Application of flueggemine B in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention discloses application of flueggeine B or pharmaceutically acceptable salt thereof in preparation of a medicine for treating pulmonary fibrosis. The chemical structure of the flueggeine B is shown as a formula (I). The flueggemine B shows an improvement effect in a bleomycin-induced pulmonary fibrosis mouse model, improves the weight and lung function parameters of a mouse, relieves the degree of pulmonary fibrosis, has an inhibition effect on lung tissue collagen deposition, can also effectively improve the phenotype of pulmonary epithelial cells and inhibit abnormal activation of myofibroblasts, and can be used for treating pulmonary fibrosis. Therefore, the traditional Chinese medicine composition has a remarkable relieving effect on bleomycin-induced pulmonary fibrosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of virosine B or a pharmaceutically acceptable salt thereof in preparation of a drug for treating pulmonary fibrosis. BACKGROUND

[0002] Pulmonary fibrosis is a chronic progressive interstitial lung disease with abnormal repair of lung tissue to form scar tissue as a core feature, and its causes are complex, related to environmental exposure, drug effects, autoimmune diseases and various factors. Clinically, the main manifestations are dry cough and progressive dyspnea, and finally respiratory failure often leads to death, with a poor prognosis and a five-year survival rate of less than 30%. At present, the clinical treatment means is limited, and drug treatment is still the core means to delay disease progression and improve the quality of life of patients, but the existing treatment drugs still have many unmet medical needs such as limited efficacy, limited applicable population and significant adverse reactions, and the research and development of related drugs and patent layout have become a research hotspot in the field.

[0003] Virosine B (CAS No. 1052228-70-2), also known as vinrosine B, is a furan quinolizine alkaloid isolated from the roots of plants such as Phyllodium elegans (related plants of the genus Phyllodium), and other plants. In vitro experiments show that it has cytotoxic effects on various tumor cell lines (such as oral cancer cell KB, lung cancer cell A549, colon adenocarcinoma cell HCT-8, leukemia cell P388, etc.), can inhibit tumor cell proliferation, and the specific mechanism of action may be related to inducing apoptosis and blocking cell cycle, etc. Its half effective dose (ED 50 ) is less than 20 μg / mL, and it is a potential anti-tumor drug lead compound. As an alkaloid compound, it may have anti-inflammatory and antibacterial activities, but relevant research is less and further exploration is still needed. SUMMARY

[0004] The application provides application of virosine B or a pharmaceutically acceptable salt thereof in preparation of a drug for treating pulmonary fibrosis, and the chemical structure of the virosine B is as shown in formula (I):

[0005]

[0006] I.

[0007] Preferably, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0008] Further, virosine B or a pharmaceutically acceptable salt thereof can improve pulmonary fibrosis by improving epithelial phenotype and inhibiting abnormal activation of myofibroblasts; specifically, it can up-regulate the expression of lung epithelial cell marker E-CAD and reduce the expression of myofibroblast marker alpha-SMA.

[0009] Preferably, the administration dosage of the Moratine B is 1-100 mg / kg of body weight, more preferably 1-10 mg / kg of body weight, and the administration dosage of the pharmaceutically acceptable salt thereof is also correspondingly.

[0010] Optionally, the drug can be administered orally, intravenously, subcutaneously, inhaled or topically.

[0011] Preferably, the drug is delivered to the lung tissue by intratracheal injection or aerosol inhalation.

[0012] Further, the dosage form of the drug can be selected from one of a tablet, a capsule, a granule, an oral liquid, an injection liquid, a freeze-dried powder injection, and an inhalation aerosol.

[0013] Further, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0014] The present application finds that Moratine B has a significant improvement effect on lung fibrosis mice by the intervention of Moratine B in the bleomycin-induced lung fibrosis mouse model. First, it improves the body weight and lung function parameters of the mice. Further, it is found that it can reduce the degree of bleomycin-induced lung fibrosis, has an inhibitory effect on lung tissue collagen deposition, can effectively improve the lung epithelial cell phenotype and inhibit the abnormal activation of myofibroblasts, thereby playing a significant role in relieving bleomycin-induced lung fibrosis. Thus, Moratine B has a significant therapeutic effect in the treatment of lung fibrosis-related diseases, and has a good application prospect in the preparation of lung fibrosis treatment drugs.

[0015] The concept, specific structure and generated technical effects of the present application will be further described below in combination with the drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a schematic diagram of the experimental process of bleomycin (BLM)-induced lung fibrosis mouse model and Moratine B intervention;

[0017] Figure 2 is a graph of the body weight change of the mice in each group during the experiment (expressed as a percentage of the baseline);

[0018] Figure 3 is the statistical result of the quantitative analysis of the hydroxyproline content in the lung tissue of the mice in each group, which is used to evaluate the collagen deposition level;

[0019] Figure 4 is the forced vital capacity (FVC) of the lung function parameters of the mice in each group determined by the small animal lung function detection system;

[0020] Figure 5The forced expiratory volume in the first second (FEV1) of each group of mice was determined by a small animal lung function detection system.

[0021] Figure 6 Figure 2 is a representative image of HE staining of lung tissue paraffin sections of mice in each experimental group, scale bar: 500 pm;

[0022] Figure 7 Figure 3 is a statistical result of Ashcroft scoring of lung tissue in each group based on the results of HE staining, to quantitatively evaluate the degree of pulmonary fibrosis;

[0023] Figure 8 Figure 4 is a representative image of Masson trichrome staining of lung tissue of mice in each group, the blue area represents collagen deposition; extensive collagen deposition and thickening of the lung interstitium can be seen in the BLM group; after treatment with Alstonia B, the range and degree of collagen deposition are significantly reduced; scale bar: 50 pm;

[0024] Figure 9 Figure 5 is a statistical result of quantitative analysis of the area of collagen-positive regions in Masson staining, expressed in percentage;

[0025] Figure 10 Figure 6 is a representative image of immunofluorescence staining of lung tissue sections of mice in each group, labeling epithelial cell marker E-cadherin (E-CAD, red) and myofibroblast marker a-SMA (green), and DAPI (blue) staining of the nucleus; the E-CAD signal is significantly weakened, while the a-SMA signal is significantly enhanced in the BLM group; after intervention with Alstonia B, the expression of epithelial markers is partially restored, while the expression of myofibroblast markers is reduced; scale bar: 100 pm;

[0026] Figure 11 Figure 7 is a statistical result of quantitative analysis of the area of E-CAD-positive regions;

[0027] Figure 12 Figure 8 is a statistical result of quantitative analysis of the area of a-SMA-positive regions;

[0028] Statistical annotation: ns, no statistically significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, data are expressed as mean ± SEM. DETAILED DESCRIPTION

[0029] Example 1: Effect of Alstonia B on bleomycin-induced pulmonary fibrosis in a mouse model

[0030] The present application adopts bleomycin (Bleomycin, BLM) airway instillation (1.2 μg / g, total volume 50 μL) to establish a mouse pulmonary fibrosis model (C57BL / 6J, male, 8-10 weeks old, n=5), as shown in Figure 1 The mice received BLM or normal saline airway instillation on day 0, and were given different doses of white rice tree alkaline B intervention or control treatment by airway instillation every 5 days from the 7th day after modeling for 3 times, wherein the low dose was 1 mg / kg, the medium dose was 10 mg / kg, the high dose was 100 mg / kg, and the white rice tree alkaline B control group dose was 100 mg / kg, the drug volume was 50 μL, and the mice were sacrificed on day 21.

[0031] The body weight changes of mice in each group were dynamically monitored during the experiment, and the results are shown in Figure 2 After BLM treatment, the body weight of mice decreased significantly, and after low, medium and high dose of white rice tree alkaline B intervention, the body weight decrease trend of mice was effectively alleviated, indicating that white rice tree alkaline B can reduce the overall physiological damage induced by bleomycin.

[0032] Further detection of lung tissue hydroxyproline content to evaluate the level of collagen deposition, the results are shown in Figure 3 The hydroxyproline content in the lung tissue of BLM group mice increased significantly, and after giving white rice tree alkaline B, the 1 mg / kg, 10 mg / kg and 100 mg / kg dose groups could significantly reduce the hydroxyproline content in the lung tissue, indicating that white rice tree alkaline B at different doses could effectively inhibit lung tissue collagen deposition.

[0033] At the same time, the lung function parameters were analyzed by small animal lung function detection system. The results are shown in Figure 4 and Figure 5 BLM treatment significantly reduced the forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) of mice, and after low, medium and high dose of white rice tree alkaline B treatment, the above lung function indexes were obviously improved, indicating that white rice tree alkaline B could effectively alleviate the lung dysfunction caused by pulmonary fibrosis.

[0034] Example 2: Improvement of white rice tree alkaline B on lung tissue pathological structure and fibrosis degree

[0035] The pathological changes of lung tissue of mice in each group were observed by lung tissue HE staining. The results are shown in Figure 6 The alveolar structure of mice in the control group and the group given white rice tree alkaline B alone was complete, and no obvious abnormalities were found.

[0036] The lung tissue of the BLM group of mice showed obvious alveolar structure destruction, lung interstitial thickening and inflammatory cell infiltration. In contrast, after intervention with low, medium and high doses of quebrachamine B, the pathological damage of lung tissue was improved to varying degrees, the alveolar structure was relatively complete, and the interstitial thickening was reduced.

[0037] Based on the HE staining results, Ashcroft scoring was performed for quantitative analysis. As shown in Figure 7 , the lung fibrosis score of the BLM group was significantly increased, while after treatment with low, medium and high doses of quebrachamine B, the Ashcroft score of lung tissue was significantly reduced, indicating that quebrachamine B at various doses could effectively reduce the degree of bleomycin-induced pulmonary fibrosis.

[0038] Example 3: Inhibitory effect of quebrachamine B on collagen deposition in lung tissue

[0039] Masson trichrome staining was used to evaluate collagen deposition in lung tissue. As shown in Figure 8 , a large amount of collagen deposition occurred in the lung tissue of the BLM group of mice, and the lung interstitium was significantly thickened.

[0040] Compared with the BLM group, after treatment with low, medium and high doses of quebrachamine B, the range and degree of collagen deposition in lung tissue were significantly reduced. Quantitative analysis of collagen-positive area showed that Figure 9 low, medium and high dose groups could significantly reduce the proportion of lung tissue collagen deposition, indicating that quebrachamine B at different doses had the effect of inhibiting lung fibrosis-related collagen deposition.

[0041] Example 4: Modulatory effect of quebrachamine B on lung epithelial cell phenotype and muscle fibroblast activation

[0042] Immunofluorescence staining was used to detect the expression of epithelial cell marker E-cadherin (E-CAD) and muscle fibroblast marker a-SMA in lung tissue. As shown in Figure 10 , after BLM treatment, the expression of E-CAD in lung tissue of mice was significantly reduced, while the expression of a-SMA was significantly enhanced. After intervention with low, medium and high doses of quebrachamine B, the expression of E-CAD in lung tissue was significantly restored, and the a-SMA positive signal was significantly weakened.

[0043] The ratio of E-CAD Figure 11 to a-SMA Figure 12The results of quantitative analysis of the positive area show that the alstonic alkaloid B can effectively improve the lung epithelial cell phenotype and inhibit the abnormal activation of myofibroblasts at low, medium and high doses, thereby playing a significant role in relieving the lung fibrosis induced by bleomycin.

[0044] The preferred embodiments of the present application are described in detail above. It should be understood that those of ordinary skill in the art can make modifications and variations without departing from the concept of the present application. Therefore, any technical solutions obtained by logical analysis, reasoning or limited experiments based on the prior art according to the concept of the present application shall be within the protection scope defined by the claims.

Claims

1. The use of sclerotinia sclerotinia B or a pharmaceutically acceptable salt thereof in the preparation of pulmonary fibrosis treatment drugs, wherein the chemical structure of sclerotinia sclerotinia B is shown in formula (I): I 。 2. The use of the alkaloid B or a pharmaceutically acceptable salt thereof as described in claim 1 in the preparation of a medicament for treating pulmonary fibrosis, characterized in that, The bacon alkaloid B or its pharmaceutically acceptable salt improves pulmonary fibrosis by improving the epithelial phenotype and inhibiting abnormal activation of myofibroblasts.

3. The use of the alkaloid B or a pharmaceutically acceptable salt thereof as described in claim 1 in the preparation of a medicament for treating pulmonary fibrosis, characterized in that, The pulmonary fibrosis mentioned is idiopathic pulmonary fibrosis.

4. The use of the alkaloid B of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a pulmonary fibrosis treatment drug, characterized in that, The dosage of the alkaloid B is 1-100 mg / kg body weight, and the dosage of its pharmaceutically acceptable salts is also corresponding.

5. The use of the alkaloid B of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating pulmonary fibrosis, characterized in that, The drug is administered orally, intravenously, subcutaneously, by inhalation, or topically.

6. The use of the alkaloid B of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating pulmonary fibrosis, characterized in that, The drug is delivered to the lung tissue via airway instillation or nebulized inhalation.

7. The use of the alkaloid B of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating pulmonary fibrosis, characterized in that, The dosage form of the drug is selected from one of the following: tablets, capsules, granules, oral liquids, injections, lyophilized powder for injection, and inhaled aerosols.

8. The use of the alkaloid B of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating pulmonary fibrosis, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

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