Application of nobiletin and nintedanib combined administration in pulmonary fibrosis treatment

Abstract

The invention provides application of nobiletin and nintedanib combined administration in treatment of pulmonary fibrosis, and belongs to the technical field of combined medication. The invention provides application of nobiletin and nintedanib combined medicine in preparation of medicine for preventing and / or treating pulmonary fibrosis. According to the invention, TGF-beta1 is adopted to induce MRC-5 cells to establish an in-vitro pulmonary fibrosis model, and bleomycin is adopted to induce a C57BL / 6J mouse to establish an in-vivo pulmonary fibrosis model. Researches find that the combination of nobiletin and nintedanib can inhibit MRC-5 cell epithelial-mesenchymal transition and significantly down-regulate the expression of fibrosis marker protein. In-vivo experiments show that nobiletin and nintedanib are combined to obviously improve the pathological state of mouse pulmonary fibrosis. The nobiletin and the nintedanib are combined to play an obvious treatment role in inhibiting the pulmonary fibrosis, so that the nobiletin and the nintedanib can be used as potential medicines for preventing and treating the pulmonary fibrosis, and new clues and ideas are provided for developing medicines for treating the pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of combined medication, and particularly relates to use of nobiletin and nintedanib in the treatment of pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis (PF) is a chronic, progressive lung disease characterized by diffuse inflammation of the lung interstitium, alveoli, and pulmonary vasculature. In 2014, the US Food and Drug Administration (FDA) approved pirfenidone and nintedanib for the treatment of idiopathic pulmonary fibrosis (IPF). These two drugs have become standard treatment options worldwide, slowing the decline in forced vital capacity (FVC) and delaying disease progression. However, they cannot reverse established pulmonary fibrosis, and their effectiveness remains limited. Although nintedanib is recommended for the treatment of IPF, its common adverse effects (such as diarrhea and liver function abnormalities) force many patients to discontinue treatment, especially when the dose is increased, when these adverse effects become more pronounced. Therefore, enhancing the efficacy of existing drugs while mitigating the adverse effects of existing drugs has become an important research direction for improving treatment compliance and efficacy in patients with PF. Summary of the Invention

[0003] The present invention provides the use of nobiletin and nintedanib in the treatment of pulmonary fibrosis by combined administration. Nobiletin combined with nintedanib can exert a significant synergistic therapeutic effect on inhibiting pulmonary fibrosis and can be used as a potential drug for the prevention and treatment of pulmonary fibrosis.

[0004] The present invention provides the use of a combination of nobiletin and nintedanib in the preparation of a medicament for preventing and / or treating pulmonary fibrosis, wherein the nintedanib comprises at least one of the following: nintedanib itself, nintedanib stereoisomers, nintedanib tautomers, nintedanib polymorphs, nintedanib solvates, and pharmaceutically acceptable nintedanib salts;

[0005] The nobiletin includes at least one of the following: nobiletin itself, nobiletin stereoisomers, nobiletin tautomers, nobiletin polymorphs, solvates of nobiletin and pharmaceutically acceptable salts of nobiletin.

[0006] In a preferred embodiment of the present invention, the combination therapy comprises nobiletin 80-150 mg / kg and nintedanib 10-50 mg / kg, based on the dosage for mice.

[0007] In a preferred embodiment of the present invention, the combination therapy comprises 90-120 mg / kg of nobiletin and 20-40 mg / kg of nintedanib in terms of dosage for mice.

[0008] The present invention provides a pharmaceutical composition for preventing and / or treating pulmonary fibrosis, comprising nobiletin and nintedanib.

[0009] The present invention also provides a pharmaceutical compound preparation for preventing and / or treating pulmonary fibrosis, comprising the above-mentioned pharmaceutical composition and at least one pharmaceutically acceptable excipient or carrier.

[0010] In a preferred embodiment of the present invention, the excipient is selected from at least one of a lubricant, a filler, a disintegrant, a plasticizer, a colorant, an emulsifier, a flavoring agent, a binder, a film-forming polymer, an antioxidant, a light stabilizer, a free radical scavenger, a surfactant, a pH regulator, a drug complexing agent, and a stabilizer against microbial attack.

[0011] In a preferred embodiment of the present invention, the dosage form of the pharmaceutical compound preparation includes any one of the following: solution, granules, tablets, pills, drops, injection, capsules, powders and decoctions.

[0012] The present invention also provides use of the above-mentioned pharmaceutical composition or the above-mentioned pharmaceutical compound preparation in preparing a drug for preventing and / or treating pulmonary fibrosis.

[0013] The present invention also provides a medicine for preventing and / or treating pulmonary fibrosis, which uses the above-mentioned pharmaceutical composition as an active ingredient.

[0014] In a preferred embodiment of the present invention, the drug, measured in terms of dosage for mice, comprises 100 mg / kg of nobiletin and 30 mg / kg of nintedanib.

[0015] Beneficial effect: The present invention provides the use of a combination of nobiletin and nintedanib in the preparation of a drug for preventing and / or treating pulmonary fibrosis. In the embodiments of the present invention, the effects of nobiletin and nintedanib alone and in combination on the EMT process and extracellular matrix (ECM) deposition in myofibroblasts were respectively used. Using the TGF-β1-induced myofibroblast model, the expression of Fibronectin, Collagen I and α-SMA proteins was significantly increased (p < 0.05). Compared with the cell model group, after the administration of nobiletin and nintedanib alone, the expression of Fibronectin protein was significantly reduced (p < 0.01), and the collagen I and α-SMA proteins had a downward trend; the combined drug can significantly reduce the expression of Fibronectin and α-SMA proteins compared with the single drug (p < 0.05), and the collagen I protein has a downward trend.

[0016] In one embodiment of the present invention, immunofluorescence analysis experiments were used to verify that Nobiletin combined with Nintedanib could reverse the upregulation of a-SMA induced by TGF-β1; scratch test and Transwell experiment showed that Nobiletin combined with Nintedanib could significantly inhibit cell migration; combined drug therapy could significantly reduce N-cadherin, Vimentin, Collagen I and α-SMA protein expression (p<0.05), promote E-cadherin protein expression level (p<0.05), not only inhibit MRC-5 cell epithelial-mesenchymal transition, but also inhibit ECM deposition. In one embodiment of the present invention, animal model experiments were used to find that Nobiletin combined with Nintedanib could improve the pathological changes in mouse lung tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the effects of nobiletin alone and in combination with nintedanib on ECM proteins are shown in Figure 3, n=3, Mean±SD, * p<0.05, ** p<0.01, *** p<0.001; Nobiletin-L refers to nobiletin 3 μM; Nobiletin-H refers to nobiletin 10 μM; Nintedanib refers to nintedanib 0.3 μM;

[0018] Figure 2 The results of the effect of nobiletin alone and in combination with nintedanib on the expression of α-SMA (×200), n=3, Mean±SD, * p<0.05, ** p<0.01, *** p<0.001; Nobiletin-L refers to nobiletin 3 μM; Nobiletin-H refers to nobiletin 10 μM; Nintedanib refers to nintedanib 0.3 μM;

[0019] Figure 3 The results of the scratch test to verify the effect of nobiletin alone and in combination with nintedanib on the migration of MRC-5 cells, n=3, Mean±SD, * p<0.05, ** p<0.01, *** p<0.001; Nobiletin-L refers to nobiletin 3 μM; Nobiletin-H refers to nobiletin 10 μM; Nintedanib refers to nintedanib 0.3 μM;

[0020] Figure 4 The results of the Transwell assay to verify the effect of nobiletin alone and in combination with nintedanib on the migration of MRC-5 cells, n=3, Mean±SD,* p<0.05, ** p<0.01, *** p<0.001; Nobiletin-L refers to nobiletin 3 μM; Nobiletin-H refers to nobiletin 10 μM; Nintedanib refers to nintedanib 0.3 μM;

[0021] Figure 5 The results of the effects of nobiletin alone and in combination with nintedanib on EMT proteins are shown in Figure 3, n=3, Mean±SD, * p<0.05, ** p<0.01, *** p<0.001; Nobiletin-L refers to nobiletin 3 μM; Nobiletin-H refers to nobiletin 10 μM; Nintedanib refers to nintedanib 0.3 μM;

[0022] Figure 6 The results of the effects of nobiletin alone and in combination with nintedanib on ECM proteins are shown in Figure 5, n=5-7, Mean±SD, * p<0.05, ** p<0.01, *** p<0.001; Nobiletin-L refers to nobiletin 100 mg / kg; Nobiletin-H refers to nobiletin 150 mg / kg; Nintedanib refers to nintedanib 30 mg / kg;

[0023] Figure 7 This figure shows the results of nobiletin combined with nintedanib improving the pathological changes in mouse lung tissue, where Nobiletin-L refers to 100 mg / kg of nobiletin; Nobiletin-H refers to 150 mg / kg of nobiletin; and Nintedanib refers to 30 mg / kg of nintedanib. DETAILED DESCRIPTION

[0024] The present invention provides the use of a combination of nobiletin and nintedanib in the preparation of a medicament for preventing and / or treating pulmonary fibrosis, wherein the nintedanib comprises at least one of the following: nintedanib itself, nintedanib stereoisomers, nintedanib tautomers, nintedanib polymorphs, nintedanib solvates, and pharmaceutically acceptable nintedanib salts;

[0025] The nobiletin includes at least one of the following: nobiletin itself, nobiletin stereoisomers, nobiletin tautomers, nobiletin polymorphs, solvates of nobiletin and pharmaceutically acceptable salts of nobiletin.

[0026] The nobiletin of the present invention is a polymethoxyflavonoid compound, mainly derived from the peel, leaves and stems of citrus plants of the Rutaceae family; and the nobiletin has a wide range of pharmacological effects, such as anti-inflammatory, antioxidant, immunomodulatory, anti-cancer, neuroprotective and anti-diabetic, etc. In one embodiment, it was purchased from Yuanye Company, CAS No.: 478-01-3. The chemical name of the nintedanib of the present invention is 1H-indole-6-carboxylic acid, 2,3-dihydro-3-[[[4-[methyl[(4-methyl-1-piperazinyl)acetyl]amino]phenyl]amino]phenylmethylene]-2-oxy-, methyl ester, (3Z)-, and its molecular formula is C 31 H 33 N5O4; clinically used to treat idiopathic pulmonary fibrosis (IPF), in one embodiment purchased from Yuanye Company, CAS No.: 656247-17-5.

[0027] The present invention uses nobiletin and nintedanib as a combined drug, and based on the dosage for mice, the working concentration of nobiletin is 80-150 mg / kg, such as 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg or 150 mg / kg. In one embodiment, 100 mg / kg is used as an example for illustration, but it cannot be solely identified as the entire scope of protection of the present invention. In combined drug use, based on the dosage for mice, the working concentration of nintedanib according to the present invention is 10-50 mg / kg, such as 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg or 50 mg / kg. In one embodiment, 30 mg / kg is used as an example for illustration, but it cannot be solely identified as the entire scope of protection of the present invention.

[0028] In one embodiment of the present invention, an in vitro cell model was constructed, and TGF-β1 (10 ng / mL, purchased from offshore protein, product number CA59) was used to induce the transformation of fibroblasts MRC-5 into myofibroblasts, thereby generating fibrosis. At the same time, the effects of fibrosis cell model, nobiletin alone, nintedanib alone, and nobiletin combined with nintedanib were also compared. The results showed that after TGF-β1 induction of MRC-5 cells, the expression of Fibronectin, Collagen I and α-SMA proteins increased significantly. Compared with the induction group, after administration of nobiletin and nintedanib alone, the expression of Fibronectin protein was significantly decreased, and Collagen I and α-SMA proteins showed a downward trend. However, after combined administration, compared with single administration, nobiletin combined with nintedanib significantly reduced the expression of Fibronectin and α-SMA proteins. In the cell migration experiment, the cell migration ability was significantly enhanced after TGF-β1 induction. After administration of nobiletin or nintedanib alone, the inhibitory effect on cell migration was not obvious, while nobiletin combined with nintedanib could significantly inhibit cell migration. After TGF-β1 induction, the relative expression levels of N-cadherin and Vimentin proteins increased, while the relative expression level of E-cadherin protein decreased. Nobiletin or nintedanib alone could significantly reduce the expression of N-cadherin and Vimentin proteins. Compared with single administration, the combined administration of nobiletin and nintedanib could significantly reduce the expression of N-cadherin and Vimentin proteins and promote the expression level of E-cadherin protein, indicating that nobiletin combined with nintedanib significantly inhibited the epithelial-mesenchymal transition of MRC-5 cells.

[0029] In one embodiment of the present invention, in vivo experiments were also used to verify the effect of nobiletin combined with nintedanib on mouse lung tissue pathology. The results showed that after administration of nobiletin combined with nintedanib, it can play a synergistic role in inhibiting mouse lung fibrosis, providing new ideas and experimental basis for the treatment of pulmonary fibrosis.

[0030] The present invention provides a pharmaceutical composition for preventing and / or treating pulmonary fibrosis, comprising nobiletin and nintedanib.

[0031] The present invention also provides a pharmaceutical compound preparation for preventing and / or treating pulmonary fibrosis, comprising the above-mentioned pharmaceutical composition and at least one pharmaceutically acceptable excipient or carrier.

[0032] In a preferred embodiment of the present invention, the excipient is selected from at least one of a lubricant, a filler, a disintegrant, a plasticizer, a colorant, an emulsifier, a flavoring agent, a binder, a film-forming polymer, an antioxidant, a light stabilizer, a free radical scavenger, a surfactant, a pH regulator, a drug complexing agent, and a stabilizer against microbial attack.

[0033] In a preferred embodiment of the present invention, the dosage form of the pharmaceutical compound preparation includes any one of the following: solution, granules, tablets, pills, drops, injection, capsules, powders and decoctions.

[0034] The present invention also provides use of the above-mentioned pharmaceutical composition or the above-mentioned pharmaceutical compound preparation in preparing a drug for preventing and / or treating pulmonary fibrosis.

[0035] The present invention also provides a medicine for preventing and / or treating pulmonary fibrosis, which uses the above-mentioned pharmaceutical composition as an active ingredient.

[0036] In a preferred embodiment of the present invention, the drug, measured in terms of dosage for mice, comprises 100 mg / kg of nobiletin and 30 mg / kg of nintedanib.

[0037] To further illustrate the present invention, the use of the combined administration of nobiletin and nintedanib in the treatment of pulmonary fibrosis provided by the present invention is described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0038] In the examples of the present invention, the test methods used are conventional methods in the art unless otherwise specified:

[0039] (1) Cell scratch test method

[0040] Use a black marker to draw positioning lines on the back of the 6-well plate. 5 Cells were seeded at 100 μg / well in a 6-well plate and cultured in an incubator until the cell density reached approximately 90%. A 200 μL pipette tip was used to streak the cells at the bottom of the plate. The detached cells were washed with PBS and then treated according to the grouping. Microscopic images were taken and the plates were returned to the incubator for further culture. After 24 hours, the images were saved and the scratch area was calculated using Image J software. The relative cell migration rate was calculated as follows: relative cell migration rate = (scratch width at 0 h - scratch width at 24 h) / cell width at 0 h × 100%.

[0041] (2) Transwell experimental method

[0042] Prepare the cell suspension: Starve the cells with incomplete culture medium (purchased from Wuhan Punosai Life Science Technology Co., Ltd., catalog number PM150410) the day before the experiment to remove the effects of serum on the experiment. Disintegrate the cells with trypsin until the cells begin to slip. Centrifuge and discard the supernatant. Wash the cells one to two times with PBS and resuspend them in an appropriate amount of incomplete culture medium. After counting the cells, dilute them to a density of 30,000 cells / 200 μL using incomplete culture medium containing the appropriate drug concentration.

[0043] Seeding cells: Add 600 μL of complete medium (89% incomplete medium and 10% serum + 1% double antibody) containing 10% FBS to a 24-well plate. After gently mixing the cells, take 200 μL of the cell suspension and drop it into the Transwell chamber. Place the 24-well plate in a cell culture incubator.

[0044] Cell fixation and result statistics:

[0045] After 24 hours, remove the 24-well plate, remove the chamber with forceps, and place it in a beaker of PBS for three washes. Then, transfer the chamber to a beaker of 4% paraformaldehyde and fix the cells for approximately 15 minutes. After the timer expires, remove the chamber and air-dry it. Place it in crystal violet solution for staining at room temperature for 25 minutes. Wash the chamber again with PBS several times. Gently wipe the inner chamber with a cotton swab to remove any cells that have not passed through the wells. Photograph the chamber under a microscope, save the image, and analyze the results using Image J software.

[0046] (3) Western blot experimental method

[0047] (1) Solution preparation

[0048] 10% ammonium persulfate (AP): Accurately weigh 1 g of ammonium persulfate using an analytical balance, add 10 mL of double-distilled water and dissolve by ultrasonication.

[0049] Preparation of electrophoresis buffer: 3.03 g Tris, 18.77 g glycine and 1 g SDS were weighed and dissolved in a 1000 mL volumetric flask by ultrasonication.

[0050] Preparation of electrotransfer buffer: Weigh 5.8 g of Tris and 2.9 g of glycine, add 200 mL of methanol and dissolve in a 1000 mL volumetric flask under ultrasonication.

[0051] 10×TBS: Weigh 44.0 g of sodium chloride, add 50 mL of Tris-HCl pH 7.5 solution, and add double-distilled water to make up to 1000 mL.

[0052] 1×TBST: Take 100 mL of TBS solution, add 1 mL of Tween 20, and add 900 mL of double-distilled water and sonicate.

[0053] (2) Extraction of total cell protein

[0054] Remove the cell culture plate, wash the culture medium with PBS, add protein lysis buffer, fully contact the cells, and place on ice for 30 minutes. Use a pipette to blow the bottom of the plate every 8 minutes to fully lyse it. After the last blow, collect the lysate in a 2 mL centrifuge tube, centrifuge at 4°C (12000 rpm, 10 minutes), collect the supernatant, and store it at -80°C.

[0055] Protein concentration determination: Protein concentration was determined using the BCA assay. Solution A and reagent B in the BCA kit were mixed at a volume ratio of 50:1 to prepare an appropriate volume of BCA working solution. Mix thoroughly using a vortexer. Using the BCA kit, dilute the protein standard (5 mg / mL) in the BCA kit to a final concentration of 0.5 mg / mL. Add PBS to a 96-well plate at volumes of 0, 4, 8, 12, 16, 18, and 20 μL. Add 0.5 mg / mL standard to each well to a volume of 20 μL per well. Set up three replicate wells for protein samples. Add 18 μL of PBS to each well to dilute 2 μL of the protein stock solution. Set up two replicate wells. Add 200 μL of the prepared BCA working solution to each well. Incubate in a 37°C incubator for 30 min. Measure the OD value at 562 nm with a microplate reader, and plot the standard curve. Calculate the protein solution concentration, dilute to the same concentration, add 4× loading buffer, heat in a metal bath for 12 minutes to denature the protein, and store at low temperature after cooling.

[0056] (3) Electrophoresis

[0057] Table 1 Separation gel formulas of different concentrations

[0058]

[0059] Prepare an appropriate volume of separating gel (Table 1) at the appropriate concentration in a 50 mL centrifuge tube. Add AP and TEMED and mix quickly. Using a 5 mL pipette, add the appropriate amount of separating gel. Seal with double-distilled water. After the gel solidifies, remove any remaining liquid with filter paper. Prepare 5% stacking gel according to the table below (Table 2). Add the appropriate amount of stacking gel and insert a sample comb. After solidification, add electrophoresis buffer, remove the comb, and load the sample.

[0060] Table 2 Stacking gel formulas of different volumes

[0061]

[0062] Use a 5mL syringe to adjust the sample well to make it flat. Vortex the protein sample and add it to the sample well in the order of loading. Change the pipette tip when loading different samples to prevent sample contamination. Add markers on both sides as indicators. Add electrophoresis solution to the electrophoresis tank, install the electrophoresis tank, turn on the power, adjust the initial voltage to 55V, and adjust the voltage to 90V when the bromophenol blue dye reaches the separation gel and continue electrophoresis. Stop the electrophoresis operation and transfer step when the bottom of the glass plate is 0.5cm.

[0063] (4) Transfer

[0064] After electrophoresis, remove the gel glass plate, gently peel it off, and remove any excess separation gel. Pre-cut a PVDF membrane to fit the gel size, activate it with methanol for 1 minute, and transfer it to 1× transfer buffer. Prepare a transfer clamp by placing the sponge, filter paper, and PVDF membrane in that order. Use a roller to remove any air bubbles between the filter paper and PVDF membrane, then gently place the separation gel, filter paper, and sponge on top and clamp securely. Place the transfer clamp into the electroporator, ensuring the entire clamp is submerged in the transfer buffer. Connect the power cord to the positive and negative terminals, and transfer the membrane at a constant current of 300 mA for 120 minutes. After transfer, remove the PVDF membrane with tweezers, soak it in 5% skim milk powder, and incubate on a shaker at room temperature for 120 minutes.

[0065] (5) Antibody incubation

[0066] After blocking the PVDF membrane with skim milk powder, wash it six times with TBST (4 minutes each time). Using a knife, cut the PVDF membrane according to protein molecular weight and insert the primary antibody solution corresponding to the target protein. Incubate at 4°C overnight. The next day, rinse the membrane four times with TBST (6 minutes each time). After washing, prepare the secondary antibody according to the antibody manufacturer's instructions and incubate at 25°C for 60 minutes.

[0067] (6) ECL color imaging

[0068] Prepare an appropriate amount of chemiluminescent solution (ECL), place the strips in a glass dish, add ECL solution, and place the strips in a gel imager for color development. Save the results and analyze them using Image J software.

[0069] (IV) Immunofluorescence experimental method

[0070] After the cells were treated with drugs on slides, they were fixed with 4% paraformaldehyde solution, permeabilized with TritonX-100 and treated with blocking solution, and then incubated with a-SMA (1:50) primary antibody at 4°C overnight. After washing with PBS buffer, they were incubated with secondary antibody at room temperature for 60 minutes. After sealing, the cells were photographed with a Leica optical microscope.

[0071] (V) Animal experiment grouping and animal model establishment

[0072] Sixty male C57BL / 6JJ mice (8-10 weeks old, weighing 20 ± 2 g) were purchased and acclimated for one week. Pulmonary fibrosis was induced in mice using a single intratracheal instillation of bleomycin. A 1 mL syringe and a 20G intravenous catheter were prepared. After the mouse is anesthetized, it is fixed on the operating table with its back facing up. The light source is irradiated on the mouse's throat. The tongue is gently pulled out with tweezers to expose the glottis. The mouse's breathing can be seen opening and closing the glottis at this time. When a white bright spot appears in the pharynx, the indwelling needle with the needle removed is quickly inserted and the BLM working solution (bleomycin solution, purchased from Beijing Solebow, item number IB0871, dose of 5 mg / kg) is drawn up with a 1 ml syringe. It is slowly injected into the mouse's trachea through a 20G intravenous indwelling needle. Immediately inject 0.5 mL of air. At this time, the anesthetized mouse is breathing rapidly and accompanied by wheezing and coughing. The mouse is rotated upright for about 2 minutes to evenly disperse the drug in the lungs. The mouse is placed on a constant temperature heating pad and returned to the cage after waking up. The mouse's condition is carefully observed and the mouse's weight is recorded every day after numbering. All mice were randomly divided into 6 groups, with 10 mice in each group. The experimental groups were divided into the control group, the BLM group, the nobiletin 100 mg / kg group, the nobiletin 150 mg / kg group, the nintedanib 30 mg / kg group, and the nobiletin 100 mg / kg combined with nintedanib 30 mg / kg group. The drugs were dissolved in sodium carboxymethylcellulose and administered orally once daily for 28 days before samples were collected.

[0073] (VI) Hematoxylin-eosin (HE) staining

[0074] After fixation, the tissue was dehydrated using graded alcohols. The tissue blocks were then transparentized, wax-impregnated, and embedded. Sections were then prepared from the embedded wax blocks. The sections were oven-dried and immersed in xylene I for approximately 20 minutes, xylene II for 20 minutes, then transferred to anhydrous ethanol for approximately 4 minutes, 90% ethanol for approximately 6 minutes, 80% ethanol for 6 minutes, and 70% ethanol for 6 minutes. The sections were then rinsed twice in distilled water. The sections were stained with Harris hematoxylin for 6 minutes, rinsed in water for 1 minute, and then placed in 1% hydrochloric acid-ethanol solution for 2 minutes. After bluing with the bluing solution, the sections were rinsed in ultrapure water for approximately 5 minutes until the nuclei appeared blue. The sections were then stained in 0.5% eosin solution for 5 minutes, dehydrated in 95% ethanol for 8 minutes, transferred to anhydrous ethanol for approximately 8 minutes, and rinsed in tap water. The sections were then dehydrated in anhydrous ethanol and transparentized by xylene treatment. After air-drying, add neutral gum to the tissue slide and seal it with a cover glass. Leave it to air-dry at room temperature for subsequent microscopic observation and image acquisition.

[0075] (7) Masson staining

[0076] The tissue was dehydrated with a gradient of alcohol, then cleared and embedded in paraffin. Sections were then sliced using a Leica pathology microtome. Paraffin sections were dewaxed sequentially with xylene and ethanol and washed with distilled water. Nuclei were stained with Weigert's iron hematoxylin solution, differentiated with acidic ethanol, and then blued with Masson's blue solution. The sections were stained with acidic Ponceau red stain for 5 minutes, followed by a weak acid stain for several minutes and a rinse under running water for several minutes. The sections were differentiated with 0.5% phosphomolybdic acid solution for approximately 5 minutes, followed by immediate restaining with aniline blue solution for 5 minutes. After a quick rinse, the sections were dehydrated and cleared with ethanol and xylene, sealed with neutral gum, and photographed under a microscope.

[0077] (8) Immunohistochemical staining

[0078] Tissue embedding, sectioning, dewaxing, and hydration were performed in the same manner as previously described for HE staining. First, antigen retrieval was performed. Tissue sections were placed in a retrieval box containing pH 6.0 citric acid solution. The box was microwaved for 10 minutes and then cooled to room temperature. Endogenous peroxidase was removed by incubation with 3% H₂O₂ solution for 10 minutes. Tissue sections were then washed four times with PBS, repeating this process for 4 minutes. Excess liquid was blotted off with filter paper. Tissue areas were outlined with an immunohistochemistry pen. 10% goat serum was added to the outlined areas and incubated at 37°C for 1 hour. Diluted Collagen I primary antibody solution was applied to the sections and incubated overnight at 4°C. The following day, sections were removed, rewarmed for 30 minutes, and washed with PBS for 5 minutes, repeating this process four times. Secondary antibodies of the appropriate species were added and incubated at 37°C for 30 minutes. Afterwards, sections were washed with PBS for 5 minutes, repeating this process three times. Freshly prepared DAB color development solution was then added. The sections were observed under a microscope for color development. The color development was terminated by rinsing the solution several times with pure water. The sections were counterstained with hematoxylin for 2 minutes, rinsed with tap water, and then differentiated in 1% hydrochloric acid alcohol for 5-10 seconds. The sections were then blued overnight. After dehydration with graded alcohol, the sections were transparentized with xylene and finally mounted with neutral gum. The sections were air-dried at room temperature and observed under a microscope.

[0079] Example 1 Effect of Nobiletin Combined with Nintedanib on TGF-β1-induced ECM Protein Expression in MRC-5 Cells

[0080] TGF-β1 induces the transformation of fibroblasts into myofibroblasts, which then secrete large amounts of ECM components, such as fibronectin and collagen, leading to structural destruction and fibrosis in lung tissue. Excessive ECM production can further induce fibroblast activation, creating a positive feedback loop that exacerbates fibrosis. To investigate whether nobiletin combined with nintedanib synergistically inhibits the expression of ECM proteins, Western blot assays were performed to examine the effects of nobiletin alone and in combination with nintedanib on the expression of fibronectin, collagen I, and α-SMA proteins in MRC-5 cells.

[0081] The results are as follows Figure 1 As shown in the figure, compared with the control group, after TGF-β1 induction in MRC-5 cells, the expression of Fibronectin, Collagen I and α-SMA proteins in the TGF-β1 group was significantly increased (p<0.05). Compared with the TGF-β1 group, the expression of Fibronectin protein in the nobiletin 10μM group and the nintedanib 0.3μM group was significantly decreased (p<0.01), and the expression of Collagen I and α-SMA proteins showed a decreasing trend. Compared with the groups treated with nobiletin 10μM and nintedanib 0.3μM alone, the combination of nobiletin and nintedanib significantly decreased the expression of Fibronectin and α-SMA proteins (p<0.05), and the expression of Collagen I protein showed a decreasing trend.

[0082] Example 2 Effect of Nobiletin Combined with Nintedanib on TGF-β1-induced α-SMA

[0083] Immunofluorescence analysis further verified that nobiletin combined with nintedanib reversed the upregulation of a-SMA induced by TGF-β1. Figure 2 As shown in the figure, compared with the control group, the fluorescence intensity of α-SMA was significantly enhanced after MRC-5 cells were induced by TGF-β1 (p<0.001). Compared with the TGF-β1 group, the fluorescence intensity of α-SMA was significantly weakened after nobiletin combined with nintedanib (p<0.001).

[0084] Example 3 Effect of Nobiletin Combined with Nintedanib on TGF-β1-induced MRC-5 Cell Migration

[0085] Under the induction of TGF-β1, fibroblasts are activated and differentiated into myofibroblasts with stronger migration ability. The effects of nobiletin and nintedanib alone or in combination on TGF-β1-induced migration of MRC-5 cells were determined by wound healing assay and Transwell assay.

[0086] Scratch test Figure 3 As shown, in MRC-5 cells, the TGF-β1 group significantly enhanced cell migration compared to the control group (p<0.001). Compared with the model group, nobiletin 10μM or nintedanib 0.3μM alone had no significant inhibitory effect on cell migration, while nobiletin combined with nintedanib significantly inhibited cell migration (p<0.05). Compared with the nobiletin 10μM or nintedanib 0.3μM alone group, the combined administration significantly inhibited cell migration (p<0.01).

[0087] Transwell assay was used to further investigate the inhibitory effects of nobiletin 10 μM and nintedanib 0.3 μM alone or in combination on the migration of MRC-5 cells. Figure 4 As shown in the results, in MRC-5 cells, TGF-β1-induced cell migration ability was enhanced, and the number of migrating cells increased significantly (p<0.001). Compared with the TGF-β1 group, cell migration was inhibited in the groups treated with either 10μM nobiletin or 0.3μM nintedanib alone (p<0.05), and the combination of nobiletin and nintedanib inhibited cell migration more significantly (p<0.001). Compared with the groups treated with either nobiletin or nintedanib alone, the combination of nobiletin and nintedanib showed a trend of decreasing the inhibition of cell migration.

[0088] Example 4 Effect of Nobiletin Combined with Nintedanib on TGF-β1-induced EMT in MRC-5 Cells

[0089] TGF-β1 plays an important role in the occurrence and development of pulmonary fibrosis by inducing EMT. TGF-β1 is the main inducer of EMT and promotes epithelial-mesenchymal transition of MRC-5 cells by activating its downstream signaling pathway. In order to explore the effect of nobiletin combined with nintedanib on myofibroblast MET, the present invention observed the effect of nobiletin alone and in combination with nintedanib on the expression of E-cadherin, N-cadherin and Vimentin proteins in MRC-5 cells by Western blot experiments.

[0090] The results are as follows Figure 5 As shown, compared with the control, the relative expression levels of N-cadherin and Vimentin proteins increased after TGF-β1 induction (p<0.05), while the relative expression level of E-cadherin protein decreased (p<0.05). Compared with the TGF-β1 group, nobiletin 10μM and nintedanib 0.3μM significantly reduced the expression of N-cadherin and Vimentin proteins (p<0.05). Compared with the group treated with nobiletin 10μM and nintedanib 0.3μM alone, nobiletin 10μM combined with nintedanib 0.3μM significantly reduced the expression of N-cadherin and Vimentin proteins (p<0.05) and promoted the expression level of E-cadherin protein (p<0.05). The results showed that nobiletin combined with nintedanib significantly inhibited the epithelial-mesenchymal transition of MRC-5 cells.

[0091] Example 5 Effect of Nobiletin Combined with Nintedanib on ECM Protein Expression in Mouse Lung Tissue

[0092] Fibroblasts secrete collagen and other ECM components in damaged lung tissue, leading to fibrosis. To investigate whether nobiletin combined with nintedanib synergistically inhibits the expression of ECM proteins, the present study used Western blot experiments to examine the effects of nobiletin combined with nintedanib on the expression of Collagen I, a direct component of lung tissue ECM, and α-SMA, a myofibroblast marker regulated by Collagen I.

[0093] The results are as follows Figure 6 As shown, compared with the Control group, the BLM group had significantly increased expression of Collagen I and α-SMA proteins (p<0.01). Compared with the BLM group, nobiletin 100 mg / kg and nintedanib 30 mg / kg reduced the expression level of Collagen I protein (p<0.001). Nobiletin combined with nintedanib significantly reduced the expression of Collagen I and α-SMA proteins (p<0.01). Compared with nobiletin 100 mg / kg and nintedanib 30 mg / kg, nobiletin combined with nintedanib significantly inhibited the expression of Collagen I and α-SMA proteins (p<0.05). These results indicate that combined administration synergistically inhibits the expression of Collagen I and α-SMA proteins.

[0094] Example 6 Nobiletin combined with nintedanib improves pathological changes in mouse lung tissue

[0095] BLM induced inflammatory response, lung tissue damage and fibrosis in mice. The present invention evaluated the alveolar inflammation, fibrosis and collagen deposition in mice by HE staining, Masson staining and IHC.

[0096] HE staining results Figure 7 As shown in the results, the alveoli of the mice in the Control group were intact, with thin walls, clear lung tissue structure, normal morphology, and no inflammatory cell infiltration. In the BLM group, the intact alveolar structure disappeared, the alveolar walls of the lung tissue were significantly thickened, the alveoli were atrophied, collapsed and disordered, and sheet-like fibrosis and connective tissue hyperplasia were observed, accompanied by inflammatory cell infiltration. The degree of alveolar structure destruction, inflammatory cell infiltration and fibrosis hyperplasia in the groups treated with nobiletin or nintedanib alone were improved compared with those in the BLM group, and the improvement in the combined treatment group was better than that in the group treated with either nobiletin or nintedanib alone.

[0097] IHC results analyzed the expression of Collagen I in lung tissue. The alveolar structure of mice in the Control group was intact, and there were almost no brown Collagen I-positive expression areas in the lung tissue. Most of the cells were stained with a large number of blue hematoxylin nuclei. The alveolar structure of the BLM group was disordered, and a large number of brown-yellow Collagen I-positive areas were visible. After intervention with nobiletin and nintedanib alone, the degree of alveolar fibrosis was improved, and the area of Collagen I-positive expression was reduced. The degree of alveolar fibrosis in the combined drug group was further improved, the brown-yellow Collagen I expression area was further reduced compared with the single drug group, and the collagen deposition condition was further improved.

[0098] Masson staining revealed that the alveolar structure of mice in the control group was relatively intact, with almost no blue collagen deposition and no fibrosis in the lung tissue. In the BLM group, lung parenchyma destruction, thickening of the alveolar septa, loss of alveolar integrity, and abundant blue collagen deposition were observed in the lung tissue. Compared with the BLM group, the groups receiving either nobiletin or nintedanib alone showed reduced blue collagen deposition in the alveolar spaces and improved alveolar wall thickening. The combination group showed greater improvement in blue collagen fiber proliferation than the single-drug group.

[0099] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of nobiletin and nintedanib in combination for the preparation of a drug for preventing and / or treating pulmonary fibrosis, characterized in that: The nintedanib comprises at least one of the following: nintedanib itself, nintedanib stereoisomers, nintedanib tautomers, nintedanib polymorphs, nintedanib solvates and pharmaceutically acceptable nintedanib salts; The nobiletin includes at least one of the following: nobiletin itself, nobiletin stereoisomers, nobiletin tautomers, nobiletin polymorphs, solvates of nobiletin and pharmaceutically acceptable salts of nobiletin.

2. The application according to claim 1, characterized in that The combined drug, calculated on a mouse dosage basis, comprises 80-150 mg / kg of nobiletin and 10-50 mg / kg of nintedanib.

3. The use according to claim 1 or 2, characterized in that: The combined drug, calculated on a mouse dosage basis, comprises 90-120 mg / kg of nobiletin and 20-40 mg / kg of nintedanib.

4. A pharmaceutical composition for preventing and / or treating pulmonary fibrosis, characterized in that: Including nobiletin and nintedanib.

5. A pharmaceutical compound preparation for preventing and / or treating pulmonary fibrosis, characterized in that: The pharmaceutical composition according to claim 4 and at least one pharmaceutically acceptable excipient or carrier.

6. The pharmaceutical compound preparation according to claim 5, characterized in that: The excipient is selected from at least one of lubricants, fillers, disintegrants, plasticizers, colorants, emulsifiers, flavoring agents, adhesives, film-forming polymers, antioxidants, light stabilizers, free radical scavengers, surfactants, pH regulators, drug complexing agents and stabilizers against microbial attack.

7. The pharmaceutical compound preparation according to claim 5, characterized in that: The dosage form of the pharmaceutical compound preparation includes any one of the following: solution, granules, tablets, pills, drops, injection, capsules, powders and decoctions.

8. Use of the pharmaceutical composition according to claim 4 or the pharmaceutical compound preparation according to any one of claims 5 to 7 in the preparation of a medicament for preventing and / or treating pulmonary fibrosis.

9. A drug for preventing and / or treating pulmonary fibrosis, characterized in that: The pharmaceutical composition according to claim 4 is used as the active ingredient.

10. The drug according to claim 9, characterized in that The drug, calculated on a mouse dosage basis, includes 100 mg / kg of nobiletin and 30 mg / kg of nintedanib.

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