Application of mulberroside A in preparation of medicine for preventing and treating pulmonary fibrosis diseases

Abstract

The invention relates to the technical field of medicines, in particular to application of mulberroside A in preparation of medicines for preventing and treating pulmonary fibrosis diseases. The new application of the traditional Chinese medicine monomer component mulberroside A is found, the mulberroside A can be applied to prevention and treatment of pulmonary fibrosis, and by reducing the transcriptional level and the protein expression level of fibronectin, alpha-actin and I-type collagen in fibroblasts, differentiation of the fibroblasts to myofibroblasts is inhibited, so that the pulmonary fibrosis process is slowed down, and the pulmonary fibrosis treatment effect is improved. And a new treatment strategy is provided for pulmonary fibrosis. Experimental results show that lung injury and fibrosis in mice treated by intraperitoneal injection of 10 mg / kg and 20 mg / kg of mulberroside A are obviously relieved. Moreover, a human primary fibroblast experiment also shows that the mulberroside A can obviously reduce the protein expression level of type I collagen, fibronectin and alpha-actin in the human primary fibroblast stimulated by TGF-beta1.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of mulberry glycoside A in preparing medicines for preventing and treating pulmonary fibrosis diseases. Background Art

[0002] 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.

[0003] 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.

[0004] 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.

[0005] Studies have shown that in the process of fibrosis, the expression of transforming growth factor-β, such as TGF-β, is upregulated in related tissues, and TGF-β can induce fibroblasts to differentiate into myofibroblasts. The differentiation of fibroblasts into myofibroblasts plays an important role in the process of fibrosis. Myofibroblasts secrete excessive amounts of fibrous ECM proteins, including type I collagen and fibronectin, which lead to increased matrix hardness and pathological matrix deposition in the lung interstitium. Although current studies have shown that TGF-β plays an important role in the process of pulmonary fibrosis, there is still a lack of drugs that can be used clinically to effectively block the TGF-β signaling pathway and thereby prevent or reverse the process of pulmonary fibrosis.

[0006] 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

[0007] In view of this, the technical problem to be solved by the present invention is to provide the use of mulberry glycoside A in the preparation of drugs for preventing and treating pulmonary fibrosis diseases. The present invention discloses that mulberry glycoside A can effectively slow down the progress of pulmonary fibrosis.

[0008] The present invention provides the use of mulberry glycoside A in preparing medicine for preventing and / or treating pulmonary fibrosis.

[0009] In some embodiments, the pulmonary fibrosis disease is interstitial lung disease.

[0010] In some specific embodiments, the pulmonary fibrosis disease comprises bleomycin-induced pulmonary fibrosis.

[0011] In some specific embodiments, the drug includes a drug that inhibits the transcription level and / or protein expression level of fibronectin.

[0012] In some specific embodiments, the drug includes a drug that inhibits the transcription level and / or protein expression level of α-actin.

[0013] In some specific embodiments, the drug includes a drug that inhibits the transcription level and / or protein expression level of type I collagen.

[0014] In some specific embodiments, the drug includes a drug that inhibits the differentiation of fibroblasts into myofibroblasts.

[0015] In some embodiments, the medicament is used to slow, stop or reverse the extent of pulmonary fibrosis.

[0016] In some embodiments, the dosage of the drug, calculated as a dose for mice, is 10-20 mg / kg once.

[0017] In some specific embodiments, the dosage of the drug includes a single injection of 10 mg / kg or 20 mg / kg, injected once a day.

[0018] In some embodiments, the medicament comprises an inert, non-toxic, pharmacologically suitable excipient.

[0019] In some embodiments, the drug is in the form of tablets, capsules, granules, drops, lyophilisates, granules, ointments or injections.

[0020] In some embodiments, the drug further includes an excipient, which is a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a stabilizer, a colorant, or a flavoring agent.

[0021] Compared with the prior art, the present invention has discovered a new use of the Chinese medicine monomer component mulberry glycoside A, which can be applied to the prevention and treatment of pulmonary fibrosis, by reducing the transcription level and protein expression level of fibronectin, α-actin and type I collagen in fibroblasts, inhibiting the differentiation of fibroblasts into myofibroblasts, thereby slowing down the process of pulmonary fibrosis, and providing a new treatment strategy for pulmonary fibrosis. Experimental results show that lung injury and fibrosis in mice treated with 10 mg / kg and 20 mg / kg mulberry glycoside A intraperitoneal injection were significantly reduced, and the transcription level and protein expression level of fibronectin, type I collagen and α-SMA in mice were reduced. Cell experiments also show that mulberry glycoside A can significantly reduce the protein expression levels of type I collagen, fibronectin and α-actin in human primary fibroblasts stimulated by TGF-β1. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The schematic diagram of the structure of Mulberiside A and the schematic diagram of the administration of the mouse experiment of the present invention are shown, wherein Figure A is a schematic diagram of the chemical molecular structure of Mulberiside A, and Figure B is a schematic diagram of the administration, that is, after two weeks of bleomycin-induced fibrosis formation, different concentrations of Mulberiside A are intraperitoneally injected daily;

[0023] Figure 2 The figure shows the evaluation of the biosafety of the drug and its corresponding concentration after the intervention of pulmonary fibrosis modeling and mulberry glycoside A, wherein Figure A and Figure B are the liver function test results in the plasma of each group of mice, Figure C and Figure D are the renal function test results in the plasma of each group of mice, Figure E and Figure F are the cardiac function test results in the plasma of each group of mice, Figure A is the ALT concentration test result, Figure B is the AST concentration test result, Figure C is the CR concentration test result, Figure D is the BUN concentration test result, Figure E is the LDH concentration test result, and Figure F is the CK concentration test result;

[0024] Figure 3 Show the changes in lung function of mice in each group, where A is Rrs (respiratory system resistance), B is Ers (respiratory system elasticity), and C is Crs (respiratory system compliance), *p<0.05, **p<0.01, ***p <0.001;

[0025] Figure 4 Figure 2 shows the staining results of lung tissues of mice in each group, the Ashcroft score statistics, the hydroxyproline content and the weight change results of mice, wherein Figure A shows the H&E, Sirius Red and Masson staining results of mouse lung tissues, Figure B shows the Ashcroft score statistics of pulmonary fibrosis in mice in each group, Figure C shows the results of hydroxyproline content in lung tissues of mice in each group, and Figure D shows the weight change of mice in each group, *p<0.05, **p<0.01, ***p <0.001;

[0026] Figure 5 The figure shows the effect of intraperitoneal injection of mulberry glycoside A on the mRNA and protein levels of fibronectin, type I collagen and α-SMA in the lung tissue of mice, wherein, in Figure A, from left to right, the mRNA levels of fibronectin, type I collagen and α-SMA in the lung tissue of each group of mice are shown, and in Figure B, from left to right, the WB representative graphs and semi-quantitative statistical graphs of the protein levels of fibronectin, type I collagen and α-SMA in the lung tissue of each group of mice are shown;

[0027] Figure 6 The figure shows the effect of mulberry glycoside A on the mRNA and protein expression levels of type I collagen, fibronectin and α-actin in human primary fibroblasts stimulated by TGF-β1, wherein, in Figure A, from left to right, the mRNA levels of fibronectin, type I collagen and α-SMA in each group of cells are shown, and in Figure B, from left to right, the WB representative graphs and semi-quantitative statistical graphs of the protein levels of fibronectin, type I collagen and α-SMA in each group of cells are shown. DETAILED DESCRIPTION

[0028] The present invention provides the use of mulberry glycoside A in the preparation of a drug for preventing and treating pulmonary fibrosis. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0029] Mulberry glycoside A is one of the main bioactive components in mulberry. Mulberry glycoside A can reduce the expression of TNF-α, IL-1β and IL-6, inhibit the activation of NALP3, caspase-1 and NF-κB, and the phosphorylation of ERK, JNK and p38. Mulberry glycoside A has anti-inflammatory and anti-apoptotic effects and has been widely studied in inflammatory diseases and tumor diseases, but its role in pulmonary fibrosis is still unclear.

[0030] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0031] Example 1 Effects of intraperitoneal injection of mulberry glycoside A on the degree of pulmonary fibrosis, lung function and lung pathology in mice

[0032] Experimental animals and materials:

[0033] 1. Experimental animals:

[0034] Source, strain, and line: Wild-type mice (WT, C57BL / 6) ordered from the Animal Experiment Center of Chongqing Medical University;

[0035] Reproductive age: 8-10 weeks;

[0036] 2. Experimental materials:

[0037] Bleomycin: purchased from Beijing Solebao Technology Co., Ltd.;

[0038] Isoflurane: purchased from Beijing Solebow Technology Co., Ltd.;

[0039] Physiological saline: purchased from Beijing Solebow Technology Co., Ltd.;

[0040] Mulberry glycoside A: purchased from MedChemExpress, its structure is as follows Figure 1 As shown in Figure A. Dissolution method: Add each solvent in sequence: 10% DMSO → 90% (20% SBE-β-CD in Saline) Solubility: ≥25 mg / mL (4.40 mM) clear solution. This solution can obtain a clear solution of ≥2.5 mg / mL (unknown saturation). Taking 1mL working solution as an example, take 100uL25.0 mg / mL of clear DMSO stock solution and add it to 900uL 20% SBE-β-CD saline solution and mix well. 2 g SBE-β-CD (sulfobutyl ether β-cyclodextrin) powder is diluted to 10mL of saline and completely dissolved until clear and transparent.

[0041] 3. Experimental methods:

[0042] 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. Mice injected with the same volume of normal saline through the airway were used as controls. 14 days after airway injection of bleomycin or normal saline, the mice were given continuous intraperitoneal injections of mulberry glycoside A at a final concentration of 10-20mg / kg for 7 consecutive days. Mice injected with the same volume of normal saline through the airway were used as controls. After pulmonary fibrosis modeling and superimposed intervention with mulberry glycoside A, the biosafety of the drug and its corresponding concentration was evaluated, that is, the liver function (ALT and AST), renal function (CR and BUN) and cardiac function (LDH and CK) in the plasma of each group of mice were detected. The mice were killed 21 days after bleomycin modeling, and the degree of pulmonary fibrosis in each mouse was analyzed (the dosing diagram is shown in the figure). Figure 1 (as shown in Figure B).

[0043] The severity of interstitial fibrosis in each serial area was independently assessed by two pathologists in a blinded manner using the Ashcroft scoring system.

[0044] 4. Experimental results:

[0045] Depend on Figure 2 As shown in the results, in the dosing regimen of this experiment, there were no significant differences in the liver function (i.e., ALT and AST), kidney function (i.e., CR and BUN), and heart function (i.e., LDH and CK) in the peripheral plasma of each group of mice, indicating the biosafety of the drug concentration; Figure 3 It can be seen that in the bleomycin-induced pulmonary fibrosis mouse model, administration of 10 mg / kg or 20 mg / kg of mulberry glycoside A can significantly improve the lung function indicators of mice; combined with Figure 4 It can be seen that after bleomycin stimulation and induction, compared with mice injected with normal saline, the degree of lung damage and fibrosis in mice treated with mulberry glycoside A intraperitoneal injection was significantly reduced, and the content of hydroxyproline in lung tissue was significantly reduced.

[0046] Example 2 Effects of intraperitoneal injection of mulberry glycoside A on protein and mRNA levels of fibronectin, type I collagen and α-SMA in mouse lung tissue

[0047] 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, type I collagen and α-SMA in the lung tissue of each mouse by western blot and RT-PCR, respectively.

[0048] 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 type I collagen and α-SMA, were detected by Western blot. The results are as follows: Figure 5 As shown in Figure 2. Western blot was based on the reference Wang et al., 2017, Journal of Allergy and Clinical Immunology 40:1550-1561.

[0049] 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 5 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.

[0050] Table 1 List of primers corresponding to target genes

[0051]

[0052] Combination Figure 5 It can be seen that compared with wild-type mice, the transcription levels and protein expression levels of fibronectin, type I collagen and α-SMA in mice injected intraperitoneally with mulberry glycoside A were reduced, which indicates that mulberry glycoside A can significantly reduce the degree of pulmonary fibrosis in mice.

[0053] Example 3 Effect of Mulberoside A on Differentiation of Fibroblasts into Myofibroblasts

[0054] 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 mulberry glycoside A on the differentiation of fibroblasts into myofibroblasts after TGF-β1 stimulation, the protein and mRNA levels of fibronectin, α-actin and type I collagen in human primary fibroblasts were detected by western blot and RT-PCR, respectively. Among them, the primers corresponding to each target gene are listed in Table 2 below.

[0055] Table 2 List of primers corresponding to target genes

[0056]

[0057] Next, the present invention further explores whether mulberry glycoside A has a reversal effect on the phenotype of activated human primary fibroblasts. Figure 6 The results showed that compared with human primary fibroblasts stimulated with 10 ng / mL TGF-β1, the transcription levels and protein expression levels of fibronectin, type I collagen and α-actin in cells pretreated with mulberry glycoside A for 1 h were significantly reduced.

[0058] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of mulberry glycoside A in the preparation of drugs for preventing and / or treating pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The pulmonary fibrosis disease is an interstitial lung disease.

3. The use according to claim 1 or 2, characterized in that: The pulmonary fibrosis disease includes bleomycin-induced pulmonary fibrosis.

4. The use according to any one of claims 1 to 3, characterized in that: The drugs include drugs that inhibit the transcription level and / or protein expression level of fibronectin.

5. The use according to any one of claims 1 to 3, characterized in that: The drugs include drugs that inhibit the transcription level and / or protein expression level of α-actin.

6. The use according to any one of claims 1 to 3, characterized in that: The drugs include drugs that inhibit the transcription level and / or protein expression level of type I collagen.

7. The use according to any one of claims 1 to 3, characterized in that: The drugs include drugs that inhibit the differentiation of fibroblasts into myofibroblasts.

8. The use according to any one of claims 1 to 7, characterized in that: The drug is used to slow, stop or reverse the extent of pulmonary fibrosis.

9. The use according to any one of claims 1 to 8, characterized in that: the dosage of the drug, The dosage for mice is 10~20 mg / kg once.

10. The use according to any one of claims 1 to 9, characterized in that: The dosage form of the drug includes tablets, capsules, granules, drops, lyophilized products, granules, ointments or injections.

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