CN117159536A - Application of compound Fraxetin in preparation of medicines for preventing and treating pulmonary fibrosis - Google Patents
Application of compound Fraxetin in preparation of medicines for preventing and treating pulmonary fibrosis Download PDFInfo
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- CN117159536A CN117159536A CN202311083343.4A CN202311083343A CN117159536A CN 117159536 A CN117159536 A CN 117159536A CN 202311083343 A CN202311083343 A CN 202311083343A CN 117159536 A CN117159536 A CN 117159536A
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- GMRNMZUSKYJXGJ-UHFFFAOYSA-N Fraxetin Natural products C1=CC(=O)C(=O)C2=C1C=C(OC)C(O)=C2O GMRNMZUSKYJXGJ-UHFFFAOYSA-N 0.000 title claims abstract description 40
- HAVWRBANWNTOJX-UHFFFAOYSA-N fraxetin Chemical compound C1=CC(=O)OC2=C1C=C(OC)C(O)=C2O HAVWRBANWNTOJX-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 208000005069 pulmonary fibrosis Diseases 0.000 title claims abstract description 27
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an application of a compound Fraxetin in preparing medicines for preventing and treating pulmonary fibrosis, and belongs to the field of biological medicines. The invention provides a novel effective inhibitor for producing ROS by alveolar epithelial cells, and the Fraxetin is verified by using in vitro and in vivo model experiments to improve pulmonary fibrosis by inhibiting the alveolar epithelial cells from producing ROS.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of a compound Fraxetin in preparation of a medicine for preventing and treating pulmonary fibrosis.
Background
Idiopathic Pulmonary Fibrosis (IPF) is a chronically fibrotic interstitial pneumonia of unknown etiology, whose main pathological feature is alveolar structural destruction accompanied by diffuse alveolitis. The incidence has increased year by year in recent years and the prognosis is poor. Once diagnosed, patients not only affect quality of life, but also die from respiratory failure within a few years.
Many studies suggest that pulmonary tissue is elevated in levels of related inflammatory factors due to prolonged tissue damage and inflammatory stimuli, leading to tissue remodeling. Recent literature suggests that the pathogenesis of IPF is due to alveolar epithelial cell damage, and wound healing is deregulated, thus triggering fibroblast activation. When injury occurs, alveolar epithelial cells fail to maintain physiological regeneration of the lung and can cause abnormal epithelial-mesenchymal interactions and abnormal epithelial-fibroblast communication, such that abnormal extracellular matrix (ECM) accumulation and scar formation occur, resulting in lung fibrosis and irreversible damage to lung tissue.
In recent years, reactive Oxygen Species (ROS) have been found to be involved in the pathological processes of pulmonary fibrosis. Oxidative stress due to excessive increases in ROS is an important mechanism for alveolar epithelial cell death. And ROS can induce alveolar epithelial cell death through a variety of pathways, ROS can induce fibroblast to myofibroblast transformation, causing extracellular matrix (ECM) deposition, causing pulmonary fibrosis. Applicants have shown that alveolar epithelial cells produce large amounts of ROS during in vitro simulation of IPF. Thus, the development of inhibition of alveolar epithelial cell production of ROS is a potential method for improving pulmonary fibrosis.
Disclosure of Invention
The invention aims to provide application of a compound Fraxetin in preparing medicines for preventing and treating pulmonary fibrosis, and the medicines can improve the pulmonary fibrosis by inhibiting alveolar epithelial cells from generating ROS.
The technical scheme of the invention is as follows:
use of a compound Fraxetin or a pharmaceutically acceptable salt thereof for the preparation of an inhibitor of the production of ROS by alveolar epithelial cells.
The application of the compound Fraxetin or the pharmaceutically acceptable salt thereof in preparing the medicines for preventing and treating the pulmonary fibrosis is to inhibit the alveolar epithelial cells from generating ROS by using the compound Fraxetin or the pharmaceutically acceptable salt thereof.
In one embodiment of the invention, the compound Fraxetin has the formula:
in one embodiment of the invention, the pharmaceutically acceptable salts include sodium, calcium, potassium, magnesium, silver, lithium salts.
The compound Fraxetin inhibits the production of ROS by alveolar epithelial cells and is thus useful for inhibiting pulmonary fibrosis.
In one embodiment of the present invention, the medicament comprises a pharmaceutical excipient.
The pharmaceutical excipients comprise: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents.
The pharmaceutical excipients further comprise: osmotic pressure regulator, stabilizer, glidant, flavoring agent, preservative, suspending agent, coating material, fragrance, anti-adhesive, integrator, permeation enhancer, pH regulator, buffer, plasticizer, surfactant, foaming agent, defoamer, thickener, inclusion agent, humectant, flocculant and deflocculant, filter aid, and release retarder.
In one embodiment of the invention, the medicament comprises a pharmaceutically acceptable carrier.
The drug carrier is selected from the group consisting of microcapsules, microspheres, nanoparticles, and liposomes.
In one embodiment of the invention, the dosage form of the medicament comprises: injection, freeze-dried powder for injection, suspension, implant, suppository, capsule, tablet, pill and oral liquid.
In one embodiment of the invention, the medicament is administered by intraperitoneal injection. The cell amount of the drug was 20. Mu.M. The dosage of the medicine experimental animal is 10mg/kg.
The beneficial effects are that:
the Fraxetin disclosed by the invention is a novel effective inhibitor for generating ROS by alveolar epithelial cells. By using in vitro and in vivo models, applicants systematically investigated the potential therapeutic effects of Fraxetin on pulmonary fibrosis, which was experimentally verified to improve pulmonary fibrosis by inhibiting the production of ROS by alveolar epithelial cells.
Drawings
FIG. 1 is a fluorescence plot of Fraxetin for cells that inhibit ROS production by mouse alveolar epithelial cells;
FIG. 2 is a graph of H & E staining of mouse lung tissue following induction of Fraxetin-reduced bleomycin
FIG. 3 is a chart of Masson staining of mouse lung tissue after Fraxetin reduction bleomycin induction;
FIG. 4 is a graph showing that Fraxetin reduces mRNA expression levels of alpha-SMA and fibractin in mouse lung tissue after bleomycin induction.
FIG. 5 shows the results of pathological evaluation of the degree of pulmonary fibrosis in mice.
FIG. 6 is a fluorescence plot of Uroliithin A for cells FITC that inhibit ROS production by mouse alveolar epithelial cells.
Detailed Description
The experimental reagent related to the invention is as follows:
bleomycin (BIOTANG Co., USA), ROS (Biyun Tian Co., ltd.), hematoxylin-eosin (H & E) (Nanjing built technology Co., ltd.), masson dye liquor Kit (Nanjing built technology Co., ltd.), qPCR RT Kit (TOYOBO Co., japan), qPCR Master mix (DBI Co., germany), fraxetin (Tao Shu Bio Co.).
Other reagents are biological grade or analytically pure reagents.
Example 1 effective inhibition of alveolar epithelial cell production of ROS using Fraxetin
Cell experiment treatment:
bleomycin+fraxetin group: mouse alveolar epithelial cells MLE-12 cells were placed in a cell culture dish, and DMEM medium containing 10% fetal bovine serum, penicillin (100. Mu.g/ml) and streptomycin (100. Mu.g/ml) was added. After adding 20. Mu.M Fraxetin for 15min, bleomycin (0.5 nM) was administered and 24 hours later, DCFH-DA was added to achieve a final concentration of 10. Mu.M under normal growth conditions. Cells were incubated in a cell incubator for 20min. After the cells were cleaned by DMEM, the cells were digested and collected, and then purified by a flow cytometer (BDC6Plus; becton Dickinson, USA) to detect FITC fluorescence.
Physiological saline group: fraxetin and bleomycin were not added and only physiological saline was added.
Bleomycin group: fraxetin is not added, and only bleomycin is added.
The results are shown in FIG. 1, and it can be seen that small MLE-12 cells have significantly reduced ROS levels following Fraxetin treatment.
Example 2 improvement of pulmonary fibrosis Using Fraxetin
Experimental animal treatment:
abdominal injection of pentobarbital sodium (50 mg/kg) anesthetized 6 to 8 week old C57BL/6 mice, and the trachea is exposed through a longitudinal incision in the neck skin of 0.5 to 1 cm. The indwelling needle is inserted into the bronchus through the oral cavity. Bleomycin solution (pulmonary fibrosis model 1.4U/kg body weight, survival assay 1.5U/kg body weight) or saline was added to the trachea via an indwelling needle and 500mL of air was rapidly injected. The indwelling catheter was removed and the skin was closed. Mice were kept for 7 days and 21 days, respectively. The model mice were randomly divided into two groups (6 mice per group), and physiological saline was used as a control group.
Control group: control mice were only tracheal infused with saline.
Bleomycin group: mice were given 1.4U/kg bleomycin solution via the trachea.
Bleomycin+fraxetin group: bleomycin-induced pulmonary fibrosis mice received Fraxetin treatment every other day starting at day 2.
Fraxetin was dissolved in 5% dmso+30% PEG 300+dd Water, 10mg/kg Fraxetin was intraperitoneally injected into rats every other day from day 2, after 21 days, the mice were anesthetized, skin was sterilized with 75% ethanol, the chest and abdomen were opened from the middle with scissors, the chest cavity was exposed, and the inferior vena cava was cut off, the lungs were flushed with PBS from the right ventricle until the lungs became white, the tissue where the heart and left lung cross-linked was ligated, the left lung was ligated after 4% paraformaldehyde was perfused through the trachea, and the left lung was cut off and fixed in 4% paraformaldehyde; the remaining lung tissue was rapidly placed in liquid nitrogen for cryopreservation. After the left lung is fixed by 4% paraformaldehyde solution for 24 hours, the tissues are sequentially immersed in 70%, 80%, 95% I, 95% II, 100% I and 100% II ethanol solutions for 1 hour respectively for dehydration, then respectively immersed in xylene solution I, II for transparent 20 minutes, respectively immersed in wax cylinders I, II and III for 1 hour respectively, and then the tissues are embedded into wax blocks by wax liquid.
A. Fraxetin reduction bleomycin post-induction mice lung tissue H & E staining assay:
(1) Slicing the embedded mouse lung tissue wax block, wherein the slice thickness is 4 mu m;
(2) Baking the cut white slices, immersing the white slices in xylene for 5min, and then adding gradient ethanol (100%, 95%, 90%, 80%, 70%) and distilled water for dewaxing;
(3) Hematoxylin staining is carried out on the dewaxed slice for 5min, and tap water is used for flushing;
(4) Color separation and transparency are realized by using a 1% hydrochloric acid ethanol solution, and a 1% ammonia water solution returns to blue;
(5) Eosin staining was performed for 10s-1min (staining time was determined based on color), and the flooding was washed off with tap water.
(6) Gradient ethanol dehydration, xylene permeation and airing, neutral resin sealing, and observation and photographing under a normal microscope.
The results are shown in FIG. 2, and it can be seen that the mice showed a significant decrease in both the degree of pulmonary fibrosis and the area of pulmonary fibrosis following Fraxetin treatment.
B. Fraxetin reduction bleomycin post-induction mouse lung tissue Masson staining assay:
(1) Slicing the embedded mouse lung tissue wax block, wherein the slice thickness is 4 mu m;
(2) Baking the cut white slices, immersing the white slices in xylene for 5min, and then adding gradient ethanol (100%, 95%, 90%, 80%, 70%) and distilled water for dewaxing;
(3) Section Masson staining procedure staining was performed according to the instructions of the kit of the biological company of south Beijing: r1 nuclear dye liquor is used for dyeing for 60s, and flushing liquor is used for flushing for 30s;
(4) R2 slurry dyeing is carried out for 30-60 s, and flushing liquid is used for flushing for 30s;
(5) The R3 yellow color separation liquid separates colors for 6-8 min, the color separation liquid is discarded, and the color separation liquid is directly dyed with the R4 blue counterstain liquid for 5min;
(6) Washing the slices with absolute ethyl alcohol, drying with a blower, sealing the slices with neutral resin, and observing and photographing under a normal microscope.
The results are shown in FIG. 3, and it can be seen that the mice have significantly reduced pulmonary fibrosis area and pulmonary fibrosis degree following Fraxetin treatment.
C. Fraxetin reduces mRNA expression levels of α -SMA and fibrinectin in mouse lung tissue following bleomycin induction:
mice lung tissue 21 days after administration of physiological saline, bleomycin and Fraxetin treatment were homogenized by adding 1mL Trizol to ice. Standing for 5min, and centrifuging at 12000rpm at 4deg.C for 5min. The supernatant was aspirated by a pipette, 200. Mu.L of chloroform was added thereto, mixed by shaking, left at room temperature for 2 minutes, and centrifuged at 12000g for 15 minutes at 4 ℃. At this time, the solution was divided into three layers, the upper aqueous phase was carefully sucked into another centrifuge tube, 500. Mu.L of isopropyl alcohol was added to each centrifuge tube, and the mixture was uniformly mixed, allowed to stand for 5 to 10 minutes, and subjected to centrifugation at 12000g at 4℃for 10 minutes. The supernatant was discarded, 1ml of 75% ethanol in DEPC water was added, and the mixture was washed twice, and centrifuged at 12000g for 5min at 4 ℃. The supernatant was discarded, and after leaving to stand and air-dried, 50. Mu.L of RNase-free water was added to each tube to dissolve RNA. And (3) measuring the ratio of OD260/OD280 by using an enzyme-labeled instrument, identifying the RNA purity, and preserving the sample at-80 ℃. Subsequently, reverse transcription was performed, 1ng of RNA, 0.5. Mu.l of Random primer, 0.5. Mu.l of Enzyme mix cassette 2. Mu.l of 5 Xbuffer were taken, the volume was fixed to 10. Mu.l by RNase-free, and the reaction was carried out in a PCR apparatus at 37℃for 1 hour, followed by reaction at 98℃for 5 minutes to inactivate the Enzyme, which was cDNA. Then, a fluorescent quantitative PCR experiment was performed, 2ng of the above cDNA was used as a template, and 1. Mu.L of the specific primer and 10. Mu.L of 2 XMasterMix (SYBR Green) were continuously added thereto, and the volume was fixed to 20. Mu.L with distilled water. In a fluorescent quantitative PCR instrument, the reaction is performed at a temperature set to: 2min at 95 ℃,15 s at 95 ℃,55 ℃ and annealing for 30s (annealing temperature is correspondingly adjusted according to the value of the primer TM), and extending for 20s at 72 ℃ for 40 cycles; and then the reaction was carried out at 95℃for 15s and at 60℃for 20min. The sequence of the mouse gene primer detected by the experiment is as follows:
fibrinectin: the amount of the upstream primer, 5'-TCTGGGAAATGGAAAAGGGGAATGG-3',
a downstream primer, 5'-CACTGAAGCAGGTTTCCTCGGTTGT-3';
alpha-SMA: the amount of the upstream primer, 5'-GACGCTGAAGTATCCGATAGAACACG-3',
a downstream primer, 5'-CACCATCTCCAGAGTCCAGCACAAT-3'.
The experimental data were processed using Graphpad Prism 8.0 software, and the comparison of the data between groups was expressed as mean ± SEM of more than 6 independent experiments using one-way ANOVA (one-way ANOVA) or two-way repeat ANOVA (two-way ANOVA). P <0.05 indicates that the results are statistically significant. The results are shown in FIG. 4, and it can be seen that both alpha-SMA and fibratecin were significantly reduced in the lung tissue of mice after Fraxetin treatment.
D. Pathological evaluation of pulmonary fibrosis degree in mice
The Ashcroft score was used to show the lung fibrosis score between groups, the experimental data were processed using Graphpad Prism 8.0 software, and the comparison of the data between groups was expressed as mean.+ -. SEM for more than 6 independent experiments using one-way ANOVA or two-way repeated ANOVA. P <0.05 indicates that the results are statistically significant.
The results are shown in fig. 5, which shows that the pathological scores in the lung tissues of mice are significantly reduced after Fraxetin treatment by the bleomycin group.
The above results indicate that Fraxetin is able to reduce bleomycin-induced pulmonary fibrosis.
Comparative example 1
Structure of Urolithin a:
cell assay:
mouse alveolar epithelial cells MLE-12 cells were placed in a cell culture dish, and DMEM medium containing 10% fetal bovine serum, penicillin (100. Mu.g/ml) and streptomycin (100. Mu.g/ml) was added. After adding 20. Mu.M Urolithin A for 15min, BLM (0.5 nM) was administered and 24 hours later, DCFH-DA was added under normal growth conditions to achieve a final concentration of 10. Mu.M. Placing cells in a cell incubatorIncubated for 20min. After the cells were cleaned by DMEM, the cells were digested and collected, and then purified by a flow cytometer (BDC6Plus; becton Dickinson, USA) to detect FITC fluorescence.
The results are shown in FIG. 6, which shows that small MLE-12 cells did not significantly change ROS levels after treatment with the bleomycin group given Urolithin A, indicating that Urolithin A was not effective in inhibiting ROS production by lung epithelial cells.
The above examples are not intended to limit the scope of the invention nor the order of execution of the steps described. The present invention is obviously modified by a person skilled in the art in combination with the prior common general knowledge, and falls within the scope of protection defined by the claims of the present invention.
Claims (10)
1. Use of a compound Fraxetin or a pharmaceutically acceptable salt thereof for the preparation of an inhibitor of the production of ROS by alveolar epithelial cells.
2. The application of the compound Fraxetin or pharmaceutically acceptable salt thereof in preparing medicines for preventing and treating pulmonary fibrosis is characterized in that the compound Fraxetin or pharmaceutically acceptable salt thereof is utilized to inhibit alveolar epithelial cells from generating ROS.
3. Use according to claim 1 or 2, characterized in that the compound Fraxetin has the formula:
4. the use according to claim 1 or 2, wherein the pharmaceutically acceptable salts comprise sodium, calcium, potassium, magnesium, silver, lithium salts.
5. The use according to claim 2, wherein the medicament comprises pharmaceutical excipients.
6. The use according to claim 5, wherein the pharmaceutical excipients comprise: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents.
7. The use according to claim 5, wherein the pharmaceutical excipients further comprise: osmotic pressure regulator, stabilizer, glidant, flavoring agent, preservative, suspending agent, coating material, fragrance, anti-adhesive, integrator, permeation enhancer, pH regulator, buffer, plasticizer, surfactant, foaming agent, defoamer, thickener, inclusion agent, humectant, flocculant and deflocculant, filter aid, and release retarder.
8. The use according to claim 2, wherein the medicament comprises a pharmaceutically acceptable carrier.
9. The use according to claim 8, wherein the pharmaceutical carrier is selected from the group consisting of microcapsules, microspheres, nanoparticles and liposomes.
10. The use according to claim 2, wherein the dosage form of the medicament comprises: injection, freeze-dried powder for injection, suspension, implant, suppository, capsule, tablet, pill and oral liquid.
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