Application of Danthron in preparation of medicine for preventing or treating pulmonary fibrosis

Abstract

The invention belongs to the technical field of biological pharmacy, and discloses application of Danthron in preparation of a medicine for preventing or treating pulmonary fibrosis. The Danthron inhibits the fibroblasts from differentiating to myofibroblasts by reducing the transcriptional level and protein expression level of fibronectin, alpha-actin and I-type collagen in the fibroblasts in lung tissues, so that the pulmonary fibrosis process is slowed down, and a new treatment strategy is provided for pulmonary fibrosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to the application of Danthron in preparing medicines for preventing or treating pulmonary fibrosis. Background Art

[0002] Danthron, also known as 1,8-Dihydroxyanthraquinone, is a natural product extracted from the traditional Chinese medicine Danshen. Increasing evidence shows that it is an effective small molecule, and its anti-angiogenic, anti-tumor and antioxidant activities show its potential role in cancer treatment, but it is still unclear what role Danthron plays in the pathogenesis of pulmonary fibrosis.

[0003] Pulmonary fibrosis is a chronic, progressive lung disease. It is an end-stage lung change of interstitial lung disease characterized by fibroblast proliferation, large amounts of extracellular matrix deposition, and destruction of lung tissue structure. It is characterized by the gradual replacement of lung tissue by scars, leading to gradual loss of lung function.

[0004] Symptoms of pulmonary fibrosis include persistent dry cough, dyspnea, fatigue and weakness. The main pathological features include proliferation of mesenchymal cells in lung tissue, proliferation and deposition of extracellular matrix and remodeling of lung parenchyma. These symptoms will gradually worsen and affect the patient's quality of life. In severe cases, pulmonary fibrosis may lead to heart disease, respiratory failure and other serious complications. There are many factors that cause pulmonary fibrosis, such as occupational or home environment factors, drugs, radiation therapy, high-concentration oxygen therapy, smoking, disease factors and genetic factors.

[0005] At present, the treatments for pulmonary fibrosis mainly include drug therapy and organ transplantation. As the only final treatment option for patients with fibrosis, the application and promotion of organ transplantation is limited by the difficulty in obtaining donors, high surgical risks, and high surgical costs. Existing drugs cannot reverse the course of fibrosis, and their clinical treatment effects and safety cannot meet the treatment needs.

[0006] Studies have shown that in the process of fibrosis, the expression of transforming growth factor-β, such as TGF-β, in related tissues is upregulated, and TGF-β can induce fibroblasts to differentiate into myofibroblasts (Su et al., 2015, Nature communications 6: 8523). The differentiation of fibroblasts into myofibroblasts plays an important role in the process of fibrosis (Chen et al., 2016, Nature communications 7: 12564; Hung et al., 2013, American journal of respiratory and critical care medicine 188: 820-830). Myofibroblasts secrete excessive amounts of fibrous ECM proteins, including type I collagen and fibronectin, leading to increased matrix stiffness and pathological matrix deposition in the lung interstitium (Chen et al., 2016, Nature communications 7:12564; Pedroza et al., 2016, FASEB journal: official publication of the Federation of American Societies for Experimental Biology 30:129-140). Although current studies have shown that TGF-β plays an important role in the process of pulmonary fibrosis, there is still a lack of clinically applicable drugs that can effectively block the TGF-β signaling pathway and thereby prevent or reverse the process of pulmonary fibrosis.

[0007] Therefore, there is an urgent need to conduct in-depth research on the pathogenesis of pulmonary fibrosis, find new therapeutic targets, fully combine innovative medical development trends, and develop new drugs that can effectively treat pulmonary fibrosis and are highly safe. Summary of the invention

[0008] In order to solve the above technical problems, the present invention discloses the use of Danthron in the preparation of drugs for preventing or treating pulmonary fibrosis. Danthron inhibits the differentiation of fibroblasts into myofibroblasts, thereby slowing down the process of pulmonary fibrosis.

[0009] In order to achieve the above purpose, this application adopts the following technical solutions:

[0010] The present invention provides application of Danthron in preparing a medicine for preventing or treating pulmonary fibrosis. Danthron is 1,8-dihydroxyanthraquinone.

[0011] In the above technical solution, Danthron is extracted from Salvia miltiorrhiza.

[0012] In the above technical scheme, Danthron inhibits the differentiation of fibroblasts into myofibroblasts by reducing the transcription level and protein expression level of fibronectin, α-actin and type I collagen in fibroblasts in lung tissue, thereby slowing down the progression of pulmonary fibrosis.

[0013] The beneficial effects of the present invention are:

[0014] The present invention has discovered a new use of Danthron, a monomer component of traditional Chinese medicine, which can be applied to the prevention and treatment of pulmonary fibrosis. It can inhibit the differentiation of fibroblasts into myofibroblasts by reducing the transcription level and protein expression level of fibronectin, α-actin and type I collagen in fibroblasts, thereby slowing down the process of pulmonary fibrosis and providing a new treatment strategy for pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of H&E, Sirius Red, and Masson's trichrome staining of lung tissues of mice treated with Danthron and saline after bleomycin-induced fibrosis;

[0016] Figure 2 Schematic diagram of the Ashcroft score for fibrosis in the above mouse models, where **p<0.01, ***p<0.001;

[0017] Figure 3 The figure is a schematic diagram of the western blot results of fibronectin and type I collagen in the above mouse model, where *p<0.05, **p<0.01, ****p<0.0001; Figure 3 a is the test result display diagram, Figure 3 b is Figure 3 a histogram;

[0018] Figure 4 Schematic diagram of the quantitative RT-PCR results of fibronectin and type I collagen in the above mouse model, among which, **p<0.01, ***p<0.001, ****p<0.0001;

[0019] Figure 5 This is a schematic diagram of the western blot and quantitative RT-PCR results of fibronectin, type I collagen and α-SMA in human primary lung fibroblasts after Danthron pretreatment and TGF-β stimulation for 24 hours, among which, **p<0.01, ***p<0.001, ****p<0.0001; among which, Figure 5 a is the test result display of western blot. Figure 5 b is Figure 5a is a bar chart, 5c is a display chart of the test results of quantitative RT-PCR;

[0020] Figure 6 This is a schematic diagram of the western blot results of fibronectin and α-SMA in human primary lung fibroblasts after TGF-β stimulation for 24 hours and then Danthron treatment for 24 hours, where *p<0.05, ****p<0.0001; Figure 6 a is the test result display diagram, Figure 6 b is Figure 6 A bar chart of a. DETAILED DESCRIPTION

[0021] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0022] The term "prevention" as used in the present invention refers to preventing or reducing the occurrence of fibrosis in the presence of possible profibrotic factors. The term "treatment" as used in the present invention refers to reducing the degree of fibrosis, or curing fibrosis to normalize it, or slowing down the progression of fibrosis.

[0023] The present invention demonstrates through the following examples that Danthron can significantly reduce the differentiation of fibroblasts into myofibroblasts. The differentiation of fibroblasts into myofibroblasts plays an extremely important role in the pathogenesis of pulmonary fibrosis.

[0024] In order to better explain the present invention, it is described in detail below in conjunction with specific embodiments.

[0025] Example 1 Effect of Danthron intragastric administration on the degree of pulmonary fibrosis;

[0026] Experimental animals and materials:

[0027] 1. Experimental animals:

[0028] Source, strain, and species: Wild-type mice (WT, C57BL / 6) were ordered from the Hubei Branch of Beijing Weitonglihua Experimental Animal Technology Co., Ltd.;

[0029] Reproductive age: 8 to 10 weeks;

[0030] 2. Experimental materials:

[0031] Bleomycin: purchased from Wuhan Promoter Biological Co., Ltd.;

[0032] Isoflurane: purchased from Wuhan Promoter Biological Co., Ltd.;

[0033] Physiological saline: purchased from Wuhan Promoter Biological Co., Ltd.;

[0034] Danthron: purchased from MedChemExpress.

[0035] 3. Experimental methods:

[0036] Wild-type mice were anesthetized by isoflurane inhalation, and then bleomycin with a final concentration of 1.7U / kg was injected through the airway. The bleomycin was dissolved in 50μL of normal saline, and mice injected with the same volume of normal saline were used as controls. 14 days after airway injection of bleomycin or normal saline, mice were given Danthron with a final concentration of 60mg / kg for 7 consecutive days, and mice gavaged with the same volume of normal saline were used as controls. Mice were killed 21 days after bleomycin modeling, and the degree of lung fibrosis in each mouse was analyzed. The severity of interstitial fibrosis in each continuous area was independently evaluated by two pathologists in a blinded manner using the Ashcroft scoring system.

[0037] 4. Experimental results:

[0038] Specifically, the results of H&E, Sirius red, and Masson's trichrome staining of mouse lung tissue are as follows Figure 1 As shown, combined Figure 1 It can be seen that after stimulation and induction with bleomycin, lung injury and fibrosis were significantly reduced in mice treated with Danthron by oral gavage compared with mice gavaged with normal saline.

[0039] Ashcroft rating results Figure 2 As shown by Figure 2 It can be seen that the Ashcroft score of mice gavaged with Danthron was lower than that of mice gavaged with normal saline, which indicates that the degree of pulmonary fibrosis was greatly reduced.

[0040] Example 2 Effect of Danthron intragastric administration on protein and mRNA levels of fibronectin, type I collagen and α-SMA in lung tissue

[0041] In order to further evaluate the degree of fibrosis in each mouse after bleomycin injection, the present invention detected the protein and mRNA levels of fibronectin and type I collagen in the lung tissue of each mouse by western blot and RT-PCR, respectively.

[0042] Specifically, the mouse lung tissues after the experiment in Example 1 were collected, the proteins in the tissues were extracted by RIPA lysis buffer, and the expression levels of target proteins, such as fibronectin and type I collagen, were detected by Western blot. The results are as follows: Figure 3 Among them, Western blot is based on Wang et al., 2017, Journal of Allergy and Clinical Immunology 40: 1550-1561.

[0043] At the same time, fluorescence quantitative RT-PCR was performed using SYBR Premix Ex Taq (TaKaRa), and GAPDH was used as an internal reference to standardize the relative expression of each target gene. The results are shown in Figure 4 As shown. The primers corresponding to each target gene are listed in Table 1 below. The specific test expression method is described in Chen et al., 2015, International journal of clinical and experimental pathology 8: 6700-6707.

[0044] Table 1 List of primers corresponding to target genes

[0045]

[0046] Combination Figure 3 and Figure 4 It can be seen that compared with wild-type mice, the transcription level and protein expression level of fibronectin and type I collagen in mice treated with Danthron by gavage were reduced, which indicates that Danthron can significantly reduce the degree of pulmonary fibrosis in mice.

[0047] Example 3 Effect of Danthron on the differentiation of fibroblasts into myofibroblasts

[0048] Evidence shows that the process of fibroblast differentiation into myofibroblasts is necessary for maintaining pulmonary fibrosis. Therefore, in order to further evaluate the effect of Danthron on the differentiation of fibroblasts into myofibroblasts after stimulation with TGF-β1, the present invention detected the protein and mRNA levels of fibronectin, α-actin and type I collagen in human primary fibroblasts by western blot and RT-PCR, respectively. Among them, the primers corresponding to each target gene are listed in Table 2 below.

[0049] Table 2 List of primers corresponding to target genes

[0050]

[0051] As shown in the western blot and quantitative RT-PCR results of Example 2 above, compared with human primary fibroblasts stimulated with 10 ng / ml TGF-β1 for 24 h, the transcription and protein expression levels of fibronectin, α-actin and type I collagen in human primary fibroblasts stimulated with TGF-β1 after pretreatment with Danthron for 1 h were significantly reduced. Figure 5 shown.

[0052] Next, the present invention further explored whether Danthron has a reversal effect on the phenotype of activated human primary fibroblasts. Specifically, human primary fibroblasts were first stimulated with 10 ng / ml TGF-β1 for 24 hours, and then treated with Danthron for 24 hours. As shown in the western blot results of Example 2 above, compared with human primary fibroblasts stimulated with TGF-β1, the protein expression levels of fibronectin and α-actin in human primary fibroblasts treated with Danthron were significantly reduced. Figure 6 shown.

[0053] In summary, the above experimental data indicate that Danthron can slow down the process of fibrosis by inhibiting the differentiation of fibroblasts into myofibroblasts.

[0054] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Application of Danthron in the preparation of drugs for preventing or treating pulmonary fibrosis, wherein Danthron is 1,8-dihydroxyanthraquinone.

2. The application according to claim 1, characterized in that: Danthron is extracted from Salvia miltiorrhiza.

3. The application according to claim 1, characterized in that: Danthron inhibits the differentiation of fibroblasts into myofibroblasts by reducing the transcription and protein expression levels of fibronectin, α-actin and type I collagen in fibroblasts in lung tissue, thereby slowing down the progression of pulmonary fibrosis.

Images