Application of icariside II in preparation of medicine for treating chronic asthma

Abstract

The invention relates to the field of biological medicines, and discloses application of icariside II in preparation of a medicine for treating chronic asthma. The icariside II plays a role in treating chronic asthma by improving lung functions, relieving airway inflammation, relieving airway remodeling and inhibiting lung epithelium-mesenchymal transition. The invention discloses the new application of icariside II in treating chronic asthma for the first time, and provides a brand-new drug choice with multi-target treatment potential for clinical treatment of chronic asthma through a multi-way synergistic effect.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, to the use of icariin II in the preparation of a medicament for treating chronic asthma. Background Technology

[0002] Asthma is a common respiratory disease, with over 30 million patients in my country, and its incidence and mortality rates are increasing year by year. Clinical features of asthma include cough, wheezing, chest tightness, and shortness of breath. Pathologically, airway inflammation is considered a core element, triggering subsequent airway hyperresponsiveness, mucus secretion, and airway remodeling. Airway remodeling caused by chronic inflammation is directly proportional to the severity and duration of asthma and is considered a significant contributing factor to refractory asthma.

[0003] Previous studies have suggested that epithelial-mesenchymal transition (EMT) is associated with cancer cell metastasis. Current research has found that type II EMT is closely related to tissue remodeling and participates in the pathological progression of chronic asthma. Studies have shown that inflammation can induce type II EMT, which further exacerbates airway remodeling in chronic asthma, thus leading to refractory asthma. EMT, as a transitional stage from inflammation to remodeling in asthma, plays a crucial role in the exacerbation of chronic asthma. Effectively controlling inflammation and inhibiting EMT are effective means to combat airway remodeling and prevent refractory asthma.

[0004] Currently, modern medicine's treatment of asthma is relatively limited, primarily using medications such as glucocorticoids, β2-receptor agonists, and leukotriene modifiers to treat symptoms, suppress asthma inflammation, relax bronchial smooth muscle, and dilate the bronchi. However, long-term use of these medications can easily cause adverse reactions, such as central obesity, elevated blood sugar, gastric ulcers, and hypertension. Furthermore, modern medicine currently lacks specific drug treatments for the pathological changes in chronic asthma, such as airway remodeling and EMT (extracorporeal membrane oxygenation), and there is an urgent need to find effective drugs.

[0005] Icariside II (IS) is a flavonoid compound isolated from the traditional Chinese medicine Epimedium. Flavonoids themselves possess potent anti-inflammatory and anti-allergic effects, and according to literature reports, icariin II also exhibits antioxidant and anti-tumor metastasis effects. A review of domestic and international research progress shows that icariin II has played a positive role in the treatment of lung cancer EMT, airway remodeling, and myocardial fibrosis. However, in the field of chronic asthma EMT, no studies have yet confirmed its efficacy. Summary of the Invention

[0006] The purpose of this invention is to explore the use of icariin II in the preparation of drugs for treating chronic asthma.

[0007] To achieve the above-mentioned objectives, this application adopts the following technical solution: In the first aspect, this application discloses the use of icariin II in the preparation of a medicament for treating chronic asthma. Furthermore, the icariin II exerts its therapeutic effect on chronic asthma by improving lung function.

[0008] Furthermore, the icariin II exerts its therapeutic effect on chronic asthma by reducing airway inflammation.

[0009] Furthermore, the icariin II exerts its therapeutic effect on chronic asthma by alleviating airway remodeling.

[0010] Furthermore, the icariin II exerts its therapeutic effect on chronic asthma by inhibiting pulmonary epithelial-mesenchymal transition.

[0011] Secondly, this application discloses a pharmaceutical composition comprising a therapeutically effective amount of icariin II, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0012] Furthermore, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers or excipients.

[0013] In summary, this application has the following beneficial effects: This application discloses for the first time a novel use of icariin II in the treatment of chronic asthma. It exerts its therapeutic effect on chronic asthma through multiple synergistic pathways, by improving lung function, reducing airway inflammation, alleviating airway remodeling, and inhibiting pulmonary epithelial-mesenchymal transition. This provides a novel drug option with multi-target therapeutic potential for the clinical treatment of chronic asthma. Attached Figure Description

[0014] Figure 1 : Molecular structure of icariin II and construction of an OVA-induced chronic asthma mouse model; A: Molecular structure of icariin II; B: Construction of an OVA-induced chronic asthma mouse model.

[0015] Figure 2 Effects of icariin II on lung function in mice with chronic asthma: NC: normal control group; Asthma: chronic asthma model group; Asthma + Dex 2mg / kg: positive control group; Asthma + IS 20mg / kg: low-dose icariin II group; Asthma + IS 40mg / kg: medium-dose icariin II group; Asthma + IS 80mg / kg: high-dose icariin II group. A: Airway resistance (RL) test results; B: Lung compliance (Cdyn) test results; C and D: Comparison of RL and Cdyn among groups at a Mch concentration of 25 mg / ml; Experimental data are expressed as mean ± SEM, N=5-7 birds / group, ** p<0.01, *** p<0.001 vs NCgroup, # p<0.05, ## p<0.01, ### p<0.001 vs Asthma group.

[0016] Figure 3 Effects of icariin II on white blood cell count in BALF of chronic asthmatic mice: NC: normal control group; Asthma: chronic asthma model group; Asthma + Dex 2mg / kg: positive control group; Asthma + IS 20mg / kg: low-dose icariin II group; Asthma + IS 40mg / kg: medium-dose icariin II group; Asthma + IS 80mg / kg: high-dose icariin II group. AE: Results of white blood cell count, eosinophil count, neutrophil count, lymphocyte count, and monocyte count in BALF; Experimental data are expressed as mean ± SEM, N=4-8 birds / group, *** p<0.001 vs NC group, #p<0.05, ## p<0.01, ### p<0.001 vs Asthma group.

[0017] Figure 4 Effects of icariin II on airway inflammation in mice with chronic asthma: NC: normal control group; Asthma: chronic asthma model group; Asthma + Dex 2mg / kg: positive control group; Asthma + IS 20mg / kg: low-dose icariin II group; Asthma + IS 40mg / kg: medium-dose icariin II group; Asthma + IS 80mg / kg: high-dose icariin II group. AD: Detection results of IL-4, IL-13, IL-1β, and TNF-α levels in BALF; E: HE staining image of mouse lung tissue; F: Quantitative analysis of HE staining in mouse lung tissue; Experimental data are expressed as mean ± SEM, N=5-8 birds / group, *** p<0.001 vs NC group, #p<0.05, ## p<0.01, ### p<0.001 vs Asthma group.

[0018] Figure 5 Effects of icariin II on airway remodeling in mice with chronic asthma: NC: normal control group; Asthma: chronic asthma model group; Asthma + Dex 2mg / kg: positive control group; Asthma + IS 20mg / kg: low-dose icariin II group; Asthma + IS 40mg / kg: medium-dose icariin II group; Asthma + IS 80mg / kg: high-dose icariin II group. AB: PAS and MASSON staining images of mouse lung tissue; CD: Quantitative analysis of PAS and MASSON staining in mouse lung tissue; EF: Detection results of TGF-β1 and MMP-9 levels in BALF; Experimental data are expressed as mean ± SEM, N=5-8 birds / group, *** p<0.001 vs NC group, #p<0.05, ## p<0.01, ### p<0.001 vs Asthma group.

[0019] Figure 6 Effects of icariin II on pulmonary EMT in mice with chronic asthma: NC: normal control group; AST: chronic asthma model group; DEX: positive control group; IS: high-dose icariin II group. AD: Western blot analysis of the expression levels and relative densities of Occludin, E-cadherin, α-SMA, and N-cadherin proteins in mouse lung tissue; Results are expressed as ratios of ocludin, E-cadherin, α-SMA, and N-cadherin relative to β-actin. Experimental data are expressed as mean ± SEM, N=5 individuals / group, * p<0.05, *** p<0.001 vs NC group, ## p<0.01, ### p<0.001 vs AST group.

[0020] Figure 7Effects of icariin II on TGF-β1-induced EMT in alveolar epithelial cells: NC: normal control group; Model: TGF-β1-induced EMT model group; IS 5: 5 μM icariin II intervention group; IS 10: 10 μM icariin II intervention group; A: CCK8 assay to detect the effect of different concentrations of icariin II on the viability of A549 alveolar epithelial cells; BF: Western blotting analysis to detect the expression levels and relative densities of ocludin, E-cadherin, α-SMA, and N-cadherin in total protein of A549 cells. Results are expressed as the ratios of ocludin, E-cadherin, α-SMA, and N-cadherin to β-actin. Experimental data are expressed as mean ± SEM, N=4-5 / group, * p<0.05, ** p<0.01, *** p<0.001 vs NC group, # p<0.05 vs Model group. Detailed Implementation

[0021] 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.

[0022] (1) Source of materials: Icariin II was purchased from Shanghai Ronghe Company, with a purity >98%, molecular formula: C27H30O10, molecular weight: 514.53. Figure 1 A); Healthy, clean-grade female BALB / c mice aged 6-8 weeks, weighing 16-20g, were provided by Shanghai Slack Laboratory Animal Co., Ltd.

[0023] (2) Construction and grouping of mouse models: Healthy, clean-grade female BALB / c mice aged 6-8 weeks, weighing 16-20g, were randomly divided into 6 groups of 10 mice each, for a total of 60 mice. The groups were as follows: normal control group (NC), chronic asthma model group (Asthma), positive control group (Asthma + Dex 2mg / kg), low-dose icariin II group (Asthma + IS 20mg / kg), medium-dose icariin II group (Asthma + IS 40mg / kg), and high-dose icariin II group (Asthma + IS 80mg / kg). Figure 1B). BALB / c mice were placed in a clean room simulating diurnal cycles (12 hours of daylight, 12 hours of darkness) at a temperature of 20–30°C and humidity of 50%. Mice in the chronic asthma model group were sensitized by a single intraperitoneal injection of 20 μg of OVA and 0.2 ml of a saline suspension containing 2 mg of aluminum hydroxide starting on day 0. Sensitization was repeated on days 7 and 14, for a total of 3 sensitizations (lasting 3 weeks). Starting on day 21, mice were administered 3% OVA via ultrasonic nebulization for approximately 30 minutes each time, continuously for 8 weeks, 3 times a week (3 consecutive days of nebulization followed by 4 days of rest). Successful model establishment was indicated by the appearance of rapid breathing, cyanosis of the lips, abdominal muscle spasms, head nodding, and unsteady gait. Mice in the icariin II intervention group were administered 20 mg / kg, 40 mg / kg, and 80 mg / kg icariin II suspension by gavage one hour before each nebulization, starting from day 21, for 8 consecutive weeks, once daily. The positive control group was administered 2 mg / kg dexamethasone by gavage one hour before each nebulization, once daily. Mice in the normal control group were sensitized, challenged, and then 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 77.

[0024] Example 1: Effects of icariin II on pulmonary function in mice with chronic asthma Within 24 hours of completing their last nebulized OVA inhalation, all animals were nebulized with acetylcholine chloride (Mch) at concentration gradients (0, 6.25 mg / mL, 12.5 mg / mL, 25 mg / mL) using a Buxco small animal pulmonary function instrument. Airway resistance (RL) and lung dynamic compliance (Cdyn) were directly measured using the FinePointe RC system to evaluate the lung function of the mice.

[0025] The results are as follows Figure 2 As shown, compared with the normal control group, the model group had increased airway resistance and decreased lung compliance. After intervention with icariin II, airway resistance was significantly reduced and lung compliance recovered. The efficacy of the high-dose icariin II group was close to that of the positive control group.

[0026] Example 2: Effect of icariin II on the number of white blood cells in the BALF of chronic asthmatic mice After centrifugation of BALF, the supernatant was aspirated, and the cell pellet was retained. The pellet was resuspended in 50 µl of PBS for cell classification and counting. The resuspended cell pellet was then analyzed using a hematology analyzer to classify and count inflammatory cells such as eosinophils, neutrophils, lymphocytes, and monocytes in the BALF.

[0027] The results are as follows Figure 3 As shown, the white blood cell count results in BALF revealed a significant increase in inflammatory cells such as eosinophils, neutrophils, lymphocytes, and monocytes in the model group compared to the normal control group. After intervention with icariin II, these inflammatory cells were significantly downregulated.

[0028] Example 3: Effects of icariin II on airway inflammation in mice with chronic asthma After pulmonary function testing, the mouse trachea and lungs were separated. An lavage apparatus was inserted into the left bronchus via the trachea, and PBS solution was injected. The lavage was repeated three times, 0.3 ml each time. BALF was collected (recovery rate >80%), centrifuged at 2000 rpm, 4°C, for 15 min, and the supernatant was collected and stored at -80°C for later use. The levels of cytokines IL-4, IL-13, TNF-α, and IL-1β in BALF were detected using ELISA.

[0029] The middle lobe of the left lung was taken, fixed by immersion in 4% neutral formaldehyde, dehydrated by routine gradient ethanol, impregnated with paraffin, embedded, and prepared into paraffin sections. The sections were stained with hematoxylin and eosin (HE) and inflammatory cells were scored under a light microscope.

[0030] The results are as follows Figure 4 As shown, the levels of inflammatory factors IL-4, IL-5, IL-1β, and TNF-α in BALF were detected. The levels of IL-4, IL-5, IL-1β, and TNF-α in the model group were significantly higher than those in the normal control group. After intervention with icariin II, the levels of IL-4, IL-5, IL-1β, and TNF-α were significantly downregulated. HE staining results showed that the model group had significant peri-airway inflammatory cell infiltration, which was reduced after intervention with icariin II.

[0031] Example 4: Effects of icariin II on airway remodeling in chronic asthmatic mice The levels of TGF-β1 and MMP-9 cytokines in BALF were detected by ELISA, using the same method as in Example 3.

[0032] The middle lobe of the left lung was taken, fixed by immersion in 4% neutral formaldehyde, dehydrated by routine gradient ethanol, paraffin-embedded, and embedded to prepare paraffin sections. PAS and MASSON staining was performed, and goblet cell proliferation, mucus secretion, and collagen volume were scored under a light microscope.

[0033] The results are as follows Figure 5As shown, the detection results of remodeling-related cytokines TGF-β1 and MMP-9 in BALF showed that the levels of TGF-β1 and MMP-9 in the model group were significantly higher than those in the normal control group. After intervention with icariin II, the levels of TGF-β1 and MMP-9 were significantly downregulated. PAS and MASSON staining results showed that peri-airway goblet cell metaplasia, mucus secretion, and collagen deposition were significantly increased in the model group. After intervention with icariin II, peri-airway goblet cell metaplasia, mucus secretion, and collagen deposition were significantly reduced.

[0034] Example 5: Effects of icariin II on EMT in the pulmonary tract of mice with chronic asthma Mouse lung tissue was minced and added to RIPA reagent containing protease and phosphatase inhibitors. Homogenization was performed at 800g, -20°C, for 10 min. The homogenized tissue suspension was centrifuged at 12000g, 4°C, for 10 min, and the supernatant was collected. The extracted protein concentration was detected using the Pierce BCAProtein Assay Kit. Sample buffer and RIPA were added to standardize the protein concentration. Denatured protein was obtained by boiling at 100°C for 10 min. Western blot analysis was performed to detect the expression level of the target protein. Separation was achieved by electrophoresis using 10% SDS-PAGE, and the protein was transferred to a 0.25 μm PVDF membrane. The membrane was blocked at 25°C with 5% skim milk for 2 h. Primary antibodies Occludin, E-cadherin, α-SMA, and N-cadherin were incubated overnight at 4°C. HRP-conjugated secondary antibody was incubated at 25°C for 1.5 h. Image exposure was performed using a LAS-4000 imaging system to detect protein signal intensity. Finally, the protein signals were analyzed using ImageJ software.

[0035] The results are as follows Figure 6 As shown, Western blotting results revealed that epithelial markers Occludin and E-cadherin were significantly downregulated and interstitial markers α-SMA and N-cadherin were significantly upregulated in the lung tissue of the model group mice, indicating EMT changes. After intervention with icariin II, epithelial markers Occludin and E-cadherin were significantly upregulated and interstitial markers α-SMA and N-cadherin were significantly downregulated in the lung tissue, inhibiting EMT.

[0036] Example 6: Effects of icariin II on TGF-β1-induced EMT in alveolar epithelial cells An EMT cell model was constructed using type II alveolar epithelial cells (A549). The safe concentration of icariin II in A549 cells was investigated using a CCK8 assay. Logarithmic growth phase A549 cells were prepared into a cell suspension and seeded into 96-well plates, ensuring each well contained 5-6 × 10⁶ cells.3 Cells were treated with different concentrations of icariin II and incubated in an incubator. After treatment, 10 μl of CCK8 was added to each well of the 96-well plate, and the plates were incubated for 1-2 hours, adjusting the time according to the actual number of cells in each well. After treatment, the OD value of the samples in the 96-well plate was measured at 450 nm using a microplate reader, and the absorbance of each well was measured to calculate cell viability. Cell viability = [Absorbance of drug-treated group - Absorbance of blank group] / [Absorbance of control group (absorbance of non-drug-treated group) - Absorbance of blank group] × 100%. A cell viability of not less than 90% after drug intervention was considered a usable concentration.

[0037] Alveolar epithelial cell EMT model was constructed using growth factor TGF-β1, with the following groups: normal control group (NC), TGF-β1-induced EMT model group (Model), icariin II 5 μM intervention group (IS 5), and icariin II 10 μM intervention group (IS 10). A549 cell suspension was seeded into 6-well plates. After cell adhesion and growth, except for the NC group, 10 ng / ml TGF-β1 was added to induce modeling, while the treatment groups were given 5 μM and 10 μM icariin II, respectively. After 24 h, when the cells expanded and covered the 6-well plates, the culture medium was aspirated from the wells, washed twice with PBS, and RIPA reagent containing protease and phosphatase inhibitors was added. Cells were scraped from the wells and collected into EP tubes, incubated on ice for 10 min, centrifuged at 12000 rpm for 10 min at 5°C, and the supernatant was collected. Protein concentrations were adjusted, and the proteins were denatured by boiling. Western blotting was used to detect the expression levels of Occludin, E-cadherin, α-SMA, and N-cadherin proteins.

[0038] The results are as follows Figure 7 As shown in the CCK8 results, the cell viability of alveolar epithelial cells was above 90% after 24 hours of intervention with 5 μM and 10 μM icariin II, which were used as usable concentrations. Western blotting results showed that in the Model group, the epithelial markers Occludin and E-cadherin were downregulated, while the mesenchymal markers α-SMA and N-cadherin were upregulated, indicating the occurrence of EMT. After intervention with icariin II, the epithelial markers Occludin and E-cadherin were upregulated, while the mesenchymal markers α-SMA and N-cadherin were downregulated, inhibiting EMT.

[0039] The beneficial effects of this invention are: it discloses for the first time a new use of icariin II in the treatment of chronic asthma. Through multi-pathway synergistic action, it exerts its therapeutic effect on chronic asthma by improving lung function, reducing airway inflammation, alleviating airway remodeling, and inhibiting pulmonary epithelial-mesenchymal transition, providing a brand-new drug option with multi-target therapeutic potential for the clinical treatment of chronic asthma.

[0040] 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. Use of icariin II in the preparation of drugs for treating chronic asthma.

2. The use according to claim 1, characterized in that, The icariin II exerts its therapeutic effect on chronic asthma by improving lung function.

3. The use according to claim 1, characterized in that, The icariin II exerts its therapeutic effect on chronic asthma by reducing airway inflammation.

4. The use according to claim 1, characterized in that, The icariin II exerts its therapeutic effect on chronic asthma by alleviating airway remodeling.

5. The use according to claim 1, characterized in that, The icariin II exerts its therapeutic effect on chronic asthma by inhibiting the transformation of pulmonary epithelial mesenchymal tissue.

6. A pharmaceutical composition, characterized in that, This includes therapeutically effective amounts of icariin II, or its pharmaceutically acceptable salts, hydrates, solvates, and prodrugs.

7. The pharmaceutical composition according to claim 6, characterized in that, It also includes one or more pharmaceutically acceptable carriers or excipients.

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