Application of 7, 8-dihydroxyflavone in preparation of medicine for preventing and treating pulmonary fibrosis

Abstract

The invention discloses application of 7, 8-dihydroxyflavone in preparation of a medicine for preventing and treating pulmonary fibrosis, and belongs to the technical field of medicines. Pharmacological experiments of the 3, 7, 8-dihydroxyflavone show that the 3, 7, 8-dihydroxyflavone can significantly inhibit the activation of fibroblasts in fibrotic animals and cell models, and has an obvious pulmonary fibrosis resistance effect; meanwhile, the 7, 8-dihydroxyflavone can also reverse transformation from alveolar epithelial cells to mesenchymal cells in a fibrosis model and inhibit occurrence and development of pulmonary fibrosis. The 7, 8-dihydroxyflavone is adopted as an effective component of the medicine for preventing and treating pulmonary fibrosis, has a remarkable pulmonary fibrosis resisting effect, low toxicity and low price, is convenient to produce, transport, store and take, and has a wide application prospect when being used as the pulmonary fibrosis resisting medicine.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a new use of 7,8-dihydroxyflavone, and specifically relates to the use of 7,8-dihydroxyflavone in the preparation of a drug for treating pulmonary fibrosis. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease (ILD) of unknown etiology. It is one of the most common interstitial lung diseases and is characterized by persistent and irreversible fibrotic scarring of the lung parenchyma, leading to respiratory failure with loss of respiratory function and ultimately death.

[0003] The exact cause of IPF remains unknown. Current studies have shown that genetics, viral infection, environmental factors (such as smoking, dust) and age are potential risk factors. With the acceleration of global population aging and the impact of the post-epidemic era, the occurrence of IPF and its social impact are expected to increase steadily in the future. In clinical practice, IPF is more common in men and people over 60 years old. Fibrosis is defined as excessive pathological deposition of extracellular matrix (ECM) in the process of dysregulated wound healing. But the exact mechanism of fibrosis is still poorly understood. Fibrosis is a highly coordinated process that integrates multiple cell types and signal transduction mechanisms across organ systems. Epithelial cells are the first line of defense to protect the lungs from external damage. In pathological conditions, it seems to drive physiological wound healing towards an uncontrolled fibrotic process. Pathological lung epithelium (caused by genetics, environmental exposure, aging, etc.) promotes chronic activation of immune cells and fibroblasts, which are key cells that produce extracellular matrix (ECM) components. Among them, impaired cell crosstalk is an important mechanism in the fibrotic process. It is currently believed that the onset of the disease is associated with recurrent micro-injury of the alveolar epithelium, followed by an abnormal repair response characterized by excessive collagen deposition, with the final result being progressive scarring of the lung parenchyma and irreversible functional loss. Myofibroblasts play an important role in scarring, which aggregate into fibroblast foci after injury and produce excessive ECM components through a TGF-β-dependent mechanism. Eventually, scar tissue spreads and replaces alveoli, resulting in impaired gas exchange and ultimately respiratory failure. Importantly, IPF is universally progressive, and there is no effective treatment to prevent and control the exacerbation of the disease. After diagnosis, the average life expectancy of patients is only 3-5 years. Existing anti-pulmonary fibrosis drugs have limited efficacy in preventing disease progression and improving quality of life, and have problems such as poor tolerability and high treatment costs.

[0004] Therefore, it is urgent to develop new, safe and effective drugs for the prevention and treatment of pulmonary fibrosis.

[0005] More and more studies have focused on the anti-fibrosis benefits of active ingredients of traditional Chinese medicine in this field, suggesting that compounds from traditional Chinese medicine and natural medicines may be a huge treasure trove for the discovery of anti-pulmonary fibrosis drugs. Naturally derived 7,8-dihydroxyflavone (7,8-DHF) belongs to the flavonoid active molecule with a molecular formula of C 15 H 10 O4, with a molecular weight of 254.24, has small toxic and side effects, definite efficacy, and is suitable for long-term treatment. It is in line with the medication characteristics of patients with pulmonary fibrosis, has a wide range of clinical applications, and has significant preventive and therapeutic effects. Studies in recent years have shown that 7,8-dihydroxyflavone is a specific agonist of tyrosine kinase B receptors, which can activate TrkB receptors and play an important neuroprotective role. In addition, 7,8-dihydroxyflavone also has antioxidant, anti-inflammatory and vasodilation functions. However, the role of 7,8-dihydroxyflavone in the prevention and treatment of pulmonary fibrosis has not been reported at home and abroad. Summary of the invention

[0006] The purpose of the present invention is to provide a new use of 7,8-dihydroxyflavone, that is, to use 7,8-dihydroxyflavone monomer as an active ingredient for treating pulmonary fibrosis diseases, and to apply it to the prevention and treatment of clinical pulmonary fibrosis.

[0007] The technical solution adopted by the present invention is: application of 7,8-dihydroxyflavone in the preparation of drugs for preventing and treating pulmonary fibrosis.

[0008] Among them, the structural formula of 7,8-dihydroxyflavone is:

[0009]

[0010] In the present invention, 7,8-dihydroxyflavone inhibits the proliferation and migration of lung fibroblasts and alveolar epithelial cells; at the same time, 7,8-dihydroxyflavone inhibits the transformation of fibroblasts to myofibroblasts (FMT) and the transformation of alveolar epithelial cells to mesenchymal cells (EMT), thereby achieving the purpose of preventing and treating pulmonary fibrosis. In addition, 7,8-dihydroxyflavone improves the sclerosis of lung tissue and the abnormality of alveolar structure caused by fibrosis, improves the deposition of collagen in lung tissue and scar formation, thereby achieving the purpose of preventing and treating pulmonary fibrosis.

[0011] The proliferation and migration of lung fibroblasts and alveolar epithelial cells were expressed as the proportion of MRC5 cell scratch healing and the ratio of EdU positive cells.

[0012] FMT was performed to evaluate the levels of myofibroblast markers α-SMA and fibronectin (FN) in MRC5 cells.

[0013] EMT was manifested by the levels of mesenchymal cell markers α-SMA, vimentin, and extracellular matrix type I collagen (Col-I), fibronectin (FN) in MLE-12 cells.

[0014] Lung tissue sclerosis and alveolar structural abnormalities are manifested as histopathological changes in lung weight, lung weight to body weight ratio, and tissue sections.

[0015] Collagen deposition and scarring in lung tissue were manifested by the collagen volume ratio (the percentage of collagen-positive blue area to the total tissue area) and the levels of Col-I, α-SMA, and Vimentin proteins in Masson-stained tissue sections.

[0016] In the present invention, the drug is composed of 7,8-dihydroxyflavone and one or more pharmaceutically acceptable carriers or excipients.

[0017] Among them, the drug can be made into various dosage forms such as granules (granules), pills, tablets, capsules, dripping pills, mixtures (oral liquids), syrups, powders, solutions, emulsions, suspensions, effervescent tablets, microsphere preparations, microcapsule preparations, nanocapsule preparations and injection solutions and other advanced dosage forms, which are conducive to absorption and improve oral bioavailability.

[0018] Among them, pulmonary fibrosis disease is pulmonary fibrosis disease or silicosis caused by chronic pneumonia;

[0019] or,

[0020] Pulmonary fibrosis caused by asbestos, minerals, dust, chemotherapy drugs, and radiation damage;

[0021] or,

[0022] It is a pulmonary fibrosis disease caused by rheumatic autoimmune diseases.

[0023] The invention also discloses a medicine for preventing and treating pulmonary fibrosis, wherein the effective component of the medicine is 7,8-dihydroxyflavone.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. 7,8-Dihydroxyflavone can significantly inhibit the proliferation and migration of lung fibroblasts and lung epithelial cells in the fibrosis model, and can significantly reverse the FMT and EMT processes induced by TGF-β1. It can be used as a safe and effective drug for the prevention and treatment of pulmonary fibrosis.

[0026] 2. 7,8-Dihydroxyflavone can be obtained through synthesis and extraction from traditional Chinese medicine. It has strong drugability. Compared with other existing marketed drugs, it is cheap, cost-effective, and easy for patients to accept.

[0027] 3. Easy to transport and store, seal and place in a cool and dry place. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the effect of 7,8-dihydroxyflavone on TGF-β1-induced MRC5 cell proliferation and migration (n=6, mean±SEM), where Figure 1 A is a representative image of MRC5 cells under an optical microscope; Figure 1 B is EdU immunofluorescence staining and its statistical results; Figure 1 C is the result of MRC5 cell scratch wound healing assay;

[0029] Figure 2 Schematic diagram of the effect of 7,8-dihydroxyflavone on TGF-β1-induced MRC5 cell activation (n=6, mean±SEM), where: Figure 2 A is the immunofluorescence and statistical results of α-smooth muscle actin (α-SMA) in MRC5 cells; Figure 2 B is the immunoblotting results and statistics of α-smooth muscle actin (α-SMA) in MRC5 cells; Figure 2 C is the immunofluorescence and statistical results of MRC5 fibronectin (FN); Figure 2 D is the immunoblotting results and statistics of fibronectin (FN) in MRC5 cells;

[0030] Figure 3 Schematic diagram of the effect of 7,8-dihydroxyflavone on TGF-β1-induced MLE-12 cells (n=6, mean±SEM), where: Figure 3 A is a representative image of MLE-12 cells under an optical microscope; Figure 3 B is the result of MLE-12 cell scratch wound healing assay; Figure 3 C Statistical results of cell scratch wound healing assay.

[0031] Figure 4 Schematic diagram of the effect of 7,8-dihydroxyflavone on extracellular matrix production of MLE-12 cells (n=6, mean±SEM), where: Figure 4 A is the immunofluorescence and statistical results of type I collagen (Col-I) in MLE-12 cells; Figure 4 B is the immunoblotting results and statistics of type I collagen (Col-I) in MLE-12 cells;

[0032] Figure 5 Schematic diagram of the effect of 7,8-dihydroxyflavone on EMT of MLE-12 cells (n=6, mean±SEM), where: Figure 5 A is the immunofluorescence and statistical results of MLE-12 (Vimentin); Figure 5B is the immunoblotting results and statistics of α-smooth muscle actin (α-SMA) and vimentin (Vimentin) in MLE-12 cells;

[0033] Figure 6 Schematic diagram of the effect of 7,8-dihydroxyflavone on bleomycin-induced pulmonary fibrosis in mice (n=6, mean±SEM), where: Figure 6 A is a schematic diagram of mouse model induction; Figure 6 B is a representative image of lung tissue; Figure 6 C. Statistical graph of lung weight of mice in each group; Figure 6 D is the statistical result of the ratio of lung weight to body weight of mice; Figure 6 F is a representative image of lung tissue sections stained with HE and Masson; Figure 6 G is the statistical graph of collagen volume fraction; Figure 6 HJ shows the western blot results and statistics of Col-I, α-SMA and Vimentin in mouse lung tissue. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below by performing animal and cell experiments, but this should not be construed as limiting the scope of the present invention to the following examples.

[0035] Example 1

[0036] Effects of 7,8-dihydroxyflavone on proliferation and migration of lung fibroblasts in a fibrosis model

[0037] Experimental methods:

[0038] A pulmonary fibroblast fibrosis model was established. Transforming growth factor-β1 (TGF-β1, 20 ng / mL, 48 h) was used to induce the pulmonary fibrosis cell model, and different concentrations of 7,8-DHF (10, 30, 100 μM) were used for treatment during 24 h-48 h. At the same time, the marketed anti-pulmonary fibrosis drug pirfenidone (PFD) was selected as a positive control. First, the cell morphology and growth status were observed under a light microscope, and the proliferation and migration ability of MRC5 cells were evaluated using an EDU kit and a scratch test.

[0039] Experimental results:

[0040] Observation under a light microscope revealed that compared with the control group, the morphology of MRC5 cells became elongated and the proliferation ability was enhanced after TGF-β1 induction, and these changes were inhibited by 7,8-DHF in a concentration-dependent manner ( Figure 1 A). EdU cell proliferation assay and wound healing assay were used to further clarify the effect of 7,8-DHF on TGF-β1-induced MRC-5 cell proliferation and migration. As shown in the figure ( Figure 1B, C), different concentrations of 7,8-DHF significantly inhibited TGF-β1-induced MRC5 cell migration, while 30μM and 100μM significantly inhibited MRC5 cell proliferation.

[0041] Example 2

[0042] Effects of 7,8-dihydroxyflavone on activation of fibroblasts

[0043] Experimental methods:

[0044] A pulmonary fibroblast fibrosis model was established. Transforming growth factor-β1 (TGF-β1, 20 ng / mL, 48 h) was used to induce the pulmonary fibrosis cell model, and different concentrations of 7,8-DHF (10, 30, 100 μM) were used for treatment during 24 h-48 h. The marketed anti-pulmonary fibrosis drug pirfenidone (PFD) was selected as a positive control. Western blot and immunofluorescence techniques were used to detect the expression of fibrosis-related marker proteins in MRC5 cells to evaluate the effect of 7,8-DHF on the transformation of fibroblasts to myofibroblasts in the fibrosis model.

[0045] Experimental results:

[0046] The results showed that compared with the control group, TGF-β1 significantly increased the protein expression of α-smooth muscle actin (α-SMA) and fibronectin (FN) in MRC5 cells, and both decreased in a concentration-dependent manner after 7,8-DHF treatment ( Figure 2 ).

[0047] Example 3

[0048] Regulatory effect of 7,8-dihydroxyflavone on epithelial-mesenchymal transition (EMT) of lung cells

[0049] Experimental methods:

[0050] MLE-12 mouse lung epithelial cell line was cultured, and TGF-β1 (20 ng / mL, 48 h) was used to induce the pulmonary fibrosis cell model as above, and 7,8-DHF (10 μM) was used for intervention during 24 h-48 h, and PFD was given as a positive control. The morphological changes of MLE-12 cells were observed under a light microscope, and the effect of 7,8-DHF on the migration of MLE-12 cells was evaluated by scratch test. Western blot and immunofluorescence techniques were used to detect the production of fibroblast markers vimentin (Vimentin), α-SMA and extracellular matrix in MLE-12 cells to evaluate the effect of 7,8-DHF on TGF-β1-induced EMT.

[0051] Experimental results:

[0052] Under light microscopy, it was found that compared with the control group, the morphology of MLE-12 cells induced by TGF-β1 transformed into fibroblast-like cells and their proliferation ability was enhanced. These changes were reversed to a certain extent by 7,8-DHF ( Figure 3 A). Meanwhile, 7,8-DHF inhibited TGF-β1-induced MLE-12 cell migration ( Figure 3 B). 7,8-DHF inhibits the production of extracellular matrix type I collagen (Col-I) and fibronectin in MLE-12 cells induced by TGF-β1 ( Figure 4 ). At the same time, the expression of α-SMA and Vimentin, mesenchymal cell markers of MLE-12 cells, was detected. The results showed that 7,8-DHF effectively inhibited the upregulation of α-SMA and Vimentin induced by TGF-β1 ( Figure 5 The above results indicate that 7,8-DHF can inhibit the generation of extracellular matrix and transdifferentiation of MLE-12 cells induced by TGF-β1.

[0053] Example 4

[0054] Effects of 7,8-dihydroxyflavone on bleomycin-induced pulmonary fibrosis in mice

[0055] Experimental methods:

[0056] Male C57BL / 6N mice (8 weeks old) with an average body weight of 19-20 g were randomly assigned to four groups: sham group (Sham), bleomycin treatment group (BLM), 7,8-DHF (5 mg / kg) + BLM treatment group, pirfenidone (PFD, 300 mg / kg) + BLM treatment group. During the experiment, mice had free access to food and water and were housed in a suitable environment (temperature 25°C; relative humidity 45-55% humidity; room with 12-hour light / dark cycle). The mouse pulmonary fibrosis model was induced by a single intratracheal instillation of BLM. The mice were anesthetized by intraperitoneal injection of tribromoethanol and the neck skin was opened to expose the trachea. Bleomycin (3 mg / kg) or an equal volume of saline was injected intratracheally. From 1 day after BLM administration to day 21, mice in the 7,8-DHF group were intraperitoneally injected (ip) with 7,8-DHF (5 mg / kg / d) and mice in the PFD group were intragastrically administered (ig) with PFD (300 mg / kg / d) as a positive control group. After 21 days, the mice were killed, their body weights and lung weights were recorded, and lung tissues were collected for pathological examination.

[0057] Experimental results:

[0058] Schematic diagram of BLM mouse model induction and 7,8-DHF intervention ( Figure 6A). The morphological changes of the lung tissues of the mice were observed after separation. The lung tissues of the PF model mice were enlarged and dark red, and lacked a spongy texture, while the lungs of the control mice were pink and soft. The color and texture of the lungs were improved after treatment with 7,8-DHF and PFD ( Figure 6 B). Meanwhile, 7,8-DHF treatment significantly attenuated the increase in lung weight and lung weight / body weight (LW / BW) index induced by BLM ( Figure 6 C, D). Assessment of collagen deposition in the whole lung by measuring hydroxyproline content showed that 7,8-DHF effectively reversed the increase in lung collagen deposition caused by BLM ( Figure 6 E). In addition, H&E staining results of lung tissue sections showed that the alveolar structure of mice treated with BLM was destroyed and inflammatory cells were significantly infiltrated, and 7,8-DHF treatment could effectively reverse this situation ( Figure 6 F). Masson staining of lung tissue showed that collagen fiber deposition (indicated by blue) was significantly reduced after 7,8-DHF treatment ( Figure 6 F, G). Western blot was used to detect the protein levels of fibrosis-related factors, including Col-I, α-SMA, and Vimentin. The results showed that the above fibrosis markers were significantly upregulated in the lung tissues of IPF mice compared with the Sham group, and this result was reversed after treatment with 7,8-DHF ( Figure 6 H, I, J).

[0059] Based on the above research, the present invention proposes the use of 7,8-dihydroxyflavone in the preparation of pathological pulmonary fibrosis. The above is only a preferred embodiment of the present invention, which is only illustrative and not restrictive to the present invention; those skilled in the art understand that many changes, modifications, and even equivalent changes can be made within the spirit and scope defined by the claims of the present invention, but they will all fall within the scope of protection of the present invention.

Claims

1. Use of 7,8-dihydroxyflavone in the preparation of drugs for preventing and treating pulmonary fibrosis.

2. The use according to claim 1, characterized in that The medicine consists of 7,8-dihydroxyflavone and one or more pharmaceutically acceptable carriers or excipients.

3. The use according to claim 1, characterized in that The medicine is a granule, a pill, a tablet, a capsule, a dripping pill, a mixture, a syrup, a powder, a solution, an emulsion, a suspension, an effervescent tablet, a microsphere preparation, a microcapsule preparation, a nanocapsule preparation or an injection.

4. The use according to claim 1, characterized in that Pulmonary fibrosis is caused by chronic pneumonia, chemotherapy drugs, radiation damage, rheumatic autoimmune diseases, or silicosis caused by asbestos, minerals, and dust.

5. A drug for pulmonary fibrosis, characterized in that: The active ingredient is 7,8-dihydroxyflavone.

Images