Application of the compound Fraxetin in the preparation of drugs for the prevention and treatment of pulmonary fibrosis

Abstract

This invention discloses the application of the compound Fraxetin in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, belonging to the field of biomedicine. This invention proposes Fraxetin as a novel and effective inhibitor of reactive oxygen species (ROS) produced by alveolar epithelial cells. Through in vitro and in vivo model experiments, it has been verified that Fraxetin improves pulmonary fibrosis by inhibiting ROS production in alveolar epithelial cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to the application of a compound, Fraxetin, in the preparation of drugs for the prevention and treatment of pulmonary fibrosis. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic interstitial pneumonia of unknown etiology, characterized by alveolar structural destruction accompanied by diffuse alveolitis. Its incidence has been increasing annually in recent years, and the prognosis is poor. Once diagnosed, patients not only experience a significant impact on their quality of life but also often die from respiratory failure within a few years.

[0003] Many studies suggest that long-term tissue damage and inflammatory stimulation in lung tissue lead to elevated levels of related inflammatory factors, triggering tissue remodeling. Recent literature indicates that the pathogenesis of IPF (intrapulmonary fibrosis) is due to damage to alveolar epithelial cells and disordered wound healing, which in turn triggers fibroblast activation. When damage occurs, alveolar epithelial cells are unable to maintain physiological lung regeneration, leading to abnormal epithelial-mesenchymal interactions and epithelial cell-fibroblast communication. This results in abnormal extracellular matrix (ECM) accumulation and scar formation, ultimately causing pulmonary fibrosis and irreversible lung tissue damage.

[0004] Recent studies have found that reactive oxygen species (ROS) are involved in the pathological process of pulmonary fibrosis. Excessive ROS-induced oxidative stress is a crucial mechanism for alveolar epithelial cell death. Furthermore, ROS can induce alveolar epithelial cell death through multiple pathways, including inducing the transformation of fibroblasts into myofibroblasts, causing extracellular matrix (ECM) deposition, and ultimately leading to pulmonary fibrosis. The applicant's research has shown that alveolar epithelial cells produce large amounts of ROS during in vitro simulations of interstitial lung disease (IPF). Therefore, developing methods to inhibit ROS production by alveolar epithelial cells is a potential approach to improve pulmonary fibrosis. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the compound Fraxetin in the preparation of a drug for the prevention and treatment of pulmonary fibrosis, which improves pulmonary fibrosis by inhibiting the production of ROS by alveolar epithelial cells.

[0006] The technical solution of this invention is:

[0007] The use of the compound Fraxetin or a pharmaceutically acceptable salt thereof in the preparation of an inhibitor of ROS production from alveolar epithelial cells.

[0008] The use of the compound Fraxetin or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of pulmonary fibrosis involves utilizing the compound Fraxetin or a pharmaceutically acceptable salt thereof to inhibit the production of ROS by alveolar epithelial cells.

[0009] In one embodiment of the present invention, the structural formula of the compound Fraxetin is:

[0010]

[0011] In one embodiment of the present invention, pharmaceutically acceptable salts include sodium salts, calcium salts, potassium salts, magnesium salts, silver salts, and lithium salts.

[0012] The compound Fraxetin inhibits the production of ROS by alveolar epithelial cells, thereby inhibiting pulmonary fibrosis.

[0013] In one embodiment of the present invention, the drug contains pharmaceutical excipients.

[0014] The pharmaceutical excipients include: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, and wetting agents.

[0015] The pharmaceutical excipients also include: osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, encapsulating agents, humectants, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0016] In one embodiment of the present invention, the drug comprises a pharmaceutical carrier.

[0017] The drug carrier is selected from microcapsules, microspheres, nanoparticles and liposomes.

[0018] In one embodiment of the present invention, the dosage form of the drug includes: injection, lyophilized powder for injection, suspension, implant, embolization, capsule, tablet, pill and oral liquid.

[0019] In one embodiment of the invention, the drug is administered via intraperitoneal injection. The cellular dosage of the drug is 20 μM. The experimental animal dosage of the drug is 10 mg / kg.

[0020] Beneficial effects:

[0021] Fraxetin, as described in this invention, is a novel and effective inhibitor of reactive oxygen species (ROS) produced by alveolar epithelial cells. Using in vitro and in vivo models, the applicant systematically investigated the potential therapeutic effects of Fraxetin on pulmonary fibrosis. Experimental results demonstrated that Fraxetin improves pulmonary fibrosis by inhibiting ROS production in alveolar epithelial cells. Attached Figure Description

[0022] Figure 1FITC fluorescence image of mice alveolar epithelial cells inhibiting ROS production by Fraxetin;

[0023] Figure 2 Image showing H&E staining of mouse lung tissue after bleomycin induction with Fraxetin.

[0024] Figure 3 Masson staining of mouse lung tissue after bleomycin induction with Fraxetin-reduced bleomycin;

[0025] Figure 4 Fraxetin was used to reduce the mRNA expression levels of α-SMA and Fibronectin in the lung tissue of mice after bleomycin induction.

[0026] Figure 5 The results show the pathological evaluation of the degree of pulmonary fibrosis in mice.

[0027] Figure 6 FITC fluorescence image of mice alveolar epithelial cells inhibiting ROS production by Urolithin A. Detailed Implementation

[0028] The experimental reagents involved in this invention are as follows:

[0029] Bleomycin (BIOTANG, USA), ROS (Beyotime Biotech), Hematoxylin and Eosin (H&E) (Nanjing Jiancheng Technology Co., Ltd.), Masson staining kit (Nanjing Jiancheng Technology Co., Ltd.), qPCR RT Kit (TOYOBO, Japan), qPCR MasterMix (DBI, Germany), Fraxetin (Taoshu Biotechnology Co., Ltd.).

[0030] All other reagents are biological grade or analytical grade.

[0031] Example 1: Fraxetin effectively inhibits ROS production in alveolar epithelial cells

[0032] Cell experimental treatment:

[0033] Bleomycin + Fraxetin group: Mouse alveolar epithelial cells (MLE-12) were placed in cell culture dishes and DMEM medium containing 10% fetal bovine serum, penicillin (100 μg / ml), and streptomycin (100 μg / ml) was added. 20 μM Fraxetin was added, and after 15 min, bleomycin (0.5 nM) was administered. 24 hours later, under normal growth conditions, DCFH-DA was added to reach a final concentration of 10 μM. Cells were incubated in a cell culture incubator for 20 min. After cleaning the cells with DMEM, they were digested and collected, and then analyzed by flow cytometry (BD). The analysis was performed using C6 Plus (Becton Dickinson, USA) to detect FITC fluorescence.

[0034] Saline group: No Fraxetin or Bleomycin were added, only normal saline was added.

[0035] Bleomycin group: No Fraxetin added, only bleomycin added.

[0036] The results are as follows Figure 1 As shown in the results, the ROS level of small MLE-12 cells was significantly reduced after treatment with Fraxetin in the bleomycin group.

[0037] Example 2: Improving Pulmonary Fibrosis with Fraxetin

[0038] Laboratory animal handling:

[0039] Six- to eight-week-old C57BL / 6 mice were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg / kg). The trachea was exposed through a 0.5 to 1 cm longitudinal incision in the neck skin. An indwelling catheter was inserted into the bronchus through the mouth. Bleomycin solution (1.4 U / kg body weight for pulmonary fibrosis model, 1.5 U / kg body weight for survival experiment) or physiological saline was added to the trachea through the indwelling catheter, and 500 mL of air was rapidly injected. The indwelling catheter was removed and the skin was closed. Mice were fed for 7 days and 21 days, respectively. The mice with the pulmonary fibrosis model were randomly divided into two groups (n=6 per group), with physiological saline serving as the control group.

[0040] Control group: Mice in the control group received only saline solution via tracheal infusion.

[0041] Bleomycin group: Mice were administered 1.4 U / kg of bleomycin solution via trachea.

[0042] Bleomycin + Fraxetin group: Mice with bleomycin-induced pulmonary fibrosis were treated with Fraxetin every other day starting on day 2.

[0043] Fraxetin was dissolved in 5% DMSO + 30% PEG 300 + dd Water. Starting from day 2, 10 mg / kg Fraxetin was injected intraperitoneally into rats every other day. After 21 days, the mice were anesthetized in the abdominal cavity, the skin was disinfected with 75% ethanol, and an incision was made in the middle of the chest and abdomen with scissors to expose the thoracic cavity. The inferior vena cava was cut off, and PBS was injected into the right ventricle to flush the lungs until the lungs turned white. The tissue at the junction of the heart and the left lung was ligated. 4% paraformaldehyde was instilled into the left lung through the trachea, and the trachea was ligated. The left lung was cut off and fixed in 4% paraformaldehyde. The remaining lung tissue was quickly placed in liquid nitrogen for cryopreservation. After fixing the left lung with 4% paraformaldehyde solution for 24 hours, the tissue was successively immersed in 70%, 80%, 95% I, 95% II, 100% I and 100% II ethanol solutions for 1 hour each to dehydrate. Then, it was immersed in xylene solutions I and II for 20 minutes each to clear the tissue. After that, it was immersed in paraffin in paraffin baths I, II and III for 1 hour each. Finally, the tissue was embedded in paraffin liquid to form a paraffin block.

[0044] A. Fraxetin reduces H&E staining of bleomycin-induced mouse lung tissue:

[0045] (1) The embedded mouse lung tissue paraffin block was sectioned, and the section thickness was 4μm;

[0046] (2) Dry the cut white slices, immerse them in xylene for 5 minutes, and then dewax them with a gradient of ethanol (100%, 95%, 90%, 80%, 70%) and distilled water.

[0047] (3) After dewaxing, the sections were stained with hematoxylin for 5 minutes and then rinsed with tap water.

[0048] (4) It is transparent when separated by 1% hydrochloric acid ethanol solution, and turns blue when separated by 1% ammonia solution;

[0049] (5) Perform eosin staining for 10s-1min (determine the staining time based on the color), and wash away the excess color with tap water.

[0050] (6) Dehydrate with graded ethanol, air dry with xylene, mount with neutral resin, and observe and photograph under an upright microscope.

[0051] The results are as follows Figure 2 As shown in the results, after treatment with Fraxetin, the degree and area of ​​pulmonary fibrosis in mice were significantly reduced in the bleomycin group.

[0052] B. Fraxetin reduces Masson staining of bleomycin-induced mouse lung tissue:

[0053] (1) The embedded mouse lung tissue paraffin block was sectioned, and the section thickness was 4μm;

[0054] (2) Dry the cut white slices, immerse them in xylene for 5 minutes, and then dewax them with a gradient of ethanol (100%, 95%, 90%, 80%, 70%) and distilled water.

[0055] (3) Masson staining procedure for sections: staining with R1 nuclear staining solution for 60s, rinsing solution for 30s;

[0056] (4) Stain with R2 paste dye for 30-60 seconds, then rinse with rinsing solution for 30 seconds;

[0057] (5) Use R3 yellow separating solution to separate colors for 6-8 minutes, discard the separating solution, and directly use R4 blue counterstain solution to stain for 5 minutes;

[0058] (6) Rinse the slides with anhydrous ethanol, dry them with a hair dryer, mount them with neutral resin, and observe and photograph them under an upright microscope.

[0059] The results are as follows Figure 3 As shown in the results, after treatment with Fraxetin, the area and degree of pulmonary fibrosis in mice were significantly reduced in the bleomycin group.

[0060] C. Fraxetin reduces the mRNA expression levels of α-SMA and Fibronectin in bleomycin-induced mouse lung tissue:

[0061] Lung tissue from mice treated with saline, bleomycin, and fraxetin for 21 days was homogenized on ice with 1 mL of Trizol. After standing for 5 min, the mixture was centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was aspirated and 200 μL of chloroform was added. The mixture was vortexed and incubated at room temperature for 2 min, then centrifuged at 12,000 g for 15 min at 4°C. The solution separated into three layers. The upper aqueous phase was carefully transferred to another centrifuge tube, and 500 μL of isopropanol was added to each tube. The mixture was incubated for 5-10 min, then centrifuged at 12,000 g for 10 min at 4°C. The supernatant was discarded, and the mixture was washed twice with 1 mL of 75% ethanol prepared with DEPC water. The mixture was then centrifuged at 12,000 g for 5 min at 4°C. The supernatant was discarded, and the mixture was allowed to air dry. 50 μL of Nase-free water was added to each tube to dissolve the RNA. The OD260 / OD280 ratio was measured using a microplate reader to determine RNA purity. Samples were stored at -80℃. Reverse transcription was then performed. 1 ng of RNA, 0.5 μl of random primer, 0.5 μl of Enzyme Mix, and 2 μL of 5x buffer were added, and the volume was adjusted to 10 μL with RNase-free reagent. The mixture was then incubated in a PCR instrument at 37℃ for 1 hour, followed by inactivation at 98℃ for 5 minutes, yielding cDNA. Quantitative real-time PCR was then performed. 2 ng of the cDNA was used as a template, and 1 μL of specific primers and 10 μl of 2x MasterMix (SYBR Green) were added, with the volume adjusted to 20 μL with distilled water. The reaction was performed in a real-time PCR instrument at the following temperatures: 95℃ for 2 min, 95℃ for 15 s, 55℃ for 30 s annealing (annealing temperature adjusted according to primer TM value), and 72℃ for 20 s extension, for a total of 40 cycles; followed by a 20-minute cycle of 95℃ for 15 s and 60℃ for 15 s extension. The primer sequences for detecting the mouse gene in this experiment are as follows:

[0062] Fibronectin: Upstream primer, 5'-TCTGGGAAATGGAAAAGGGGAATGG-3',

[0063] Downstream primer, 5'-CACTGAAGCAGGTTTCCTCGGTTGT-3';

[0064] α-SMA: Upstream primer, 5'-GACGCTGAAGTATCCGATAGAACACG-3',

[0065] Downstream primer: 5'-CACCATCTCCAGAGTCCAGCACAAT-3'.

[0066] The experimental data were processed using Graphpad Prism 8.0 software. One-way ANOVA or two-way repeated ANOVA was used to compare data between groups. Experimental data are expressed as mean ± SEM of at least six independent experiments. P < 0.05 was considered statistically significant. Results are as follows... Figure 4 As shown in the results, after treatment with Fraxetin, the levels of α-SMA and Fibronectin in the lung tissue of mice were significantly reduced in the bleomycin group.

[0067] D. Pathological evaluation of the degree of pulmonary fibrosis in mice

[0068] The Ashcroft score was used to display pulmonary fibrosis scores between groups. Data were processed using GraphpadPrism 8.0 software. One-way ANOVA or two-way repeated ANOVA was used for comparisons between groups. Experimental data are expressed as mean ± SEM of at least six independent trials. P < 0.05 was considered statistically significant.

[0069] The results are as follows Figure 5 As shown in the figure, after treatment with Fraxetin, the pathological scores in the lung tissue of mice in the bleomycin group were significantly reduced.

[0070] These results indicate that Fraxetin can reduce bleomycin-induced pulmonary fibrosis.

[0071] Comparative Example 1

[0072] Structure of Urolithin A:

[0073]

[0074] Cellular experiments:

[0075] Mouse alveolar epithelial cells (MLE-12) were placed in cell culture dishes and filled with DMEM medium containing 10% fetal bovine serum, penicillin (100 μg / ml), and streptomycin (100 μg / ml). 20 μM Urolithin A was added, and after 15 min, BLM (0.5 nM) was administered. After 24 hours, DCFH-DA was added under normal growth conditions to reach a final concentration of 10 μM. The cells were incubated in a cell culture incubator for 20 min. After cleaning the cells with DMEM, they were digested and collected, and then analyzed by flow cytometry (BD). The analysis was performed using C6Plus (Becton Dickinson, USA) to detect FITC fluorescence.

[0076] The results are as follows Figure 6 As shown in the results, after treatment with Urolithin A, the ROS level of small MLE-12 cells in the bleomycin group did not change significantly, indicating that Urolithin A could not effectively inhibit the production of ROS by lung epithelial cells.

[0077] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. The use of the compound Fraxetin or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and treatment of pulmonary fibrosis, characterized in that, It utilizes the compound Fraxetin or its pharmaceutically acceptable salts to inhibit the production of ROS by alveolar epithelial cells; The structural formula of the compound Fraxetin is: 。 2. The application according to claim 1, characterized in that, Pharmaceutically acceptable salts include sodium salts, calcium salts, potassium salts, magnesium salts, silver salts, and lithium salts.

3. The application according to claim 1, characterized in that, The drug contains pharmaceutical excipients.

4. The application according to claim 3, characterized in that, The pharmaceutical excipients include: solvents, propellants, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, and wetting agents.

5. The application according to claim 3, characterized in that, The pharmaceutical excipients also include cosolvents.

6. The application according to claim 3, characterized in that, The pharmaceutical excipients also include: osmotic pressure regulators, stabilizers, flavoring agents, preservatives, suspending agents, coating materials, integrators, penetration enhancers, plasticizers, surfactants, foaming agents, defoamers, thickeners, humectants, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

7. The application according to claim 3, characterized in that, The pharmaceutical excipients also include: pH adjusters and inclusion agents.

8. The application according to claim 3, characterized in that, The pharmaceutical excipients also include buffers.

9. The application according to claim 3, characterized in that, The pharmaceutical excipients also include fragrances.

10. The application according to claim 3, characterized in that, The pharmaceutical excipients also include flow aids.

11. The application according to claim 3, characterized in that, The pharmaceutical excipients also include anti-adhesion agents.

12. The application according to claim 1, characterized in that, The drug contains a pharmaceutical carrier.

13. The application according to claim 12, characterized in that, The pharmaceutical carrier is selected from microcapsules, microspheres, nanoparticles and liposomes.

14. The application according to claim 1, characterized in that, The dosage forms of the drugs include: injections, lyophilized powder for injection, suspensions, implants, embolic agents, capsules, tablets, pills, and oral liquids.

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