving fibrosis-like changes caused by long-term chronic inflammation, suggesting that it has a mitigating effect on macrophage-related tissue remodeling.

[0018] Furthermore, this invention can reduce abnormal mucus secretion. PAS staining results showed that the number of goblet cells decreased and the mucus staining area was reduced in the ICAII treatment group, thereby helping to improve airway obstruction caused by excessive mucus secretion and further improving airway patency.

[0019] More importantly, the technical solution of this application has a specific advantage in improving pathological phenotypes involving abnormal macrophage involvement. In a chronic asthma model, the application of ICAII can not only reduce the overall degree of inflammation, but also significantly improve pathological changes such as macrophage infiltration and macrophage-related tissue damage, giving it significant therapeutic potential over existing technologies in chronic asthma under the background of immune imbalance.

[0020] In summary, the use of ICAII described in this application in the preparation of drugs for improving macrophage-related airway pathological phenotypes can achieve multiple beneficial effects, including improving lung function, reducing inflammatory infiltration, decreasing collagen deposition, alleviating mucus secretion, and alleviating macrophage-related pathological phenotypes. This invention effectively addresses the shortcomings of existing technologies in providing targeted interventions for the pathological processes of chronic asthma involving immune cells, demonstrating significant technical advantages and application value. Attached Figure Description

[0021] Figure 1 ICAII can alleviate pulmonary dysfunction and airway inflammation in a mouse model of chronic asthma.

[0022] (A) A chronic asthma model was established in BALB / c mice by sensitization with ovalbumin (OVA) and repeated challenge. ICAII (20, 40, 80 mg / kg) or dexamethasone (2 mg / kg) was administered by gavage after each OVA nebulization challenge for 6 weeks. Changes in lung resistance (RL) during methacholine (Mch) challenge were observed in each group. (C) Changes in dynamic lung compliance (Cdyn) during Mch challenge were observed in each group. (D, E) RL and Cdyn at a Mch concentration of 25 mg / mL; ICAII40, ICAII80, and DEX showed reduction in acute asthma remission (AHR). (F, G) Semi-quantitative scores of peribronchial inflammation and representative HE-stained images of lung tissue (scale bar = 100). (H) Levels of Th2 cytokines IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid (BALF); (I) Serum OVA-specific IgE levels; (J) Levels of IFN-γ, IL-10, and IL-17 in BALF. Data are presented as mean ± SEM (n = 6, IgE n = 3). Statistical markers: # indicates comparison with the NC group. Indicates comparison with the AST group; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001; P<0.05, P<0.01, P<0.001, P<0.0001.

[0023] Figure 2 ICAII can reduce airway remodeling in a mouse model of chronic asthma.

[0024] (A) Representative PAS-stained images of goblet cell metaplasia and mucus hypersecretion in the airway epithelium of different experimental groups (scale bar = 100µm); (B) Semi-quantitative analysis of PAS+ bronchial epithelial cells, expressed as a percentage of the total number of airway epithelial cells; (C) Representative images of Masson's trichrome staining, showing peribronchial collagen deposition in different groups (scale bar = 100µm); (D) Semi-quantitative analysis of airway collagen volume fraction. Data are expressed as mean ± SEM (n = 6). Statistical markers: # indicates comparison with the NC group. Indicates comparison with the AST group; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001; P<0.05, P<0.01, P<0.001, P<0.0001.

[0025] Figure 3 ICAII reduces M2 macrophage infiltration in chronic asthma.

[0026] (A, B) Representative IHC images and quantification of ARG1+ macrophages in lung tissue from different treatment groups (scale bar = 50 μm); (C, D) Representative IHC images and quantification of CD206+ macrophages (scale bar = 50 μm), M2 infiltration was observed in the AST group, and reduced M2 infiltration was observed in the ICAII group; (E) Western blot analysis and quantification showed reduced expression of CD206 and Arg1 proteins in ICAII-treated mice. β-actin was used as an internal control; (F) Analysis of CD206 and ARG1 in BALF and serum confirmed the reduction of M2-related cytokines; (G) Flow cytometry analysis of M2 markers (CD206+) in lung macrophages. + The proportion of M2 macrophages was increased in the CD86- group and decreased in the ICAII40 group, suggesting macrophage repolarization. Data are expressed as mean ± SEM (n = 3-6). Statistical significance: Compared with the NC group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001; P<0.05, P<0.01, P<0.001, P<0.0001 vs AST.

[0027] Figure 4 ICAII exhibits different regulatory effects on M1 macrophage markers in vivo.

[0028] (A, B) Representative IHC images and quantitative analysis of CD86+ macrophages showed that CD86+ cell infiltration was reduced in the AST group, and slightly recovered after ICAII treatment; (C) Representative Western blot images of iNOS, CD80, and CD86; (D) Quantitative analysis of iNOS protein expression. β-actin was used as an internal control; (E) Quantitative analysis of CD80 and CD86 protein expression. β-actin was used as an internal control; (F) The relative expression levels of iNOS and CD86 mRNA and the corresponding cytokine concentrations in BALF were increased in the AST group, and downregulated after ICAII treatment; (G) Representative iNOS immunostaining images and quantification showed that iNOS+ infiltration was increased in the AST group and decreased after ICAII treatment; (H) M1 type (CD86) + Flow cytometry analysis of CD206 macrophages showed that ICAII40 could restore the proportion of M1 macrophages in lung tissue. Data are expressed as mean ± SEM (n = 3–6). Statistical significance: Compared with the NC group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001; P<0.05, P<0.01, P<0.001, P<0.0001 vs AST.

[0029] Figure 5 ICAII inhibits the polarization of macrophages to the M2 type in vitro.

[0030] (A, B) Representative flow cytometry images and quantitative analysis of CD206 and CD163 expression in RAW264.7 and MHS macrophages under different conditions (M0, M0+ICAII, M2, M2+ICAII); (C, D) Real-time quantitative qPCR analysis and protein expression levels of CD206, Arg1, and TGF-β in RAW264.7 and MHS macrophages. β-actin was used as an internal control; (E, F) Real-time quantitative PCR analysis of protein expression levels of CD86, IL-1β, and iNOS in RAW264.7 and MHS macrophages. β-actin was used as an internal control; (G, H) Representative flow cytometry images and quantitative analysis of CD86 expression in RAW264.7 and MHS macrophages under different conditions (M0, M0+ICAII, M1, M1+ICAII). Data are expressed as mean ± SEM (n = 3–6). Statistical significance: Compared with the NC group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001; P<0.05, P<0.01, P<0.001, P<0.0001 vs AST. Detailed Implementation

[0031] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0032] Example 1: ICAII alleviates pulmonary dysfunction and airway inflammation in a mouse model of chronic asthma. Animal model establishment, grouping, and drug administration: A chronic asthma model was established in BALB / c mice using ovalbumin (OVA) sensitization and repeated challenge. Mice were randomly divided into a normal control group (NC), a chronic asthma model group (AST), a dexamethasone positive control group (AST+DEX, 2 mg / kg), and low, medium, and high dose ICAII groups (AST+ICAII20, AST+ICAII40, and AST+ICAII80, 20, 40, and 80 mg / kg, respectively). On days 0, 7, and 14, except for the NC group, mice in all groups were sensitized by intraperitoneal injection of 0.2 mL of a saline suspension containing 20 μg OVA and 2 mg aluminum hydroxide adjuvant; the NC group received an equal volume of saline instead of the OVA suspension. From day 21, except for the NC group, mice in all groups were challenged by ultrasonic nebulization with 3% (w / v) OVA solution for 30 minutes each time, 3 times a week, for 6 consecutive weeks (3 consecutive days of nebulization followed by 4 days of rest). Successful model establishment was defined as the appearance of rapid breathing, cyanosis of the lips, abdominal muscle spasms, head nodding, and unsteady standing. Mice in the icariin II intervention group were administered 20 mg / kg, 40 mg / kg, and 80 mg / kg icariin II suspension by gavage 1 hour before each nebulization, respectively, for 6 consecutive weeks, once daily. The positive control group was administered 2 mg / kg dexamethasone by gavage 1 hour before each nebulization, once daily. Mice in the normal control group were sensitized, challenged, and gavaged with an equal volume of physiological saline, while mice in the model group were gavaged with an equal volume of physiological saline. Samples were collected on day 63.

[0033] Lung function tests: Twenty-four hours after the last nebulization challenge, the mice were weighed and anesthetized by intraperitoneal injection of 2% sodium pentobarbital at 50 mg / kg. After endotracheal intubation, the mice were connected to a mechanical ventilation device and a Buxco FinePointe system. Nebulization challenges were administered sequentially at concentrations of 0, 6.25, 12.5, and 25 mg / mL methacholine (Mch), and lung resistance (R) was measured. L ) and dynamic lung compliance (Cdyn) are used to evaluate airway hyperresponsiveness.

[0034] The results are as follows Figure 1 As shown in (BE). Compared with the NC group, the AST group mice showed increased R under gradually increasing Mch concentration stimulation. L Elevated levels of Cdyn and decreased levels of creatine kinase (Cdyn) suggest airway hyperresponsiveness. In the AST+ICAII40 group stimulated with 12.5 mg / mL Mch, respiratory rate (R) was significantly reduced. L decline( P <0.001), Cdyn increases ( P<0.01), the improvement was similar to that of the DEX group; the AST+ICAII80 group also showed improvement, but was not superior to the AST+ICAII40 group; the AST+ICAII20 group had a relatively weaker effect. Under 25 mg / mL Mch stimulation, the R values ​​of the AST+ICAII40, AST+ICAII80 and AST+DEX groups were significantly higher. L All decreased ( P <0.01), Cdyn improved ( P <0.05). The above results indicate that ICAII can reduce airway hyperresponsiveness in a mouse model of chronic asthma, with the 40 mg / kg dose showing the most significant overall effect.

[0035] Airway inflammation assessment: Whole blood was collected after pulmonary function testing and allowed to coagulate at room temperature for 1 hour. The blood was then centrifuged at 2000 rpm for 10 minutes to separate the serum, which was stored at -80 °C for the detection of OVA-specific IgE. 0.8 mL of pre-cooled phosphate-buffered saline (PBS) was slowly injected into the lungs via the trachea and recovered to obtain bronchoalveolar lavage fluid (BALF). BALF was centrifuged at 2500 rpm for 15 minutes at 4 °C, and the supernatant was collected and stored at -80 °C. IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ were detected using ELISA; the cell pellet was used for white blood cell counting.

[0036] A separate sample from the left lung lobe was fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned to 4 μm. Hematoxylin-eosin (HE) staining was used to observe peribronchial inflammatory infiltration. A semi-quantitative scoring system was established based on the number of layers of peribronchial inflammatory cell rings: 0 points for no lesion; 1 point for a few scattered cells; 2 points for a single-layer cell ring; 3 points for 2-4 layers of inflammatory cells; and 4 points for dense infiltration with more than 4 layers. Scoring and image analysis were performed by blinded evaluators.

[0037] The results are as follows Figure 1 As shown in (FJ), the AST group exhibited significant peribronchial dense inflammatory cell infiltration, airway wall thickening, mucosal edema, and focal luminal narrowing. Compared with the AST group, the AST+ICAII40 group and the AST+DEX group showed significantly reduced inflammatory infiltration and airway wall thickening, and significantly lower inflammation scores. P <0.0001); the inflammation scores of the AST+ICAII20 group and the AST+ICAII80 group also decreased ( P <0.001). IL-4, IL-5, and IL-13 levels were elevated in the BALF of the AST group ( P <0.0001), the above-mentioned Th2 cytokines decreased after ICAII treatment, with the 40 mg / kg group showing the most significant effect. P<0.0001). Serum OVA-specific IgE was decreased in the AST+ICAII40 group ( P <0.01). IFN-γ, IL-10, and IL-17 levels were also elevated in the BALF of the AST group ( P <0.001); ICAII 40 mg / kg can reduce IFN-γ ( P <0.001), while IL-10 and IL-17 showed no significant changes. These results indicate that ICAII can alleviate airway inflammation in a mouse model of chronic asthma, mainly by inhibiting Th2-related inflammatory responses.

[0038] Example 2: ICAII alleviates airway remodeling in a mouse model of chronic asthma Lung tissue, after fixation, embedding, and sectioning as described in Example 1, was used to evaluate airway remodeling. Periodic acid-Schiff (PAS) staining was used to observe goblet cell metaplasia and mucus hypersecretion in the airway epithelium, and the percentage of PAS-positive bronchial epithelial cells was used for quantification. Masson's trichrome staining was used to observe peribronchial and perivascular collagen deposition, and the collagen volume fraction in the airway tissue was calculated. Microscopic images were acquired, and semi-quantitative analysis was performed using ImageJ software.

[0039] The results are as follows Figure 2 As shown in (AD). Compared with the NC group, the AST group showed a significant increase in PAS-positive cells in the bronchial epithelium, indicating goblet cell metaplasia and hypersecretion of mucus; peribronchial and perivascular collagen deposition was also significantly increased, suggesting the formation of airway fibrosis-like remodeling. ICAII treatment reduced the proportion of PAS-positive epithelial cells and collagen volume fraction, with the 40 mg / kg group showing the most significant improvement. P <0.0001). The 80 mg / kg group also reduced airway remodeling, but the improvement in mucus hypersecretion and collagen deposition did not exceed that of the 40 mg / kg group. These results indicate that ICAII can reduce mucus hypersecretion and collagen deposition in a chronic asthma model.

[0040] Example 3: ICAII reduces M2 macrophage infiltration in chronic asthma To evaluate the effect of ICAII on M2 macrophage infiltration, lung tissue was analyzed using immunohistochemistry, Western blot, ELISA, and flow cytometry. For immunohistochemistry, lung tissue sections were dewaxed and hydrated, then subjected to microwave thermal retrieval of antigens using 0.01 mol / L citrate buffer (pH 6.0). Endogenous peroxidase was blocked with 3% H2O2 and then with 5% BSA at room temperature for 1 h. Sections were then incubated overnight at 4 °C with ARG1 or CD206 primary antibody, followed by DAB staining, hematoxylin counterstaining, and mounting. Microscopic images were acquired by blinded evaluators and semi-quantitatively analyzed using ImageJ software. Approximately 30 mg of lung tissue was collected and homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors. The homogenate was centrifuged at 14000×g for 15 min at 4 °C, and the supernatant was collected. After determining the protein concentration using the BCA method, 20 μg of protein was loaded onto each well for SDS-PAGE, electroporation, blocking with 5% skim milk powder, incubation with primary antibody at 4 °C overnight, incubation with HRP-labeled secondary antibody at room temperature for 1 h, and ECL imaging. CD206 and ARG1 protein expression were detected, with β-actin used as an internal control. The levels of CD206 and ARG1 in BALF and serum were detected using appropriate detection kits.

[0041] For flow cytometry analysis, the upper right lung lobe was harvested, washed with pre-cooled PBS, mechanically minced, and passed through a 300-mesh filter to prepare a single-cell suspension, which was then treated with erythrocyte lysis buffer. Approximately 1 × 10^6 cells were resuspended in 100 μL of flow cytometry buffer. The Fc receptor was first blocked for 10 min with anti-mouse CD16 / 32 antibody, followed by the addition of fluorescently labeled antibodies for CD45, CD11b, Ly6G, F4 / 80, CD86, and CD206. The cells were incubated at 4 °C in the dark for 30 min, and dead cells were excluded using live / dead cell staining. Analysis was performed sequentially using single-cell phylogenetics, debris exclusion, live cell phylogenetics, CD45-positive leukocyte phylogenetics, CD11b-positive myeloid cell phylogenetics, Ly6G-negative cell phylogenetics, and F4 / 80-positive macrophage phylogenetics. Macrophages that were CD206-positive and CD86-negative were defined as M2 macrophages. Data were acquired using a CytoFLEX S flow cytometer and analyzed using FlowJo software.

[0042] The results are as follows Figure 3 As shown in (AG). The AST group showed a significant increase in peribronchial and perivascular ARG1-positive and CD206-positive cells in the lung tissue, suggesting increased infiltration of M2 macrophages. After ICAII treatment, the infiltration of both ARG1-positive and CD206-positive cells decreased, with the 40 mg / kg group showing the most significant inhibitory effect. P<0.0001. Western blot showed that the expression of ARG1 and CD206 proteins in lung tissue decreased after ICAII treatment. P <0.01%, consistent with the results of BALF and serum tests ( P <0.01). Flow cytometry results showed that the proportion of M2 macrophages was approximately 30% in the NC group, increased to approximately 85% in the AST group, and decreased to approximately 39% after treatment with ICAII 40 mg / kg. P <0.001). The above results indicate that ICAII can reduce the infiltration of M2 macrophages in a chronic asthma model.

[0043] Example 4: ICAII exhibits different regulatory effects on M1 macrophage markers in vivo. To analyze the effects of ICAII on M1 macrophage-related indicators, immunohistochemistry was used to detect CD86 and iNOS-positive cell infiltration in lung tissue, and Western blot was used to detect the expression of iNOS, CD80, and CD86 proteins in lung tissue. Approximately 20 mg of lung tissue was used to extract total RNA, and after RNA purity testing, cDNA was synthesized via reverse transcription and subjected to real-time quantitative PCR. Using GAPDH or β-actin as internal controls, the relative mRNA expression of iNOS and CD86 was calculated using the 2^-ΔΔCt method. The levels of iNOS and CD86 in BALF and serum were detected using appropriate detection kits. Flow cytometry was performed according to the method described in Example 3, and macrophages that were CD86-positive and CD206-negative were defined as M1 macrophages.

[0044] The results are as follows Figure 4 As shown in (AH). Under different detection methods, M1 macrophage-related indicators showed certain differences. Immunohistochemical results showed that CD86-positive cell infiltration was reduced in the AST group, and recovered somewhat after ICAII treatment; iNOS immunostaining increased in the AST group (…). P <0.001), decreased after ICAII treatment ( P <0.01. Western blot showed that iNOS protein expression increased after ICAII treatment ( P <0.05), while CD80 and CD86 proteins only showed an increasing trend, not reaching statistical significance. Lung tissue mRNA, BALF, and serum tests showed elevated iNOS and CD86-related indicators in the AST group; ICAII treatment reduced some of these indicators. P <0.05). Flow cytometry results showed that the proportion of M1 macrophages in the AST group was approximately 11.8%, which recovered to approximately 32.6% after treatment with ICAII 40 mg / kg. P<0.05). Therefore, the effect of ICAII on M1 macrophage markers is method-dependent; combined with the results of Example 3, the main stabilizing effect of ICAII is to inhibit M2 macrophage infiltration and improve the M1 / M2 balance.

[0045] Example 5: Inhibition of macrophage polarization towards M2 type in vitro In vitro validation was performed using RAW264.7 and MHS mouse macrophage cell lines. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 ℃ and 5% CO2. Unstimulated cells were designated as the M0 group; M1 polarization was induced by stimulation with 100 ng / mL LPS for 24 h; and M2 polarization was induced by stimulation with 20 ng / mL IL-4 and IL-13 for 24 h. ICAII cytotoxicity was evaluated using the CCK-8 assay. The results showed no significant cytotoxicity of ICAII at concentrations not exceeding 10 μmol / L, therefore 10 μmol / L was selected as the concentration for subsequent experiments. After polarization induction, ICAII was added and incubated for another 24 h. Flow cytometry was used to detect the expression of CD206, CD163, and CD86; real-time quantitative PCR and Western blot were used to detect the expression of CD206, Arg1, TGF-β, CD86, IL-1β, and iNOS.

[0046] The results are as follows Figure 5 As shown in (AH). In RAW264.7 and MHS cells, stimulation with IL-4 and IL-13 induced M2 polarization. After ICAII treatment, the proportion of CD163-positive cells decreased from 39.8% to 14.9% in RAW264.7 cells and from 21.6% to 13.3% in MHS cells. P <0.01); the proportion of CD206-positive cells in RAW264.7 cells also decreased ( P <0.05. Simultaneously, ICAII reduced the expression of M2-related indicators such as CD206, Arg1, and TGF-β ( P <0.001). Under LPS-induced M1 polarization conditions, ICAII has a mild regulatory effect on some M1-related proteins; flow cytometry showed that the proportion of CD86-positive cells after ICAII treatment reached 39.6% and 33.0% in RAW264.7 and MHS cells, respectively. P <0.01). In unstimulated M0 cells, ICAII treatment alone did not induce significant CD86 expression. These results indicate that ICAII can directly inhibit IL-4 and IL-13-induced macrophage M2 polarization, adjusting the macrophage phenotype towards a lower fibrosis-associated state.

[0047] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Application of icariin II in the preparation of drugs for improving macrophage-related airway pathological phenotypes.

2. The application according to claim 1, characterized in that, The drug is used to improve pulmonary dysfunction and airway inflammation caused by chronic asthma.

3. The application according to claim 1, characterized in that, The drug is used to improve airway remodeling caused by chronic asthma.

4. The application according to claim 1, characterized in that, The drug is used to reduce the infiltration of M2 macrophages in a chronic asthma model.

5. The application according to claim 4, characterized in that, The drug is used to downregulate the expression of M2 macrophage markers, including ARG1 and / or CD206.

6. The application according to claim 1, characterized in that, The drug is used to inhibit macrophage polarization towards the M2 type.

7. A pharmaceutical composition for improving macrophage-associated airway pathological phenotypes, characterized in that, It contains a therapeutically effective amount of icariin II, as well as a pharmaceutically acceptable carrier or excipient.

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