Use of dicumarol in the preparation of a medicament for the prevention or treatment of a fibrotic disease

Abstract

The application discloses application of dicumarol in preparation of a medicine for preventing or treating a fibrosis disease, and relates to the technical field of biological medicine. The application creatively uses dicumarol for treating and preventing a fibrosis disease, can play a strong anti-fibrosis role, has small toxic side effect and is safe to use, has an attractive application prospect in prevention and treatment of fibrosis, and has good market application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of dicumarol in the preparation of drugs for the prevention or treatment of fibrotic diseases. Background Technology

[0002] Fibrosis is an excessive and uncontrolled repair response following tissue damage caused by various factors. Cells secrete excessive amounts of extracellular matrix proteins, leading to the proliferation of fibrous connective tissue and collagen deposition within organ tissues. While the proliferating fibrous connective tissue repairs the defects, it lacks the original structure and function of the organ's parenchymal cells, resulting in organ deformation, functional decline, and even failure. Organ fibrosis is a common pathological process in many acute and chronic diseases, and a major pathological change in chronic autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, myelofibrosis, scleroderma, and chronic suppression and rejection. Organ failure caused by fibrosis in parenchymal organs such as the lungs, liver, kidneys, and heart is a significant cause of disability and death in patients.

[0003] Clinically, treatment strategies and methods for fibrosis are mainly based on the roles of immune and inflammatory responses in the fibrotic process, using a combination of anti-inflammatory drugs and immunosuppressive drugs to slow the progression of fibrosis. However, these treatments have not been widely adopted due to the uncertainty of their efficacy and numerous adverse reactions. While organ transplantation is the only effective treatment for end-stage fibrosis, it also faces many challenges, including organ donor shortages, low surgical survival rates, and difficult postoperative recovery. Antifibrotic drugs with high specificity, definite efficacy, and no significant adverse reactions have immense clinical value; however, the number of drugs currently approved for antifibrotic indications is very limited.

[0004] The pathophysiology of fibrosis is highly complex and remains largely undefined. Studies have shown that numerous cytokines and signaling pathways are involved in the progression of fibrosis. Transforming growth factor β1 (TGF-β1) is a polypeptide member of the TGF-β superfamily of cytokines and can be produced by various cell types, including monocytes / macrophages, neutrophils, osteoblasts, endothelial cells, lymphocytes, and fibroblasts. As a secreted protein, TGF-β1 has a wide range of biological functions, regulating cell growth, proliferation, differentiation, and apoptosis. Numerous studies have demonstrated that TGF-β1 and its signaling pathways are the most important known mechanisms inducing fibrosis and are also key signaling pathways and targets for the study and prevention of fibrosis in various organs.

[0005] Epithelial-mesenchymal transition (EMT) is the process by which epithelial cells, under the influence of various factors, lose their epithelial characteristics and transform into mesenchymal cells. During EMT, morphologically, cobblestone-like or polygonal epithelial cells become elongated and thin; at the molecular level, the expression of the epithelial cell marker protein E-cadherin significantly decreases, while the expression of mesenchymal cell marker proteins such as N-cadherin and Vimentin significantly increases, and the expression of the myofibroblast marker α-SMA significantly increases. There are three types of EMT, among which type 2 EMT is closely related to tissue regeneration and organ fibrosis. Numerous studies have shown that EMT is an important physiological and pathological process leading to organ fibrosis, a key focus in the prevention and treatment of fibrosis, and a hot topic in drug development; inhibiting EMT can effectively prevent and treat organ fibrosis. TGF-β1 is a key cytokine inducing EMT. It can induce EMT by stimulating epithelial cells and endothelial cells, stimulating the deposition of extracellular matrix while inhibiting its degradation, and promoting collagen deposition and connective tissue formation in tissues. Inhibition of the TGF-β1 pathway and TGF-β1-induced EMT have significant protective effects against fibrosis and are a hot topic in the research and development of anti-fibrotic drugs.

[0006] Endothelial cells are a type of epithelial cell. Upon stimulation by TGF-β1, endothelial cells can transform into mesenchymal cells, a process known as endothelial-mesenchymal transition (EndMT). During EndMT, cell morphological changes are similar to those in endometrial-mesenchymal transition (EMT), and the expression of the molecular marker VE-cadherin is significantly reduced. EndMT is an important mediator of diseases including fibrosis and cardiovascular diseases.

[0007] Numerous studies have confirmed that TGF-β and its induced EMT signaling pathway play crucial roles in the development and progression of idiopathic pulmonary fibrosis (IPF), drug-induced pulmonary fibrosis (such as bleomycin-induced), pesticide-induced pulmonary fibrosis (such as paraquat-induced), liver fibrosis, kidney fibrosis, cardiac fibrosis, and pancreatic fibrosis. Inhibition of TGF-β1 and its induced EMT signaling pathway is a major mechanism by which many compounds prevent and treat these types of fibrosis. However, currently, no drugs that directly regulate EMT for the prevention and treatment of organ fibrosis have been approved for marketing.

[0008] During tissue injury, fibroblasts, mesenchymal cells, and circulating fibroblasts can differentiate into myofibroblasts under stimulation by TGF-β, inflammatory factors, and other stimuli. Myofibroblasts initiate wound healing responses and maintain tissue homeostasis by remodeling the extracellular environment. However, persistent injury and damage lead to a sustained upregulation of myofibroblast activity and the secretion of large amounts of myosin and α-SMA, resulting in excessive deposition of ECM proteins and inducing the occurrence and development of fibrosis. Myofibroblast formation is a crucial process promoting fibrotic diseases, and inhibiting the differentiation of fibroblasts into myofibroblasts is an important strategy in the development of anti-fibrotic drugs.

[0009] Currently, only a few drugs are approved by the FDA for the treatment of organ fibrosis. Among them, nintedanib and pirfenidone are approved for the treatment of idiopathic pulmonary fibrosis, while ruxolitinib and fedratinib are approved for the treatment of myelofibrosis. Nintedanib is a small-molecule tyrosine kinase inhibitor that inhibits receptor tyrosine kinases such as platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor receptor (VEGFR), thereby inhibiting the proliferation, migration, and differentiation of fibroblasts. The mechanism of action of pirfenidone is not fully understood, but it may be related to the inhibition of proliferation, fibrosis-related protein and cytokine production, and extracellular matrix synthesis and accumulation induced by fibroblast stimulation from cell growth factors such as TGF-β and platelet-derived growth factor (PDGF). Ruxolitinib is a selective kinase inhibitor that works by inhibiting JAK1 and JAK2, thereby blocking the JAK signal transducer and activator of transcription (STAT) pathway.

[0010] However, nintedanib, pirfenidone, ruxolitinib, and fizzotinib all have toxic side effects: nintedanib can cause diarrhea, nausea, vomiting, skin ulcers, abdominal pain, elevated liver enzymes, and weight loss. Pirfenidone can cause photosensitivity, loss of appetite, stomach upset, nausea, and severe liver damage. Ruxolitinib's side effects include decreased platelet count, anemia, ecchymosis, dizziness, and headache. Fizzotinib's side effects include encephalopathy (including Wernicke's encephalopathy), decreased platelet count, anemia, nausea, vomiting, and diarrhea.

[0011] Besides the drugs mentioned above, there are currently no effective treatments for fibrosis in other organs. Therefore, there is an urgent need for highly effective and low-toxicity anti-fibrotic drugs for various types of fibrosis and related diseases in clinical practice.

[0012] In view of this, the present invention is proposed. Summary of the Invention

[0013] The purpose of this invention is to provide the application of dicumarol in the preparation of medicaments for the prevention or treatment of fibrotic diseases, with the aim of improving the efficacy of fibrotic disease treatment while reducing toxic side effects.

[0014] The present invention also aims to provide the application of dicumarol in the preparation of reagents for the analysis and testing of EMT, EndMT, and fibroblast-to-myofibroblast transformation.

[0015] This invention is implemented as follows:

[0016] In a first aspect, the present invention provides the use of dicumarol in the preparation of medicaments for the prevention or treatment of fibrotic diseases.

[0017] In an optional implementation, fibrotic diseases include those induced by pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, pancreatic fibrosis, and organ fibrosis.

[0018] Preferably, the fibrotic disease is selected from at least one of pulmonary fibrosis, liver fibrosis, and kidney fibrosis;

[0019] More preferably, the fibrotic disease is selected from renal fibrosis.

[0020] In an optional implementation, the mechanism of drug prevention and treatment of fibrotic diseases includes at least one of the following (a) to (h);

[0021] (a) By inhibiting EMT;

[0022] (b) By inhibiting EndMT;

[0023] (c) By inhibiting TGF-β1-induced conversion of fibroblasts to myofibroblasts;

[0024] (d) By reversing the decrease in E-cadherin expression in epithelial cells and VE-cadherin expression in endothelial cells induced by TGF-β1 and TGF-β2;

[0025] (e) By reversing the increased expression of interstitial cell marker proteins N-cadherin and Vimentin;

[0026] (f) By inhibiting the expression of fibronectin;

[0027] (g) By reversing the expression of TGF-β1-induced myofibroblast marker α-SMA;

[0028] (h) By inhibiting the morphological changes in epithelial cells, endothelial cells and cardiomyocytes induced by TGF-β1 and TGF-β2.

[0029] In an optional implementation, pulmonary fibrosis includes idiopathic pulmonary fibrosis, induced pulmonary fibrosis, and other pulmonary fibrosis of unknown etiology; wherein, induced pulmonary fibrosis includes sarcoidosis-induced pulmonary fibrosis, pneumoconiosis-induced pulmonary fibrosis, allergic pneumonia-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, drug-induced pulmonary fibrosis, and pesticide-induced pulmonary fibrosis.

[0030] In optional embodiments, liver fibrosis includes liver fibrosis induced by viral hepatitis, liver fibrosis induced by alcoholic hepatitis, liver fibrosis induced by autoimmune diseases, liver fibrosis induced by fatty liver, drug-induced liver fibrosis, and other liver fibrosis of unknown etiology.

[0031] In optional embodiments, renal fibrosis includes renal fibrosis induced by hypertension, renal fibrosis induced by glomerulonephritis, renal fibrosis induced by systemic lupus erythematosus, renal fibrosis induced by scleroderma, renal fibrosis induced by kidney transplant rejection, renal fibrosis induced by pyelonephritis, renal fibrosis induced by kidney stones, renal fibrosis induced by hyperlipidemia, renal fibrosis induced by diabetes, renal fibrosis induced by hyperuricemia, renal fibrosis induced by hypercalciuria, and other renal fibrosis of unknown etiology.

[0032] In optional embodiments, cardiac fibrosis includes cardiac fibrosis induced by ischemic heart disease, cardiac fibrosis induced by hypertension, cardiac fibrosis induced by viral myocarditis, cardiac fibrosis induced by metabolic cardiomyopathy, cardiac fibrosis induced by Keshan disease, cardiac fibrosis induced by dilated cardiomyopathy, cardiac fibrosis induced by restrictive cardiomyopathy, cardiac fibrosis induced by cardiac remodeling, cardiac fibrosis induced by myocardial hypertrophy, and other cardiac fibrosis of unknown etiology.

[0033] In an alternative implementation, pancreatic fibrosis includes pancreatic fibrosis induced by chronic pancreatitis and other pancreatic fibrosis of unknown etiology.

[0034] In an optional embodiment, the drug is a pharmaceutical composition, which further includes a pharmaceutical carrier or other active pharmaceutical ingredient;

[0035] Preferably, the dicumarol in the pharmaceutical composition is dicumarol or a pharmaceutical derivative thereof.

[0036] Preferably, the dosage form of the pharmaceutical composition is selected from at least one of solution, suspension, tablet, capsule, granule and injection.

[0037] Secondly, the present invention also provides the use of dicumarol in the preparation of reagents for the analysis and testing of EMT, EndMT, and fibroblast-to-myofibroblast transformation, wherein the reagents are selected from analytical reagents, biochemical reagents, or detection reagents.

[0038] The present invention has the following beneficial effects: The present invention creatively utilizes dicumarol for the treatment and prevention of fibrotic diseases, which can exert a strong anti-fibrotic effect with few toxic side effects. Its application prospects in the prevention and treatment of fibrosis are attractive and it has great market application value. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 Figure 1 shows the results of DIC's inhibition of TGF-β1-induced EMT in human lung epithelial A549 cells; DIC inhibits TGF-β1-induced cell morphology (AD) changes and reverses the expression of EMT biomarkers (EH); A, normal control group; B, TGF-β1 (5 ng / mL) group; C, TGF-β1 + DIC (2 μM) group; D, TGF-β1 + DIC (4 μM) group; E, Western blotting detection of DIC concentration-dependent reversal of TGF-β1-induced EMT signaling pathway and biomarker expression; F, Western blotting detection of DIC time-dependent reversal of TGF-β1-induced EMT signaling pathway and biomarker expression; G, Immunofluorescence detection of the effect of DIC on the expression of TGF-β1-induced EMT biomarker E-cadherin; H, Immunofluorescence detection of the effect of DIC on the expression of TGF-β1-induced EMT biomarker N-cadherin.

[0041] Figure 2 Figure 1 shows the results of the assay for the inhibition of TGF-β1-induced EMT in human hepatic epithelial HepG2 cells by dicoumarin (DIC); A, normal control group; B, TGF-β1 (5 ng / mL) group; C, TGF-β1 + DIC (2.5 μM) group; D, TGF-β1 + DIC (5.0 μM) group; E, Western blotting detection of the expression of biomarkers of TGF-β1-induced EMT by different concentrations of DIC.

[0042] Figure 3Figure 1 shows the results of the assay for the inhibition of TGF-β1-induced EMT in human pancreatic epithelial MIA PaCa-2 cells by dicoumarin (DIC); A, normal control group; B, TGF-β1 (5 ng / mL) group; C, TGF-β1 + DIC (2.5 μM) group; D, TGF-β1 + DIC (5.0 μM) group; E, Western blotting detection of the expression of biomarkers of TGF-β1-induced EMT by different concentrations of DIC.

[0043] Figure 4 Figure 1 shows the results of the assay for the inhibition of TGF-β2-induced EndMT in EA.hy926 vascular endothelial cells by dicoumarin (DIC); A, normal control group; B, TGF-β2 (5 ng / mL) group; C, TGF-β2 + DIC (5.0 μM); D, TGF-β2 + DIC (10 μM); E, Western blotting analysis of the effect of different concentrations of DIC on the expression of TGF-β2-induced EMT biomarkers.

[0044] Figure 5 Figure 1 shows the effect of dicumarol (DIC) on TGF-β1-induced H9c2 cardiomyocytes; A, normal control group; B, TGF-β1 (5 ng / mL) group; C, TGF-β1 + DIC (5.0 μM) group; D, TGF-β1 + DIC (10 μM); E, Western blotting analysis of the effect of different concentrations of DIC on the expression of TGF-β1-induced myocardial fibrosis-related proteins.

[0045] Figure 6 Figure 1 shows the results of EMT assay in human renal tubular epithelial HK-2 cells induced by dicoumarin inhibition (DIC) of TGF-β1; A, normal control group; B, TGF-β1 (5 ng / mL); C, TGF-β1 + DIC (5.0 μM); D, TGF-β1 + DIC (10 μM); E, Immunofluorescence assay of the effect of DIC on the expression of N-cadherin, a protein in TGF-β1-induced EMT; E, normal control; F, TGF-β1 (5 ng / mL); G, TGF-β1 + DIC (10 μM); H, Western blotting assay of the effect of DIC on the expression of N-cadherin induced by TGF-β1.

[0046] Figure 7Figure 1 shows the results of EMT assay in TGF-β1-induced rat renal tubular epithelial NRK-52E cells induced by dicumarol (DIC); A, normal control group; B, TGF-β1 (10 ng / mL); C, TGF-β1 + DIC (2.5 μM); D, TGF-β1 + DIC (5.0 μM); E, Western blotting analysis of the effect of different concentrations of DIC on the expression of TGF-β1-induced fibrosis-related proteins.

[0047] Figure 8 Figure 1 shows the results of DIC inhibition of TGF-β1-induced EMT in rat renal tubular epithelial NRK-52E cells; AC, Immunofluorescence detection of the effect of DIC on TGF-β1-induced α-SMA expression; A, Normal control group; B, TGF-β1 (10 ng / mL) group; C, TGF-β1 + DIC (2.5 μM); DF, Immunofluorescence detection of the effect of DIC on TGF-β1-induced E-cadherin expression; D, Normal control group; E, TGF-β1 (10 ng / mL) group; F, TGF-β1 + DIC (2.5 μM) group.

[0048] Figure 9 The image shows the test results of dicoumarin (DIC) inhibiting TGF-β1-induced myofibroblast formation in human embryonic lung fibroblasts MRC-5 and WI-38 cells;

[0049] Figure 10 Figure 1 shows the results of testing other coumarin anticoagulants (including warfarin, 3,3'-carbonylbis(7-diethylaminocoumarin), and acetocoumarin) on TGF-β1-induced EMT; AE, warfarin test group; FJ, 3,3'-carbonylbis(7-diethylaminocoumarin) test group; KO, acetocoumarin test group. Among them, A, F, and K were normal control groups; B, G, and L were TGF-β1 (5 ng / mL) groups; C was TGF-β1 + warfarin (2.0 μM); D was TGF-β1 + warfarin (4.0 μM); H was TGF-β1 + 3,3'-carbonylbis(7-diethylaminocoumarin) (2.0 μM); I was TGF-β1 + 3,3'-carbonylbis(7-diethylaminocoumarin) (4.0 μM); M was TGF-β1 + acetocoumarin (2.0 μM); and N was TGF-β1 + acetocoumarin (4.0 μM).

[0050] Figure 11 Figure 1 shows the results of H&E staining of kidneys in a rat UUO model, which showed the therapeutic effect of dicumarol (DIC) on kidney damage. A, control group; B, model group; C, dicumarol 2.5 mg / kg group; D, dicumarol 5 mg / kg group.

[0051] Figure 12 Figure 1 shows the results of Masson staining test on the therapeutic effect of dicumarol (DIC) on renal fibrosis in a rat UUO model; A, control group; B, model group; C, dicumarol 2.5 mg / kg group; D, dicumarol 5 mg / kg group.

[0052] Figure 13 Figure 1 shows the results of testing the therapeutic effect of dicumarol (DIC) on renal fibrosis in a rat UUO model (Sirius red staining of the kidney); A, control group; B, model group; C, dicumarol 2.5 mg / kg group; D, dicumarol 5 mg / kg group.

[0053] Figure 14 Figure 1 shows the results of immunohistochemical staining of kidney E-cadherin protein in the rat UUO model of renal fibrosis (the therapeutic effect of dicumarol (DIC) on renal fibrosis). A, control group; B, model group; C, dicumarol 2.5 mg / kg group; D, dicumarol 5 mg / kg group.

[0054] Figure 15 Figure 1 shows the results of the test on the therapeutic effect of dicumarol (DIC) on renal fibrosis in a rat UUO model (immunohistochemical staining of renal α-SMA protein); A, control group; B, model group; C, dicumarol 2.5 mg / kg group; D, dicumarol 5 mg / kg group.

[0055] Figure 16 Figure 1 shows the results of immunohistochemical staining of kidney vimentin protein in rats with renal fibrosis (UUO model) and the therapeutic effect of dicumarol (DIC); A, control group; B, model group; C, dicumarol 2.5 mg / kg group; D, dicumarol 5 mg / kg group.

[0056] Figure 17 The figure shows the test results of the therapeutic effect of dicoumarin (DIC) on renal fibrosis in a rat UUO model. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0058] Dicoumarol (DIC), also known as dicumarol, alfalfa phenol, and dicumarol, is a naturally occurring coumarin compound. Dicoumarol has been approved for clinical use as an anticoagulant for many years, used to prevent and treat thrombosis and thromboembolic diseases such as thrombosis and thrombophlebitis. Dicoumarol is abundant, has a high safety profile, and is already a clinically used drug, making it valuable for research and development.

[0059] Dicoumarol, as a coumarin anticoagulant, has been used clinically for decades and is a well-established drug with a clear anticoagulant effect and high safety profile. In particular, even for the bleeding side effect caused by overdose of dicoumarol, inexpensive vitamin K can effectively antagonize it.

[0060] The chemical structure of dicoumarol (DIC) (CAS No.: 66-76-2) is as follows:

[0061]

[0062] Coumarin anticoagulants also include warfarin, 3,3'-carbonylbis(7-diethylaminocoumarin), and acenitroprusside. The chemical structure of warfarin is:

[0063]

[0064] The chemical structure of 3,3'-carbonylbis(7-diethylaminocoumarin) is as follows:

[0065]

[0066] The chemical structure of acetocoumarin is as follows:

[0067]

[0068] This invention creatively applies dicumarol to the preparation of drugs for the prevention or treatment of fibrotic diseases, and discovers that dicumarol has a significant inhibitory effect on TGF-β-induced EMT, EndMT, and the transformation of fibroblasts into myofibroblasts at the cellular level; it also exhibits significant anti-fibrotic activity at the animal level, showing promising application prospects and value in the development of anti-fibrotic drugs. Given the extremely limited clinical drugs currently available for organ fibrosis, dicumarol is an "old drug" that has been used clinically for many years. It is effective orally, has good safety, and can exert a strong anti-fibrotic effect when taken orally. Its application prospects in the prevention and treatment of fibrosis are attractive, and it has very good market application value.

[0069] It should be noted that, in order to overcome the shortcomings and deficiencies of existing technologies in the development of antifibrotic drugs, the inventors conducted extensive research and practice, ultimately providing dicumarol as a potential antifibrotic drug. Further experimental findings revealed that dicumarol can regulate the TGF-β1 signaling pathway, a key signaling pathway in various fibrotic diseases, significantly inhibiting TGF-β1-induced EMT, EndMT, and the transformation of fibroblasts into myofibroblasts. This was confirmed using an in vivo rat fibrosis model. Therefore, research has shown that dicumarol is a promising drug for the prevention and treatment of fibrosis, providing a treatment option for fibrotic diseases for which there are virtually no other effective treatments.

[0070] It is worth noting that although other coumarins, such as warfarin, 3,3'-carbonylbis(7-diethylaminocoumarin), and acetocoumarin, have similar chemical structures and anticoagulant mechanisms to dicumarol, they do not inhibit TGF-β1-induced EMT. This suggests that dicumarol has a unique role in the prevention and treatment of fibrotic diseases, which is not significantly related to its anticoagulant effect.

[0071] In some embodiments, fibrotic diseases include diseases induced by pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, pancreatic fibrosis, and organ fibrosis. It can be fibrosis of any one or more of these organs, or it can be a disease induced by pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, pancreatic fibrosis, etc. Preferably, the fibrotic disease is selected from any one of pulmonary fibrosis, liver fibrosis, and kidney fibrosis; more preferably, the fibrotic disease is selected from kidney fibrosis.

[0072] Furthermore, pulmonary fibrosis includes idiopathic pulmonary fibrosis, induced pulmonary fibrosis, and other pulmonary fibrosis of unknown etiology, which can be any one or more of the above. Among them, induced pulmonary fibrosis includes pulmonary fibrosis induced by sarcoidosis, pulmonary fibrosis induced by pneumoconiosis, pulmonary fibrosis induced by allergic pneumonia, radiation-induced pulmonary fibrosis, drug-induced pulmonary fibrosis, pesticide-induced pulmonary fibrosis, and pulmonary fibrosis induced by pulmonary infection, etc. Specifically, drug-induced pulmonary fibrosis refers to drugs such as bleomycin, mitomycin, amiodarone, methotrexate, hydralazine, nitrofurantoin, dalometabolite, penicillamine, and carmustine, but is not limited to these; pesticide-induced pulmonary fibrosis refers to pesticides such as paraquat, but is not limited to these; pulmonary infection-induced pulmonary fibrosis refers to infections such as hematogenous disseminated tuberculosis, Pneumocystis carinii infection, and viral pneumonia such as severe acute respiratory syndrome (SARS), but is not limited to these.

[0073] Furthermore, liver fibrosis includes liver fibrosis induced by viral hepatitis, liver fibrosis induced by alcoholic hepatitis, liver fibrosis induced by autoimmune diseases, liver fibrosis induced by fatty liver, drug-induced liver fibrosis, and other liver fibrosis of unknown etiology, and may be any one or more of the above. Viral hepatitis may refer to hepatitis B virus, but is not limited to this; drug-induced liver fibrosis may refer to drugs such as methotrexate, methyldopa, amiodarone, chlorpromazine, and Panax notoginseng, but is not limited to these.

[0074] Furthermore, renal fibrosis includes renal fibrosis induced by hypertension, glomerulonephritis, systemic lupus erythematosus, scleroderma, kidney transplant rejection, pyelonephritis, kidney stones, hyperlipidemia, diabetes, hyperuricemia, hypercalciuria, drug-induced fibrosis, and other renal fibrosis of unknown etiology, which can be any one or more of the above. Drug-induced renal fibrosis can refer to drugs such as aristolochic acid, cisplatin, neomycin, kanamycin, gentamicin, doxorubicin, and daunorubicin, but is not limited to these.

[0075] Furthermore, cardiac fibrosis includes cardiac fibrosis induced by ischemic heart disease, cardiac fibrosis induced by hypertension, cardiac fibrosis induced by viral myocarditis, cardiac fibrosis induced by metabolic cardiomyopathy, cardiac fibrosis induced by Keshan disease, cardiac fibrosis induced by dilated cardiomyopathy, cardiac fibrosis induced by restrictive cardiomyopathy, cardiac fibrosis induced by cardiac remodeling, cardiac fibrosis induced by myocardial hypertrophy, and other cardiac fibrosis of unknown etiology, which can be any one or more of the above.

[0076] Furthermore, pancreatic fibrosis includes pancreatic fibrosis induced by chronic pancreatitis and other pancreatic fibrosis of unknown etiology. It can be pancreatic fibrosis induced by chronic pancreatitis, but is not limited to this. Chronic pancreatitis can be primary, alcoholic, obstructive, autoimmune, metabolic, idiopathic, etc.

[0077] In some embodiments, the mechanism of drug prevention and treatment of fibrotic diseases includes at least one of the following (a) to (h);

[0078] (a) By inhibiting EMT;

[0079] (b) By inhibiting EndMT;

[0080] (c) By inhibiting TGF-β1-induced conversion of fibroblasts to myofibroblasts;

[0081] (d) By reversing the decrease in E-cadherin expression in epithelial cells and VE-cadherin expression in endothelial cells induced by TGF-β1 and TGF-β2;

[0082] (e) By reversing the increased expression of interstitial cell marker proteins N-cadherin and Vimentin;

[0083] (f) By inhibiting the expression of fibronectin;

[0084] (g) By reversing the expression of TGF-β1-induced myofibroblast marker α-SMA;

[0085] (h) By inhibiting the morphological changes in epithelial cells, endothelial cells and cardiomyocytes induced by TGF-β1 and TGF-β2.

[0086] It should be noted that the mechanism by which dicumarol can prevent or treat fibrotic diseases can be based on any one or more of the above, mainly through regulating EMT and EndMT, inhibiting the transformation of fibroblasts into myofibroblasts, collagen deposition, and connective tissue formation. In addition, dicumarol can also inhibit the phosphorylation of TβRI and Smad2 / 3, and inhibit the expression of fibronectin.

[0087] At the molecular level, dicumarol can significantly upregulate the expression of E-cadherin protein in epithelial cells and downregulate the expression of N-cadherin, Vimentin, and α-SMA proteins. Dicoumarol can reverse the TGF-β1 and TGF-β2-induced decrease in the expression of E-cadherin in epithelial cells and VE-cadherin in endothelial cells, and the increase in the expression of N-cadherin and Vimentin. It can also reverse the TGF-β1-induced expression of α-SMA.

[0088] At the cellular level, dicumarol can significantly inhibit the morphological changes of epithelial cells, endothelial cells and cardiomyocytes induced by TGF-β1 and TGF-β2; dicumarol can significantly inhibit the transformation of fibroblasts into myofibroblasts induced by TGF-β1.

[0089] At the animal level, dicumarol can significantly inhibit kidney damage in a rat model of renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO); dicumarol can significantly inhibit the deposition of renal interstitial collagen in the rat UUO model; dicumarol can also significantly inhibit the expression of α-SMA, Vimentin and fibronectin in the kidneys of the rat UUO model.

[0090] In some embodiments, the medicament provided by the present invention uses dicumarol as the active ingredient and may also include other functional drugs or pharmaceutical carriers, applied in the form of a composition. The specific types of other functional drugs or pharmaceutical carriers are not limited. The dicumarol in the pharmaceutical composition is dicumarol or its pharmaceutical derivative, specifically at least one of a pharmaceutical salt or ester, its derivative, and a pharmaceutically acceptable salt thereof.

[0091] Furthermore, the medicament provided in the embodiments of the present invention may also contain pharmaceutically acceptable excipients and be formulated into a pharmaceutically acceptable dosage form. Specifically, the excipients are liquid, solid, or semi-solid excipients. The dosage form of the pharmaceutical composition is selected from at least one of solution, suspension, tablet, capsule, granule, and injection, and the solution may be an aqueous solution or a non-aqueous solution.

[0092] Based on the aforementioned technical effects and mechanisms of dicumarol, dicumarol can also be used in the preparation of analytical reagents, biochemical reagents, or detection reagents for the analysis and testing of EMT, EndMT, and the transformation of fibroblasts into myofibroblasts.

[0093] Similarly, the dicumarol in the above applications includes at least one of dicumarol, its pharmaceutically acceptable salt or ester, its derivatives, and pharmaceutically acceptable salts of its derivatives, preferably dicumarol.

[0094] It should be noted that the dicumarol in the embodiments of the present invention can be replaced by one or a combination of several of its pharmaceutically usable salts or esters, its derivatives, pharmaceutically acceptable salts of its derivatives, and nanoscale substances of its derivatives to achieve the same or similar therapeutic effects, and all of them should fall within the scope of protection of the present invention.

[0095] This invention also provides an anti-fibrotic product comprising at least one of the following: dicumarol, a pharmaceutically acceptable salt or ester thereof, a derivative thereof, and a pharmaceutically acceptable salt thereof; any one or more of the above may be present.

[0096] Specifically, the products include medicines, reagent kits, and health supplements.

[0097] In some embodiments, the drug may be an oral or injectable formulation. The antifibrotic product may also include an acceptable pharmaceutical carrier and / or pharmaceutically acceptable excipients, and can be formulated into a variety of pharmaceutically acceptable formulations, including oral formulations such as tablets (controlled-release tablets, sustained-release tablets), granules, capsules (hard capsules, soft capsules), pills, decoctions, and injectable formulations such as lyophilized powder for injection.

[0098] Specifically, pharmaceutical carriers include, but are not limited to, one or more of the following: saline, buffer solution, glucose, water, glycerol, ethanol, low molecular weight dextran, polyethylene glycol 400, polyethylene glycol 6000, cyclodextrin, mannitol, lactose, glucose, sucrose, sodium chloride, and sorbitol.

[0099] It should be noted that there are no strict limitations on the preparation method of dicumarol drugs. Under aseptic conditions, they can be formulated into injections, for example, using physiological saline or aqueous solutions containing glucose and other excipients through conventional methods; they can also be formulated into tablets and capsules, prepared through conventional methods. Dicoumarol can also be a commercially available product, with no restrictions on the specific manufacturer or model.

[0100] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0101] Cellular level experiments (Examples 1-10):

[0102] Experimental materials:

[0103] 1. Experimental cell lines:

[0104] A549 and HepG2 cells were purchased from the American College of Cell Bank (ATCC). HK-2 human renal tubular epithelial cells, NRK-52E rat renal tubular epithelial cells, EAhy926 human endothelial cells, H9c2 human cardiomyocytes, human embryonic lung fibroblasts (MRC-5 cells), and WI-38 cells were purchased from the Peking Union Medical College Cell Resource Center. The MIA PaCa-2 cell line was donated to Professor Chen Qiang of the Faculty of Health Sciences, University of Macau.

[0105] 2. Cell Culture:

[0106] A549 cells were cultured using complete medium (RPMI-1640 medium containing 10% FBS and 1% penicillin antibiotics) according to standard procedures; HepG2 cells, HK-2 cells, NRK-52E cells, H9c2 cells, and EA.hy926 cells were cultured using complete medium (DMEM medium containing 10% FBS and 1% penicillin antibiotics); MIA PaCa-2 cells were cultured using complete medium (DMEM medium containing 10% FBS, 2.5% horse serum, and 1% penicillin antibiotics); and MRC-5 cells and WI-38 cells were cultured using MEM medium (containing 10% FBS). All cultures were cultured at a constant temperature of 37°C and under 5% CO2 conditions.

[0107] 3. Experiment on preparation of drug stock solution:

[0108] Take dicumarol (purchased from Maclean's, purity >99%) powder, weigh it, and dissolve it in DMSO solution to prepare a 10 mM mother liquor.

[0109] Experimental Example 1

[0110] To investigate the effect of dicumarol on EMT in A549 cells.

[0111] Experimental methods: A549 cells were stimulated with TGF-β1, with or without dicumarol treatment. Cell morphology changes were observed under a microscope, and EMT biomarker expression was detected by Western blotting and immunofluorescence.

[0112] Test Procedure: A549 cells in logarithmic growth phase were cultured in 96-well or 6-well plates and grown overnight at 37°C with 5% CO2. Cells were then treated with TGF-β1 (5 ng / mL) in combination with different concentrations of dicumarol (0 μM, 1.0 μM, 2.0 μM, 4.0 μM) in medium containing 1% FBS for 48 h. Cell morphology changes were observed under a microscope, and the expression of EMT biomarkers was measured by routine Western blotting and immunofluorescence.

[0113] See test results Figure 1 .in, Figure 1 In the middle section, AD represents the effect of dicumarol on TGF-β1-induced cell morphology changes; E represents the changes in TGF-β1-induced EMT biomarkers by different concentrations of dicumarol; F represents the effect of dicumarol on TGF-β1-activated signaling pathway proteins, including the effects of phosphorylation activation of TGF-β receptors I and II and Smad; G and H represent the immunofluorescence results of dicumarol on TGF-β1-induced EMT biomarkers E-cadherin and N-cadherin, respectively.

[0114] according to Figure 1 Microscopic examination revealed that after TGF-β1 stimulation, A549 cells exhibited a slender, elongated morphology. Western blotting showed a significant decrease in E-cadherin expression and a significant increase in N-cadherin expression. Immunofluorescence analysis showed that both E-cadherin and N-cadherin were expressed on the cell membrane. These changes were consistent with EMT, suggesting that TGF-β1 induces EMT. Treatment with dicumarol significantly altered these changes, indicating that dicumarol can significantly inhibit TGF-β1-induced EMT in A549 cells. Furthermore, dicumarol significantly inhibited TGF-β1-induced expression of TGF-β receptor I, had no significant effect on receptor II, and also significantly inhibited phosphorylation activation of Smad2 and Smad3, suggesting that dicumarol can significantly inhibit the TGF-β pathway. Therefore, DIC can reverse the TGF-β1-induced decrease in E-cadherin and increase in N-cadherin.

[0115] Experiment Example 2

[0116] To investigate the effect of dicumarol on EMT in HepG2 cells.

[0117] Experimental methods: HepG2 cells were stimulated with TGF-β1, with or without dicumarol treatment. Cell morphology changes were observed under a microscope, and EMT biomarker expression was detected by Western blotting and immunofluorescence.

[0118] Test Procedure: HepG2 cells in logarithmic growth phase were cultured in 96-well or 6-well plates and grown overnight at 37°C with 5% CO2. Cells were then treated with TGF-β1 (5 ng / mL) in combination with different concentrations of dicumarol (0 μM, 2.5 μM, 5.0 μM, 10.0 μM) in medium containing 1% FBS for 48 h. Cell morphology changes were observed under a microscope, and the expression of EMT biomarkers was determined by routine Western blotting.

[0119] See test results Figure 2 .in, Figure 2 In the middle, AD represents the effect of dicumarol on TGF-β1-induced cell morphological changes; E represents the effect of dicumarol on TGF-β1-induced EMT biomarkers.

[0120] according to Figure 2 Microscopic examination revealed that HepG2 cells slightly elongated after TGF-β1 stimulation. Western blotting showed a significant decrease in E-cadherin expression and a significant increase in Vimentin, α-SMA, and N-cadherin. Treatment with dicumarol significantly altered these changes, suggesting that dicumarol can significantly inhibit EMT in HepG2 cells.

[0121] Experimental Example 3

[0122] To investigate the effect of dicumarol on EMT in MIA PaCa-2 cells.

[0123] Experimental methods: MIA PaCa-2 cells were stimulated with TGF-β1, with or without dicumarol treatment. Cell morphology changes were observed under a microscope, and EMT biomarker expression was detected by Western blotting and immunofluorescence.

[0124] Test Procedure: Logarithmically growing MIA PaCa-2 cells were cultured in 96-well or 6-well plates at 37°C with 5% CO2 overnight. Cells were then treated with TGF-β1 (5 ng / mL) in combination with different concentrations of dicumarol (0 μM, 2.5 μM, 5.0 μM, 10.0 μM) in a medium containing 1% FBS for 48 h. Cell morphology changes were observed under a microscope, and the expression of EMT biomarkers was determined by routine Western blotting.

[0125] See test results Figure 3 .in, Figure 3 In the middle, AD represents the effect of dicumarol on TGF-β1-induced cell morphological changes; E represents the effect of dicumarol on TGF-β1-induced cellular EMT biomarkers.

[0126] according to Figure 3 Microscopic examination revealed that MIA PaCa-2 cells showed increased cell elongation after TGF-β1 stimulation. Western blotting protein assays showed a significant decrease in E-cadherin expression and a significant increase in Vimentin, α-SMA, and N-cadherin expression. Treatment with dicumarol significantly altered these changes, suggesting that dicumarol can significantly inhibit EMT in MIA PaCa-2 cells.

[0127] Experiment Example 4

[0128] To investigate the effect of dicumarol on inhibiting EndMT in vascular endothelial cells.

[0129] Experimental methods: Human endothelial EA.hy926 cells were stimulated with TGF-β2, with or without dicoumarin treatment. Cell morphology changes were observed under a microscope, and EMT biomarker expression was detected by Western blotting.

[0130] Test procedure: EA.hy926 cells in logarithmic growth phase were cultured in 96-well or 6-well plates and grown overnight at 37°C with 5% CO2. Cells were treated with TGF-β2 (5 ng / mL) in combination with different concentrations of dicumarol (0 μM, 2.5 μM, 5.0 μM, 10.0 μM) for 48 h. Cell morphology changes were observed under a microscope, and the expression of EMT biomarkers was measured by routine Western blotting.

[0131] See test results Figure 4 .in, Figure 4 In the middle, AD represents the effect of dicumarol on TGF-β2-induced cell morphological changes; E represents the effect of dicumarol on TGF-β2-induced cellular EMT biomarkers.

[0132] according to Figure 4 Microscopic examination revealed that after TGF-β2 stimulation, EA.hy926 cells exhibited a slender, elongated morphology. Western blotting protein assays showed a significant decrease in VE-cadherin expression, a significant increase in N-cadherin and Vimentin, and no significant change in α-SMA. Treatment with dicumarol significantly altered these changes, suggesting that dicumarol can significantly inhibit EndMT in EA.hy926 cells.

[0133] Experimental Example 5

[0134] To investigate the effect of dicumarol on TGF-β1-stimulated H9c2 cells.

[0135] Experimental methods: H9c2 cells were stimulated with TGF-β1, with or without dicumarol treatment. Cell morphology changes were observed under a microscope, and the expression of fibrosis biomarkers was detected by Western blotting and immunofluorescence.

[0136] Test procedure: H9c2 cells in logarithmic growth phase were cultured in 96-well or 6-well plates and grown overnight at 37°C with 5% CO2. Cells were treated with TGF-β1 (5 ng / mL) in combination with different concentrations of dicumarol (0 μM, 2.5 μM, 5.0 μM, 10.0 μM) for 48 h. Cell morphology changes were observed under a microscope, and the expression of EMT biomarkers was measured by routine Western blotting.

[0137] See test results Figure 5 .in, Figure 5 In the middle, AD represents the effect of dicumarol on TGF-β1-induced cell morphological changes; E represents the effect of dicumarol on TGF-β1-induced cellular EMT biomarkers.

[0138] according to Figure 5 Microscopic examination revealed that H9c2 cells became elongated after TGF-β1 stimulation. Western blotting showed a significant decrease in E-cadherin expression and a significant increase in N-cadherin, Vimentin, and α-SMA expression. Treatment with dicumarol significantly altered these changes, suggesting that dicumarol can significantly inhibit the expression of EMT and fibrosis-related proteins in H9c2 cells.

[0139] Experimental Example 6

[0140] To investigate the effect of dicumarol on EMT in TGF-β1-stimulated HK-2 cells.

[0141] Experimental methods: HK-2 cells were stimulated with TGF-β1, with or without dicumarol treatment. Cell morphology changes were observed under a microscope, and EMT biomarker expression was detected by Western blotting.

[0142] Test procedure: Logarithmically growing HK-2 cells were cultured in 96-well or 6-well plates and incubated overnight at 37°C with 5% CO2. Cells were treated with TGF-β1 (5 ng / mL) in combination with different concentrations of dicumarol (0 μM, 2.5 μM, 5.0 μM, 10.0 μM) for 48 h. Cell morphology changes were observed under a microscope, and the expression of EMT biomarkers was determined by routine Western blotting.

[0143] See test results Figure 6 .in, Figure 6 In the middle section, AD represents the effect of dicumarol on TGF-β1-induced cell morphological changes; EG represents the immunofluorescence staining of dicumarol-induced TGF-β1-induced N-cadherin expression. H represents the Western blotting results of dicumarol-induced TGF-β1-induced N-cadherin expression.

[0144] according to Figure 6 Microscopic examination revealed that HK-2 cells became elongated and thinner after TGF-β1 stimulation; immunofluorescence showed that N-cadherin was expressed in the cell membrane. Western blotting protein assay showed a significant increase in N-cadherin. Treatment with dicumarol significantly altered these changes, suggesting that dicumarol can significantly inhibit EMT in HK-2 cells.

[0145] Experimental Example 7

[0146] To investigate the effect of dicumarol on EMT in TGF-β1-stimulated NRK-52E cells.

[0147] Experimental methods: NRK-52E cells were stimulated with TGF-β1, with or without dicumarol treatment. Cell morphology changes were observed under a microscope, and EMT biomarker expression was detected by Western blotting.

[0148] Test procedure: Logarithmically growing NRK-52E cells were cultured in 96-well or 6-well plates and grown overnight at 37°C with 5% CO2. Cells were treated with TGF-β1 (10 ng / mL) in combination with different concentrations of dicumarol (0 μM, 1.25 μM, 2.5 μM, 5.0 μM) for 6 days. Cell morphology changes were observed under a microscope, and the expression of EMT biomarkers was measured by routine Western blotting.

[0149] See test results Figure 7 .in, Figure 7 In the middle, AD represents the effect of dicumarol on TGF-β1-induced cell morphological changes; E represents the expression of TGF-β1-induced EMT biomarkers by dicumarol.

[0150] according to Figure 7 Microscopic examination revealed that NRK-52E cells stimulated with TGF-β1 became larger. Western blotting showed a significant decrease in E-cadherin expression and a significant increase in N-cadherin, Vimentin, and α-SMA. Treatment with dicumarol significantly altered these changes, suggesting that dicumarol can significantly inhibit TGF-β1-induced expression of EMT and fibrosis-related proteins in NRK-52E cells.

[0151] Experimental Example 8

[0152] To investigate the effect of dicumarol on EMT in TGF-β1-stimulated NRK-52E cells.

[0153] Experimental methods: NRK-52E cells were stimulated with TGF-β1, with or without dicoumarin treatment, and the expression of EMT biomarkers was detected by immunofluorescence.

[0154] Test procedure: Logarithmically growing NRK-52E cells were cultured in 96-well plates and grown overnight at 37°C with 5% CO2. After treatment with TGF-β1 (10 ng / mL) in combination with different concentrations of dicumarol (0 μM, 1.25 μM) for 6 days, the expression of fibrosis markers was measured by immunofluorescence.

[0155] See test results Figure 8 .in, Figure 8 In the middle section, AC represents the immunofluorescence staining of TGF-β1-induced α-SMA expression by dicumarol; DF represents the immunofluorescence staining of TGF-β1-induced E-cadherin expression by dicumarol.

[0156] according to Figure 8 Immunofluorescence revealed that the blank control group showed low expression of α-SMA and high expression of E-cadherin. After TGF-β1 treatment, α-SMA expression significantly increased while E-cadherin expression significantly decreased. Treatment with dicumarol significantly inhibited these TGF-β1-induced changes, suggesting that dicumarol can significantly inhibit TGF-β1-induced EMT in NRK-52E cells.

[0157] Experimental Example 9

[0158] To investigate the effect of dicumarol on the transformation of TGF-β1-stimulated human embryonic lung fibroblasts MRC-5 and WI-38 into myofibroblasts.

[0159] Experimental methods: MRC-5 cells and WI-38 cells were stimulated with TGF-β1, with or without dicoumarin treatment, and the expression of myofibroblast biomarkers was detected by Western blotting.

[0160] Test procedure: Logarithmically growing MRC-5 or WI-38 cells were cultured in 6-well plates and grown overnight at 37°C with 5% CO2. After treatment with TGF-β1 (5 ng / mL) in combination with different concentrations of dicumarol (0 μM, 4.0 μM) for 48 h, the expression of α-SMA and E-cadherin was determined by routine Western blotting.

[0161] See test results Figure 9 It can be seen that DIC (4 μM) can reverse the decrease in E-cadherin induced by TGF-β1 (5 ng / mL) and also inhibit the increase in α-SMA induced by TGF-β1 (5 ng / mL).

[0162] Experimental Example 10

[0163] To investigate the effects of other coumarin-based drugs on EMT in A549 cells.

[0164] Experimental methods: A549 cells were stimulated with TGF-β1 and treated with or without other coumarin drugs (warfarin, 3,3'-carbonylbis(7-diethylaminocoumarin), acenitrocoumarin). Cell morphology changes were observed under a microscope, and EMT biomarker expression was detected by Western blotting.

[0165] Test Procedure: A549 cells in logarithmic growth phase were cultured in 96-well or 6-well plates and grown overnight at 37°C with 5% CO2. Cells were then treated with TGF-β1 (5 ng / mL) in combination with different concentrations of warfarin, 3,3'-carbonylbis(7-diethylaminocoumarin), or acetocoumarin (0 μM, 2.0 μM, 4.0 μM) for 48 h. Cell morphology was observed under a microscope, and the expression of EMT biomarkers was determined by routine Western blotting.

[0166] See test results Figure 10 .in, Figure 10In the middle, AD represents the effect of warfarin on TGF-β1-induced cell morphology changes; E represents the changes in TGF-β1-induced EMT biomarkers induced by different concentrations of warfarin; FI represents the effect of 3,3'-carbonylbis(7-diethylaminocoumarin) on TGF-β1-induced cell morphology changes; J represents the changes in TGF-β1-induced EMT biomarkers induced by different concentrations of 3,3'-carbonylbis(7-diethylaminocoumarin); KN represents the effect of acetocoumarin on TGF-β1-induced cell morphology changes; and O represents the changes in TGF-β1-induced EMT biomarkers induced by different concentrations of acetocoumarin.

[0167] according to Figure 10 Microscopic examination revealed that after TGF-β1 stimulation, A549 cells exhibited a slender, elongated morphology. Western blotting analysis showed a significant decrease in E-cadherin expression and a significant increase in N-cadherin expression. These changes were consistent with EMT, suggesting that TGF-β1 induces EMT. However, treatment with warfarin, 3,3'-carbonylbis(7-diethylaminocoumarin), or acetocoumarin did not significantly alter these changes, indicating that warfarin, 3,3'-carbonylbis(7-diethylaminocoumarin), or acetocoumarin had no effect on TGF-β1-induced EMT in A549 cells. Therefore, compared to other anticoagulants in the same class, dicumarol exhibits a unique inhibitory effect on EMT.

[0168] Animal-level experiments

[0169] Effects of dicumarol on a rat model of renal interstitial fibrosis with urogenital dysplasia (UUO).

[0170] 1. Experimental Materials

[0171] Dicoumarin, sourced as described above. SPF-grade Wistar rats (8-10 weeks old) were obtained from the Faculty of Health Sciences, University of Macau.

[0172] 2. Main experimental reagents

[0173] Paraformaldehyde, xylene, H&E staining solution, Masson staining solution kit, and neutral resin were all purchased from Beijing Solarbio Biotechnology Co., Ltd.; the DAB colorimetric reagent kit was purchased from Nanjing Zhongshan Jinqiao Pharmaceutical Co., Ltd.; α-SMA antibody (19245s), Vimentin antibody (5741s), and E-cadherin antibody (3195s) were purchased from Cell Signaling (CST); and Fibronectin antibody (ab2413) was purchased from Abcam.

[0174] 3 Experimental Methods

[0175] 3.1 Animal grouping, model establishment, and drug administration

[0176] Wistar rats (250g±20g) were acclimatized to their environment for one week and then randomly divided into four groups: control group, model group, and drug treatment group, with six rats in each group. The rats were anesthetized with chloral hydrate (35mg / kg), fixed, and their abdominal hair was shaved. The abdominal skin was disinfected, and a 1.5-2.5cm midline incision was made in the abdomen. The left ureter was separated, double-ligated, and the incision was sutured and disinfected. The model group and drug treatment group underwent unilateral ureteral ligation (UUO), while the control group only had a surgical wound without ligation. The drug treatment groups were given dicumarol by gavage starting on day 7 after model establishment, at doses of 2.5mg / kg and 5mg / kg, respectively, once daily. The control group and model group received the same amount of solvent.

[0177] 3.2 Animal euthanasia, sampling, and sample processing

[0178] After 7 days of continuous administration to rats, they were anesthetized and euthanized. Both kidneys were quickly removed, washed in physiological saline, and the surface adipose tissue and mucosa were removed. One-third of the kidneys were fixed in 4% paraformaldehyde for histopathological examination. The remaining kidneys were labeled and placed in a -80°C freezer for other related tests.

[0179] 3.3 Histopathological examination

[0180] Kidney tissue was fixed in 4% formalin, routinely embedded in paraffin, cut into 4μm thick sections, and subjected to routine H&E staining, Masson staining, and Sirius Red staining. The tissue was observed, photographed, and analyzed under a light microscope. Routine immunohistochemical staining was used to detect the expression of E-cadherin, α-SMA, and Vimentin.

[0181] Kidney tissue was excised, total protein was extracted, protein quantification was performed using the BCA method, and the expression of fibrosis-related proteins Vimentin, α-SMA, and Fibronectin in kidney tissue was detected by routine Western blotting.

[0182] 4 Experimental Results

[0183] 4.1 The effect of dicumarol in alleviating kidney damage on the ligated side in UUO model rats

[0184] See test results Figure 11 H&E staining of kidney sections, in which, Figure 11 The groups represented by AD were the control group, the model group, and the groups treated with 2.5 mg / kg and 5.0 mg / kg of dicumarol, respectively.

[0185] from Figure 11H&E staining results showed that, compared with the control group, the model group rats exhibited moderate tubular atrophy, lumen enlargement, significant local inflammation, and epithelial cell necrosis or degeneration in their kidney tissue; moderate glomerular atrophy; and significant inflammatory cell infiltration in the renal interstitium. Dicoumarol administration significantly improved these pathological changes, with a marked reduction in tubular atrophy and lumen enlargement compared to the model group, and mild epithelial cell necrosis and mild inflammatory cell infiltration.

[0186] 4.2 Dicoumarin reduces collagen fiber deposition in the ligated kidney of UUO model rats

[0187] See test results Figure 12 and Figure 13 .

[0188] in, Figure 12 The results show the Masson trichrome staining results, with AD representing the control group, model group, and dicumarol 2.5 and 5.0 mg / kg groups, respectively. Masson trichrome staining, also known as Masson staining, is an authoritative and classic technique for staining collagen fibers. After Masson staining, muscle fibers appear red, while collagen fibers appear green (pale green) or blue (aniline blue).

[0189] from Figure 12 Masson staining results showed that in the model group, a large amount of blue-stained collagen fibers were deposited in the submucosal tissue of the main medullary part of the renal interstitium of rats, some of which fused into thick cord-like structures; collagen deposition was significantly reduced in the renal tissue of rats in the drug-treated group, with only a small amount of blue-stained collagen fibers deposited and no obvious cord-like fusion observed.

[0190] Figure 13 This is the result of Sirius Red staining. Sirius Red staining is a simple and sensitive method for detecting collagen fiber networks in tissue sections.

[0191] from Figure 13 The results of Sirius staining showed that a large amount of red-stained collagen fibers were deposited in the renal interstitium of rats in the model group, while the amount of red collagen in the renal tissue of rats in the drug treatment group was significantly reduced.

[0192] The above tissue staining results indicate that dicumarol can improve fibrotic lesions in the kidney tissue of the UUO model, inhibit collagen fiber deposition, and protect the kidney.

[0193] 4.3 Dicoumarin reduces the expression of EMT protein and fibrosis protein in the kidney tissue of UUO model rats.

[0194] Immunohistochemical staining and Western blot analysis results of EMT and fibrosis-related proteins in the kidney tissues of rats in each group are as follows: Figures 14-17 As shown.

[0195] Figure 14 Immunohistochemical staining results of E-cadherin protein in rat kidney tissue. AD represents the control group, model group, and 2.5 and 5.0 mg / kg dicumarol groups, respectively.

[0196] from Figure 14 Immunohistochemical staining results of E-cadherin showed that a large amount of E-cadherin expression was observed in the renal tubular epithelial cells of the control group; E-cadherin expression in the renal tubular epithelium of the model group was significantly decreased, with only patchy weak expression observed; and E-cadherin expression in the renal tubular epithelium of the drug-treated group was significantly increased compared with that of the model group.

[0197] Figure 15 Immunohistochemical staining results of α-SMA protein in rat kidney tissue. AD represents the control group, model group, and 2.5 mg / kg and 5.0 mg / kg dicoumarin groups, respectively.

[0198] from Figure 15 Immunohistochemical staining results of α-SMA showed that no significant expression was observed in the glomeruli, renal tubules, and renal interstitium of rats in the control group, and it was only expressed in the smooth muscle cells of the renal artery. The expression of α-SMA in the renal interstitium and renal tubular epithelial cells of the model group was significantly increased. α-SMA was observed in the drug-treated group, but the expression level was significantly reduced compared with the model group.

[0199] Figure 16 Immunohistochemical staining results of Vimentin protein in rat kidney tissue. AD represents the control group, model group, and 2.5 mg / kg and 5.0 mg / kg dicoumarin groups, respectively.

[0200] from Figure 16 Immunohistochemical staining results of Vimentin showed that, as a component of the cytoskeleton, Vimentin expression was very low in the kidney tissue of the control group, while its expression was significantly increased in the model group. Vimentin expression in the kidney tissue of the drug-treated group was significantly lower than that in the model group.

[0201] Figure 17 This is the result of Western blotting detection of EMT-related protein expression in the kidneys of a rat UUO model.

[0202] from Figure 17 The results of renal EMT-related protein expression showed that, compared with the blank group, the expression of Fibronectin, Vimentin and α-SMA in the kidneys of the model group was significantly increased, while the expression of the three proteins was significantly reduced after DIC treatment.

[0203] The pharmacological results above show that the anticoagulant dicumarol has significant antifibrotic activity and has the potential to be developed into an anti-renal fibrosis drug.

[0204] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of dicumarol as the sole active ingredient in the preparation of a medicament for the prevention or treatment of fibrotic diseases; wherein the fibrotic disease is selected from renal fibrosis.

2. The application according to claim 1, characterized in that, The mechanism by which the drug prevents and treats the fibrotic disease includes at least one of the following (a) to (h); (a) By inhibiting EMT; (b) By inhibiting EndMT; (c) By inhibiting TGF-β1-induced transformation of fibroblasts into myofibroblasts; (d) By reversing the decrease in E-cadherin expression in epithelial cells and VE-cadherin expression in endothelial cells induced by TGF-β1 and TGF-β2; (e) By reversing the increase in expression of mesenchymal cell marker proteins N-cadherin and Vimentin; (f) By inhibiting the expression of fibronectin; (g) By reversing the expression of the TGF-β1-induced myofibroblast marker α-SMA; (h) By inhibiting the morphological changes of epithelial cells, endothelial cells and cardiomyocytes induced by TGF-β1 and TGF-β2.

3. The application according to claim 1, characterized in that, The renal fibrosis includes renal fibrosis induced by hypertension, renal fibrosis induced by glomerulonephritis, renal fibrosis induced by systemic lupus erythematosus, renal fibrosis induced by scleroderma, renal fibrosis induced by kidney transplant rejection, renal fibrosis induced by pyelonephritis, renal fibrosis induced by kidney stones, renal fibrosis induced by hyperlipidemia, renal fibrosis induced by diabetes, renal fibrosis induced by hyperuricemia, and renal fibrosis induced by hypercalciuria.

4. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition, and the pharmaceutical composition further includes a pharmaceutical carrier.

5. The application according to claim 4, characterized in that, The dicumarol in the pharmaceutical composition is dicumarol or its pharmaceutical derivative.

6. The application according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is selected from at least one of solution, suspension, tablet, capsule, granule and injection.

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