Use of mulberry leaf extract in preparation of medicine for preventing and treating pulmonary fibrosis

Abstract

This invention relates to the field of pharmaceutical technology, specifically to the application of morin A in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. This invention discovers a novel use for morin A, a monomeric component of traditional Chinese medicine, which can be applied to the prevention and treatment of pulmonary fibrosis. By reducing the transcriptional and protein expression levels of fibronectin, α-actin, and type I collagen in fibroblasts, it inhibits the differentiation of fibroblasts into myofibroblasts, thereby slowing the progression of pulmonary fibrosis and providing a new treatment strategy for pulmonary fibrosis. Experimental results show that lung injury and fibrosis were significantly reduced in mice treated with intraperitoneal injections of 10 mg / kg and 20 mg / kg morin A. Furthermore, experiments on human primary fibroblasts also demonstrate that morin A can significantly reduce the protein expression levels of type I collagen, fibronectin, and α-actin in TGF-β1-stimulated human primary fibroblasts.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of morin A in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a chronic, progressive lung disease. It is the end-stage lung change of interstitial lung disease characterized by fibroblast proliferation, massive extracellular matrix deposition, and destruction of lung tissue structure. Its characteristic feature is that lung tissue is gradually replaced by scar tissue, leading to a gradual loss of lung function.

[0003] Symptoms of pulmonary fibrosis include persistent dry cough, shortness of breath, fatigue, and weakness. The main pathological features include mesenchymal cell proliferation, extracellular matrix deposition, and lung parenchymal remodeling. These symptoms gradually worsen, impacting the patient's quality of life. In severe cases, pulmonary fibrosis can lead to heart disease, respiratory failure, and other serious complications. A variety of factors can trigger pulmonary fibrosis, including occupational or home environment factors, medications, radiation therapy, high-concentration oxygen therapy, smoking, disease-related factors, and genetic factors.

[0004] Currently, the main treatments for pulmonary fibrosis include drug therapy and organ transplantation. Organ transplantation, as the only last resort for fibrosis patients, faces limitations in its application and promotion due to difficulties in donor sourcing, high surgical risks, and high costs. Furthermore, existing drugs cannot reverse the progression of fibrosis, and their clinical efficacy and safety do not meet treatment needs.

[0005] Studies have shown that during the fibrotic process, the expression of transforming growth factor-β, such as TGF-β, is upregulated in relevant tissues, and TGF-β can induce fibroblasts to differentiate into myofibroblasts. The differentiation of fibroblasts into myofibroblasts plays a crucial role in the fibrotic process. 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. Although current research indicates that TGF-β plays an important role in the progression of pulmonary fibrosis, there are still no clinically applicable drugs that can effectively block the TGF-β signaling pathway, thereby preventing or reversing the progression of pulmonary fibrosis.

[0006] Therefore, it is urgent to conduct in-depth research on the pathogenesis of pulmonary fibrosis, find new therapeutic targets, and fully integrate innovative pharmaceutical development trends to develop novel drugs that can effectively treat pulmonary fibrosis and have high safety. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide the application of morin A in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. The present invention discloses that morin A can effectively slow down the process of pulmonary fibrosis.

[0008] This invention provides the use of morin A in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis.

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

[0010] In some specific embodiments, the pulmonary fibrosis includes pulmonary fibrosis induced by bleomycin.

[0011] In some specific embodiments, the drug includes drugs that inhibit the transcriptional level and / or protein expression level of fibronectin.

[0012] In some specific embodiments, the drug includes drugs that inhibit the transcriptional level and / or protein expression level of α-actin.

[0013] In some specific embodiments, the drug includes a drug that inhibits the transcriptional 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 drug is used to slow, stop, or reverse the progression of pulmonary fibrosis.

[0016] In some embodiments, the dosage of the drug, calculated based on the mouse dosage, is 10-20 mg / kg per dose.

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

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

[0019] In some embodiments, the drug is a tablet, capsule, granule, drop, lyophilized product, granule, ointment, or injection.

[0020] In some embodiments, the medicament further includes excipients, which are carriers, solvents, emulsifiers, dispersants, wetting agents, binders, stabilizers, colorants, and fragrances.

[0021] Compared with existing technologies, this invention discovers a novel application for the traditional Chinese medicine monomer component morin A, which can be used for the prevention and treatment of pulmonary fibrosis. By reducing the transcriptional and protein expression levels of fibronectin, α-actin, and type I collagen in fibroblasts, it inhibits the differentiation of fibroblasts into myofibroblasts, thereby slowing the progression of pulmonary fibrosis and providing a new treatment strategy for pulmonary fibrosis. Experimental results show that mice treated with intraperitoneal injections of 10 mg / kg and 20 mg / kg morin A showed significantly reduced lung injury and fibrosis, and the transcriptional and protein expression levels of fibronectin, type I collagen, and α-SMA were all decreased. Cell experiments also showed that morin A can significantly reduce the protein expression levels of type I collagen, fibronectin, and α-actin in TGF-β1-stimulated human primary fibroblasts. Attached Figure Description

[0022] Figure 1 The diagram shows the structural schematic of morin A and the experimental administration diagram of the present invention in mice. In the diagram, A is the chemical molecular structure of morin A, and B is the administration diagram, that is, after two weeks of bleomycin-induced fibrosis, morin A was administered intraperitoneally daily at different concentrations.

[0023] Figure 2 This study evaluated the biosafety of the drug and its corresponding concentrations after pulmonary fibrosis modeling combined with morin A intervention. Figures A and B show the liver function test results in the plasma of mice in each group; Figures C and D show the kidney function test results in the plasma of mice in each group; Figures E and F show the cardiac function test results in the plasma of mice in each group; Figure A shows the ALT concentration test results; Figure B shows the AST concentration test results; Figure C shows the CR concentration test results; Figure D shows the BUN concentration test results; Figure E shows the LDH concentration test results; and Figure F shows the CK concentration test results.

[0024] Figure 3 The changes in lung function of mice in each group are shown in Figure A (Respiratory System Resistance), Figure B (Respiratory System Elasticity), and Figure C (Respiratory System Compliance). *p<0.05, **p<0.01, ***p <0.001;

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

[0026] Figure 5 This study shows the effects of intraperitoneal injection of morin A on the mRNA and protein levels of fibronectin, type I collagen, and α-SMA in mouse lung tissue. 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. In Figure B, from left to right, the WB representative graph and semi-quantitative statistical graph 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 This study investigated the effects of morin A on the mRNA and protein expression levels of type I collagen, fibronectin, and α-actin in human primary fibroblasts stimulated by TGF-β1. Figure A shows the mRNA levels of fibronectin, type I collagen, and α-SMA in each group of cells from left to right. Figure B shows the WB representation and semi-quantitative statistical plots of the protein levels of fibronectin, type I collagen, and α-SMA in each group of cells from left to right. Detailed Implementation

[0028] This invention provides the application of morin A in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

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

[0030] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

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

[0032] Laboratory animals and materials:

[0033] 1. Laboratory animals:

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

[0035] Childbearing age: 8-10 weeks;

[0036] 2. Experimental materials:

[0037] Bleomycin: Purchased from Beijing Solarbio Science & Technology Co., Ltd.

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

[0039] Physiological saline: purchased from Beijing Solarbio Science & Technology Co., Ltd.;

[0040] Morcinol 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 method can obtain a clear solution of ≥2.5 mg / mL (saturation unknown). Taking 1 mL of working solution as an example, take 100 μL of 25.0 mg / mL clear DMSO stock solution and add it to 900 μL of 20% SBE-β-CD physiological saline solution, and mix well. 2 g of SBE-β-CD (sulfobutyl ether β-cyclodextrin) powder is diluted to 10 mL of physiological saline and completely dissolved until clear and transparent.

[0041] 3. Experimental methods:

[0042] Wild-type mice were anesthetized with isoflurane inhalation, followed by intratracheal injection of bleomycin at a final concentration of 1.7 U / kg, dissolved in 50 μL of physiological saline. Mice that received the same volume of physiological saline via intratracheal injection served as controls. Fourteen days after intratracheal injection of bleomycin or physiological saline, mice were administered morin A at a final concentration of 10-20 mg / kg via intraperitoneal injection for seven consecutive days. Mice that received the same volume of physiological saline via intraperitoneal injection served as controls. Following pulmonary fibrosis modeling combined with morin A intervention, the biosafety of the drug and its corresponding concentrations was assessed by measuring liver function (ALT and AST), kidney function (CR and BUN), and cardiac function (LDH and CK) in plasma of each group of mice. Mice were sacrificed 21 days after bleomycin modeling, and the degree of pulmonary fibrosis in each mouse was analyzed (administration diagram shown in Figure 1). Figure 1 (As shown in Figure B).

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

[0044] 4. Experimental Results:

[0045] Depend on Figure 2 As shown, in this experiment, there were no significant differences in liver function (ALT and AST), kidney function (CR and BUN), and cardiac function (LDH and CK) in the peripheral plasma of mice in each group, indicating the biocompatibility of this drug concentration; Figure 3 It was found that in a bleomycin-induced pulmonary fibrosis mouse model, administration of morin A at 10 mg / kg or 20 mg / kg significantly improved lung function indicators in mice; combined with Figure 4 It can be seen that, after bleomycin stimulation and induction, compared with mice injected intraperitoneally with physiological saline, mice treated with intraperitoneal injection of morin A showed significantly reduced lung damage and fibrosis, and significantly reduced hydroxyproline content in lung tissue.

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

[0047] To further evaluate the degree of fibrosis in mice after bleomycin injection, the protein and mRNA levels of fibronectin, type I collagen, and α-SMA in the lung tissue of each mouse were detected by Western blot and RT-PCR, respectively.

[0048] Specifically, lung tissue from mice was collected after the experiment in Example 1. Proteins were extracted from the tissue using 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. The Western blot data is from Wang et al., 2017, Journal of Allergy and Clinical Immunology 40:1550-1561.

[0049] Simultaneously, quantitative RT-PCR was performed using SYBR Premix Ex Taq (TaKaRa), with GAPDH as an internal control, to normalize the relative expression of each target gene. The results are as follows: Figure 5 As shown in the table below, the primers corresponding to each target gene are listed in Table 1. Specific testing methods for gene expression are described in Chen et al., 2015, International journal of clinical and experimental pathology 8:6700-6707.

[0050] Table 1. List of primers corresponding to the target gene

[0051]

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

[0053] Example 3: Effect of morin A on the differentiation of fibroblasts into myofibroblasts

[0054] Evidence suggests that the differentiation of fibroblasts into myofibroblasts is essential for maintaining pulmonary fibrosis. Therefore, to further evaluate the effect of morin A on the differentiation of TGF-β1-stimulated fibroblasts into myofibroblasts, this invention detected the protein and mRNA levels of fibronectin, α-actin, and type I collagen in human primary fibroblasts using Western blot and RT-PCR. The primers corresponding to each target gene are listed in Table 2 below.

[0055] Table 2. List of primers corresponding to the target gene

[0056]

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

[0058] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of morin A in the preparation of drugs for treating pulmonary fibrosis.

2. The application according to claim 1, characterized in that, The pulmonary fibrosis mentioned refers to pulmonary fibrosis caused by bleomycin.

3. The application according to claim 1 or 2, characterized in that, The dosage forms of the drug include tablets, capsules, granules, drops, lyophilized products, granules, ointments, or injections.

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