CN113425714A - Application of indoleacetic acid in preparation of medicine for preventing and treating chronic obstructive pulmonary disease - Google Patents
Application of indoleacetic acid in preparation of medicine for preventing and treating chronic obstructive pulmonary disease Download PDFInfo
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Abstract
The invention provides a new application of a compound indoleacetic acid in preparation of a medicine for preventing and treating chronic obstructive pulmonary disease. Animal experiments prove that the compound indoleacetic acid can obviously inhibit lung inflammatory cell infiltration of chronic obstructive pulmonary disease, inhibit the generation of inflammatory factors of the chronic obstructive pulmonary disease and lung cell apoptosis, inhibit the expression of lung cell apoptosis-related protein clear caspase-3, obviously improve the lung function of a chronic obstructive pulmonary disease model mouse, reduce the proportion of lung neutrophils of the chronic obstructive pulmonary disease, and can be used for preparing medicines related to the prevention and treatment of the chronic obstructive pulmonary disease.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of indoleacetic acid in preparation of medicines for preventing and treating chronic obstructive pulmonary disease.
Background
Chronic obstructive pulmonary disease is a complex heterogeneous lung disease characterized by persistent airflow obstruction, chronic bronchitis and/or emphysema, one of the common diseases with high global morbidity and mortality, associated with abnormal inflammatory responses of the body to harmful gases or harmful particles. At present, chronic obstructive pulmonary disease is the fourth leading cause of death in China, seriously damages human health and is an important public health problem.
Indolylacetic acid was originally found in plants by Frits WarmoltWent in 1928 and is the most common auxin in plants. However, increasing research has also shown that many microorganisms are also capable of producing indoleacetic acid, such as Agrobacterium, Azospira, Bacillus, Enterobacter, and the like. Also present in mammalian cells is endogenous indoleacetic acid, which may result from the decarboxylation of tryptophan or the oxidative deamination of tryptophan. Exogenous indole acetic acid is also present in mammals and is typically produced by microorganisms in the mammalian intestinal tract. The current research shows that indoleacetic acid has a protective effect on liver cancer mice induced by diethylnitrosamine, and can relieve oxidative stress and inflammatory reaction of the liver in a non-alcoholic fatty liver mouse model. However, the role of indoleacetic acid in chronic obstructive pulmonary disease and related respiratory diseases has not been reported yet.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a new application of indoleacetic acid in preparing a medicine for preventing and treating chronic obstructive pulmonary disease.
The application of compound indole acetic acid in preparing a medicament for preventing and treating chronic obstructive pulmonary disease is disclosed, wherein the compound indole acetic acid is a compound shown in a formula (I) or a hydrate, a solvate, a metabolite and a pharmaceutically acceptable salt of the compound, and is indole acetic acid sodium salt shown in a formula (II).
Application of compound indoleacetic acid in preparation of medicines for preventing and treating chronic obstructive pulmonary diseases mainly caused by neutrophil inflammation.
The indoleacetic acid is colorless crystalline powder with molecular formula of C10H9NO2Molecular weight of 175.187, melting point of 164-165 ℃ CSoluble in water and soluble in polar organic solvents. It is easily decomposed in light and air and is not storage-stable. The aqueous solution is capable of being decomposed by ultraviolet light, but is stable to visible light. Is safe to human and livestock.
In the invention, the compound indoleacetic acid can obviously improve the lung function of chronic obstructive pulmonary disease.
According to the invention, the compound indoleacetic acid can obviously inhibit lung inflammatory cell infiltration of chronic obstructive pulmonary disease.
In the invention, the compound indoleacetic acid can obviously inhibit the generation of inflammatory factors of chronic obstructive pulmonary diseases. The chronic obstructive pulmonary disease inflammatory factors include, but are not limited to TNF-alpha, IL-1 beta, and IL-17A.
According to the invention, the indoleacetic acid compound can obviously reduce the proportion of apoptotic cells in lung tissues of chronic obstructive pulmonary diseases and inhibit the expression of proteins related to apoptosis of lung cells. The protein related to the apoptosis of the lung cells comprises but is not limited to clear caspase-3. The clear caspase-3 is considered to be the most important performer of apoptosis, and once activated, the occurrence of apoptosis is irreversible.
In the invention, the indoleacetic acid compound can obviously reduce the proportion of neutrophils in the lung of the chronic obstructive pulmonary disease. Therefore, the compound indoleacetic acid can be applied to preparation of medicaments for preventing and treating chronic obstructive pulmonary diseases related to neutrophil inflammation, particularly bronchitis type chronic obstructive pulmonary diseases related to neutrophil inflammation.
A pharmaceutical composition comprising said indoleacetic acid compound and a pharmaceutically acceptable carrier, excipient, adjuvant, or combination thereof.
The use of the compound indoleacetic acid or the pharmaceutical composition in the manufacture of a medicament for preventing, treating or ameliorating chronic obstructive pulmonary disease.
Further, the chronic obstructive pulmonary disease of the present invention includes, but is not limited to, airway inflammation, emphysema, impairment of pulmonary ventilation, and respiration and critical illness caused thereby. The chronic obstructive pulmonary disease is characterized by airflow limitation, and has clinical characteristics of expiratory airflow reduction, slow lung forced emptying, asthma, cough and expectoration, accompanied with chronic airway obstruction and lung over-expansion, and frequently occurring chronic obstructive bronchitis and obstructive emphysema at the same time.
Clinical data show that the content of indoleacetic acid in sputum of patients with chronic obstructive pulmonary disease is remarkably reduced, and the indoleacetic acid is closely related to the occurrence and development processes of the chronic obstructive pulmonary disease. According to the invention, through preparing a chronic obstructive pulmonary disease mouse model, adopting indoleacetic acid intervention, detecting and statistically analyzing mouse lung function indexes, inflammatory factors, inflammatory cell infiltration conditions, lung cell apoptosis proportion, lung cell apoptosis protein clear caspase-3 relative expression quantity and lung neutrophil proportion, the indoleacetic acid intervention is found to improve the lung function of the chronic obstructive pulmonary disease mouse model, inhibit the generation of the chronic obstructive pulmonary disease inflammatory factors, inhibit inflammatory cell infiltration, reduce the lung cell apoptosis proportion, inhibit the expression of apoptosis-related proteins and reduce the lung neutrophil proportion.
Compared with the prior art, the invention has the following beneficial effects:
1. animal experiments show that the compound indoleacetic acid can obviously inhibit inflammatory cell infiltration, inflammatory factor generation and lung cell apoptosis of chronic obstructive pulmonary disease lung on the animal level, improve the lung function of a chronic obstructive pulmonary disease model mouse, reduce the proportion of neutrophils in the lung of the chronic obstructive pulmonary disease, and can be used for preventing and treating the chronic obstructive pulmonary disease.
2. The preparation method of the compound indoleacetic acid is simple, stable in property, safe in use, easy to control and convenient for clinical application and popularization.
Drawings
FIG. 1 is a graph showing the results of indoleacetic acid improving lung function index FEV100/FVC in a chronic obstructive pulmonary disease mouse model. The data were analyzed by t-test, ns indicates no statistical difference between the two groups of time; indicates there was a statistical difference between the two groups, p < 0.05.
FIG. 2 is a graph showing the ELISA results of indoleacetic acid in inhibiting the production of inflammatory factors in pulmonary alveolar lavage fluid of a mouse model of chronic obstructive pulmonary disease. The data were analyzed by t-test, ns indicates no statistical difference between the two groups of time; indicates statistical differences between the two groups, p < 0.05; indicates there was a statistical difference between the two groups, p < 0.01.
FIG. 3 is a graph of pathological results of indoleacetic acid inhibiting lung inflammatory cell infiltration in a chronic obstructive pulmonary disease mouse model; wherein, the A picture is a HE staining picture representative picture of lung tissue morphological change of the Control group; b is a representative HE staining pattern of morphological changes of lung tissues in IAA group; panel C is a representation of HE staining for morphological changes in lung tissue in the COPD group; and the D picture is a representative HE staining picture of lung tissue morphological change of a COPD + IAA group, and the picture scale is 400 mu m.
Fig. 4 is statistics of scoring results of inflammatory cell infiltration degree in HE staining chart of morphological changes of lung tissues of each group of lung tissues of indole acetic acid inhibiting lung inflammatory cell infiltration of chronic obstructive pulmonary disease mouse model. The data were analyzed by t-test, ns indicates no statistical difference between the two groups of time; indicates statistical differences between the two groups, p < 0.05; indicates that there is a statistical difference between the two groups, p < 0.01; indicates that there was a statistical difference between the two groups, p < 0.001.
FIG. 5 is a graph showing the results of the ratio of indoleacetic acid to indoleacetic acid in reducing lung apoptosis in a mouse model of chronic obstructive pulmonary disease. Data were analyzed using t-test, ns indicates no statistical difference between the two groups; indicates there was a statistical difference between the two groups, p < 0.05.
FIG. 6 is a protein immunoblotting (Western blot) result chart and statistics of indole acetic acid inhibiting chronic obstructive pulmonary disease mouse model lung cell apoptosis protein clear caspase-3; wherein, A is the immunoblot representation of the apoptotic protein clear caspase-3 and the internal reference protein beta-actin of each group, and B is the statistical result of the grey values of the immunoblot bands of each group. The data were analyzed by t-test, ns indicates no statistical difference between the two groups of time; indicates statistical differences between the two groups, p < 0.05; indicates there was a statistical difference between the two groups, p < 0.01.
FIG. 7 is a graph showing the results of indolacetic acid reducing the proportion of neutrophils in the lung of a mouse model of chronic obstructive pulmonary disease. Data were analyzed using t-test, ns indicates no statistical difference between the two groups; indicates that there was a statistical difference between the two groups, p < 0.001.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1: preparation of chronic obstructive pulmonary disease mouse model and indoleacetic acid intervention
1. Preparation of COPD mouse model:
male C57BL/6J mice of 6-8 weeks old, weighing 18-25g, were selected and purchased from Liaoning Biotechnology GmbH. The mice were randomly divided into 4 groups, which were a Control group (Control group), an indoleacetic acid group (IAA group), a model group (COPD group), and a model plus indoleacetic acid group (COPD + IAA group). On the first day from the start of the experiment, 7 μ g of Lipopolysaccharide (LPS) +1.2U Elastase (Elastase) (dissolved in 100 μ LPBS) was titrated nasally into the lungs of each mouse in the COPD group and the COPD + IAA group, titrated once a week for 4 consecutive weeks, and PBS was titrated in the same manner in the Control group and the IAA group.
2. And (3) indoleacetic acid intervention:
the indoleacetic acid powder was completely dissolved with an appropriate amount of 1mol/L sodium hydroxide, the pH of the solution was adjusted to 7.4 by adding 0.25mol/L hydrogen chloride, the solution was diluted to 5mg/mL with PBS, and the solution was stored at-20 ℃ in the dark. On the first day from the start of the experiment, the IAA group and the COPD + IAA group were injected with indolacetic acid (dose 5mg/mL, volume 200 μ L) into mice by intraperitoneal injection, once a day for 4 weeks, while the Control group and the COPD group were injected with PBS in the same manner. After 4 weeks of continuous intervention, subsequent correlation index detection was performed.
Example 2: indoleacetic acid for improving lung function of chronic obstructive pulmonary disease mouse model
1. And (3) lung function detection:
the lung function of mice in a model group and a non-model group which are subjected to model preparation and IAA intervention is detected, the mice are anesthetized by tribromoethanol, the trachea of the mice is exposed by a sterilized surgical instrument, the lung function indexes FEV100 and FVC indexes of the mice are detected and read by an experimental animal lung function detection system (DSI company in America), and the FEV100/FVC ratio is calculated. And (5) detecting other subsequent indexes after the lung function detection is finished.
2. The experimental results are as follows:
as shown in fig. 1, in the COPD group, FEV100/FVC was significantly decreased (p <0.05) compared to the Control group, whereas in the COPD + IAA group, FEV100/FVC was significantly increased (p <0.05) compared to the COPD group, and the IAA group was not significantly different from the Control group. Experimental results show that the lung function of mice with chronic obstructive pulmonary diseases is reduced, indoleacetic acid is injected to protect the lung function of the mice with chronic obstructive pulmonary diseases, and the indoleacetic acid does not have obvious influence on the lung function of normal mice.
Example 3: indoleacetic acid can inhibit the generation of lung inflammatory factors of mice model of chronic obstructive pulmonary disease
1. Alveolar lavage fluid supernatant acquisition:
mice that completed the lung function test were subjected to alveolar lavage to obtain alveolar lavage fluid, and a sterilized 20G PE needle was inserted into the exposed trachea and the PE needle was fixed with an operating wire. The lungs of mice were lavaged with 0.8mL of ice-precooled Phosphate Buffered Saline (PBS) using a 1mL sterile syringe, connected to a fixed PE needle, and gently back and forth three times to obtain alveolar lavage fluid. The alveolar lavage fluid is centrifuged for 10min at 5000g and 4 ℃, the supernatant is transferred to a new centrifuge tube for subsequent detection, and the cell sediment is left for alveolar lavage fluid smear and HE staining.
2. ELISA for detecting inflammatory factors:
alveolar lavage supernatants were assayed using ELISA kits for TNF- α, IL-1 β, and IL-17A (Shanghai enzyme-linked Biotech, Inc.), respectively. To each well coated with the corresponding antibody, 50. mu.L of alveolar lavage supernatant, standards of various concentrations, and sample dilutions were added, 100. mu.L of horseradish peroxidase (HRP) -labeled detection antibody was added to each well, the wells were sealed with a sealing membrane, and incubated at 37 ℃ for 1 h. Pouring out the liquid in the holes, patting the liquid on absorbent paper, adding 350 mu L of washing liquid into each hole, standing for 1min, throwing off the washing liquid, patting the liquid on the absorbent paper, and repeatedly washing for 5 times. mu.L of substrate A and 50. mu.L of substrate B were added to each well and incubated at 37 ℃ for 15min in the absence of light. mu.L of stop solution was added to each well, OD values were read at a wavelength of 450nm using a multi-functional microplate reader (PerkinElmer, USA), a standard curve was drawn, and the concentration of inflammatory factor in each sample was calculated.
3. The experimental results are as follows:
as shown in FIG. 2, the ELISA results of the inflammatory factors in the mouse alveolar lavage fluid showed that the levels of the inflammatory factors (TNF-. alpha., IL-1. beta., and IL-17A) in the mouse alveolar lavage fluid were significantly increased in the COPD group compared to the Control group, and the levels of the inflammatory factors were significantly decreased in the COPD + IAA group compared to the COPD group, whereas the IAA group was not significantly different from the Control group. Experimental results show that the chronic obstructive pulmonary disease can cause the increase of the lung inflammatory factor level of mice, and the injection of indoleacetic acid can inhibit the increase of the lung inflammatory factor of mice with the chronic obstructive pulmonary disease without obvious influence on normal mice.
Example 4: indoleacetic acid can relieve lung inflammatory cell infiltration of chronic obstructive pulmonary disease model mice
1. Preparing lung tissue disease marble wax slices:
(1) tissue fixation: and separating part of fresh lung tissues of the mice which finish the lung function detection, and soaking the mice in 4% paraformaldehyde fixing solution for fixing for 24 hours.
(2) And (3) dehydrating: dehydration was carried out according to the following procedure: 3 hours of 75% alcohol, 2 hours of 85% alcohol, 1 hour of 95% alcohol, 2 hours of 95% alcohol, 1 hour of absolute ethyl alcohol, 1 hour of environment-friendly transparent agent, 2 hours of melted paraffin and 2 hours of melted paraffin.
(3) Embedding: embedding is carried out on a paraffin section embedding machine.
(4) Slicing: sections were taken on a paraffin microtome to a thickness of 4 μm.
2.HE staining
And (3) placing the paraffin sections into an environment-friendly transparent agent for 10min, absolute ethyl alcohol for 5min, 95% alcohol for 5min and 75% alcohol for 5min, and washing the paraffin sections with tap water. Staining with hematoxylin for 5min, washing with tap water, differentiating with 1% hydrochloric acid alcohol for 10s, and washing with tap water. Eosin dyeing is carried out for 2s, 95% ethanol is used for 1min, absolute ethanol is used for 1min, environment-friendly transparent agent is used for 1min, and environment-friendly transparent agent is used for 1 min. HE stained sections were mounted using neutral resin.
4. Photographing device
The prepared HE sections were placed under an EVOS fl auto-automated fluorescence inverted microscope (Thermo fisher, USA) with a 10 Xmagnification, and 10 fields were randomly selected for each section to be photographed.
5. Scoring
The inflammatory cell infiltration condition of each visual field is scored, and the specific scoring detailed formula is as follows: it is 0 point when there is no inflammatory cell around the bronchus, 1 point when there is a small amount of inflammatory cell around the bronchus, 2 point when there is 1 layer of inflammatory cell around the bronchus, 3 point when there is 2 to 4 layers of cells around the bronchus, and 4 point when there is 5 layers or more of inflammatory cell around the bronchus. The mean was calculated as the final score for each HE slice.
6. Results of the experiment
As shown in fig. 3 and 4, the lung tissue HE staining and scoring results showed that the degree of inflammatory cell infiltration around the bronchi of the lung tissue of mice in the COPD group was significantly increased (p <0.001) compared to the Control group, and that the above-mentioned condition was significantly decreased (p <0.05) in the COPD + IAA group compared to the COPD, while the IAA group was not significantly different from the Control group. Experimental results show that chronic obstructive pulmonary disease can cause inflammatory cell infiltration of mouse lung to be aggravated, and indoleacetic acid is injected to relieve inflammatory cell infiltration of chronic obstructive pulmonary disease mouse lung tissue without obvious influence on normal mice.
Example 5: indoleacetic acid can inhibit lung cell apoptosis of chronic obstructive pulmonary disease mouse
1. Tunel staining:
and (3) placing the paraffin sections into an environment-friendly transparent agent for 10min, absolute ethyl alcohol for 5min, 95% alcohol for 5min and 75% alcohol for 5min, and washing the paraffin sections with tap water. 0.1% TritonX-100 and 0.1% sodium citrate are added dropwise at room temperature and then are permeated for 8 min. Wash 3 times 5min each with TBS. And dropwise adding a mixed solution of 3% BSA and 20% normal calf serum into each slice to block the nonspecific reaction, and keeping the temperature at room temperature for 30 min. The serum was removed, 50ul of Tunel reaction mix (solution A: solution B: 1:9) was added dropwise to each section and mixed, incubated in a wet box for 60min in the dark at 37 ℃ with TBS as a negative control instead of working medium. After completion, the TBS was washed 3 times for 5min each. DAPI staining nuclei, protected from light and room temperature for 10 min. And (3) sealing the anti-fluorescence attenuation sealing agent.
2. And (3) photographing:
the prepared Tunel sections were placed under a fluorescence microscope (Leica, Germany), the magnification of 40X was selected, 10 fields were randomly selected for each section, and two types of blue and green fluorescence were photographed, with green light as apoptotic cells and blue light as total cells.
3. And (3) calculating statistics:
the Image J software was used to count the cells in the photographed images, and the ratio of the number of apoptotic cells to the total number of cells was used as the apoptosis rate.
4. The experimental results are as follows:
as shown in fig. 5, the statistical results of lung tissue Tunel staining showed that the proportion of cells undergoing apoptosis in lung tissue of mice in the COPD group was significantly increased (p <0.05) compared with the Control group, the above-mentioned condition was significantly decreased in the COPD + IAA group compared with COPD (p <0.05), and the IAA group was not significantly different from the Control group. Experimental results show that the chronic obstructive pulmonary disease can increase the proportion of apoptotic cells in the lung of a mouse, and the injection of indoleacetic acid can reduce the proportion of apoptotic cells in the lung of the mouse with the chronic obstructive pulmonary disease without obvious influence on normal mice.
Example 6: indoleacetic acid can inhibit the generation of lung apoptosis protein clear caspase-3 of mice with chronic obstructive pulmonary disease
1. Protein extraction:
50mg of lung tissue was placed in a 2mL grinding tube containing grinding beads, 0.5mL of RIPA lysate and 5. mu.L of protease inhibitor were added, the mixture was placed in a rapid sample preparation apparatus (MP, USA) and sufficiently shaken and ground, the ground homogenate was transferred to a new 1.5mL centrifuge tube, centrifuged at 12000rpm at 4 ℃ for 15min, the supernatant was aspirated and transferred to another new 1.5mL centrifuge tube, protein concentration was quantified according to the BCA protein quantification kit (Beebur Biotech Co., Ltd.), PBS and loading buffer were added to dilute the sample to 1. mu.g/. mu.L, and the protein was sufficiently denatured by heating at 98 ℃ for 10 min.
2. Western blot to detect the relative expression of clear caspase-3:
(1) preparation of 12% separation gel: 3.4mL of distilled water, 4.0mL of 30% Acr-Bis (29:1), 2.5mL of 4 XTris-HCl-SDS gel separation buffer (pH 8.8), 0.1mL of 10% ammonium persulfate solution and 0.004mL of TEMED were mixed well and poured into a gel preparation device.
(2) 3.5% concentrated glue preparation: after the gel was solidified, 2.28mL of distilled water, 0.68mL of 30% Acr-Bis (29:1), 1.0mL of 4 XTTris-HCl-SDS gel-separation buffer (pH 6.8), 0.04mL of 10% ammonium persulfate solution and 0.004mL of TEMED were mixed well, poured into a gel-making device, and inserted into a 15-well comb.
(2) Loading: and (3) after the concentrated gel is solidified, mounting the gel into an electrophoresis tank, pouring the electrophoresis solution into the electrophoresis tank, and slightly pulling out the comb. The samples were sequentially loaded with 5. mu.L of protein marker and 10. mu.L of sample.
(3) Electrophoresis: the voltage was adjusted to a constant voltage of 80V when the sample was on the concentrated gel, 120V when the sample was moving to the separation gel, and stopped when the marker was sufficiently separated.
(4) Film transfer: taking out the SDS-PAGE gel after electrophoresis, clamping the gel and the PVDF membrane soaked in methanol by using a special clamp for membrane transfer and filter paper, transferring the gel and the PVDF membrane to a membrane transfer tank filled with membrane transfer liquid, and transferring the membrane for 60min by constant current 300 mA.
(5) And (3) sealing: and taking down the PVDF membrane after the membrane conversion, converting and soaking in distilled water for 10s, cutting out a target strip by a protein Marker, adding freshly prepared 5% skimmed milk, and sealing for 1 h.
(6) Primary antibody incubation: after the completion of the blocked PVDF membrane was washed 3 times with 1 XTSSbuffer, the clear caspase-3 antibody (dilution factor 1:1000) and the β -actin antibody (dilution factor 1:1000) were added, and the mixture was incubated overnight on a shaker at 4 ℃.
(7) And (3) secondary antibody incubation: after completion of primary antibody incubation, PVDF membrane was washed 3 times with 1 XTSST buffer, goat anti-rabbit secondary antibody (dilution factor 1:5000) was added and incubated at room temperature for 1 h.
(8) Luminescence imaging: after the PVDF membrane subjected to the secondary antibody incubation was washed 3 times with 1 × TBST buffer, the protein on the PVDF membrane was imaged and photographed using a chemiluminescence solution under a fluorescence chemical imaging system (Bio-rad, usa).
(9) Counting: and (5) performing gray value calculation on the pictures by using Image J software, and counting.
3. Results of the experiment
The results of graphs A and B in FIG. 6 show that the expression of clear caspase-3 in lung tissue is significantly increased in the COPD group compared to the Control group, the expression of clear caspase-3 is significantly decreased in the COPD + IAA group compared to the COPD group, and the expression of clear caspase-3 is not significantly different in the IAA group compared to the Control group. Experimental results show that the chronic obstructive pulmonary disease can promote the expression of the mouse lung cell apoptosis-related protein, and the injection of indoleacetic acid can obviously inhibit the expression of the chronic obstructive pulmonary disease mouse lung cell apoptosis-related protein without obviously influencing normal mice.
Example 7: indoleacetic acid can reduce the proportion of neutrophils in the lung of mice with chronic obstructive pulmonary disease
1. Alveolar lavage fluid smear
The alveolar lavage fluid cell pellets obtained in example 3 were resuspended in 400. mu.L of PBS, and the total number of cells per tube was calculated by cell counting using a bovine Bowden white blood cell counting plate. Centrifuging at 2500r/s for 7min, pouring off the supernatant, resuspending with 20 μ L PBS, uniformly smearing the cell suspension on the glass slide with a pipette, naturally air drying, fixing with 10% neutral formaldehyde for 15min, washing with clear water to remove the neutral formaldehyde, and HE staining.
HE staining
And (3) staining the glass slide, wherein the staining steps are as follows: hematoxylin 60s, washing with running water, alcohol differentiation with hydrochloric acid for 10s, washing with running water, lithium carbonate anti-blue for 20s, washing with running water, eosin staining for 10s, and washing with running water. The slide is placed in a baking machine for baking, and the slide is sealed by using neutral resin.
3. Microscopic examination
The proportion of neutrophils was calculated by classifying 200-400 leukocytes using an optical microscope.
4. Results of the experiment
The results in fig. 7 show that the proportion of neutrophils in the lung tissue was significantly increased in the COPD group compared to the Control group, the proportion of neutrophils was significantly decreased in the COPD + IAA group compared to the COPD group, and there was no significant difference in the IAA group compared to the Control group. Experimental results show that the chronic obstructive pulmonary disease can promote the increase of the proportion of neutrophils in the lung of a mouse, and the injection of indoleacetic acid can obviously reduce the proportion of the neutrophils in the lung of the mouse with the chronic obstructive pulmonary disease, without obvious influence on normal mice.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. Application of compound indoleacetic acid in preparation of medicines for preventing and treating chronic obstructive pulmonary disease, wherein the compound indoleacetic acid is a compound shown as a formula (I) or a hydrate, a solvate, a metabolite and a pharmaceutically acceptable salt of the compound shown as the formula (I)
2. The use of claim 1, wherein the chronic obstructive pulmonary disease is neutrophil inflammation associated chronic obstructive pulmonary disease.
3. The use of claim 1, wherein the compound indoleacetic acid is capable of improving pulmonary function in chronic obstructive pulmonary disease.
4. The use of claim 1, wherein said compound indoleacetic acid is capable of inhibiting pulmonary inflammatory cell infiltration of chronic obstructive pulmonary disease.
5. The use of claim 1 wherein the compound indoleacetic acid is capable of inhibiting chronic obstructive pulmonary disease inflammatory factor production; the chronic obstructive pulmonary disease inflammatory factors comprise TNF-alpha, IL-1 beta and IL-17A.
6. The use of claim 1, wherein said compound indoleacetic acid is capable of reducing the proportion of apoptotic cells in lung tissue of chronic obstructive pulmonary disease and inhibiting the expression of proteins associated with apoptosis in lung cells.
7. The use of claim 6, wherein the protein associated with pulmonary apoptosis comprises cleavedcaspase-3.
8. The use of claim 2 wherein the compound indoleacetic acid is capable of reducing the proportion of neutrophils in the lung of chronic obstructive pulmonary disease.
9. A pharmaceutical composition comprising the compound indoleacetic acid and a pharmaceutically acceptable carrier, excipient, adjuvant or combination thereof; the pharmaceutical composition is used for preventing, treating or alleviating chronic obstructive pulmonary disease.
10. The pharmaceutical composition of claim 9, wherein the chronic obstructive pulmonary disease is selected from airway inflammation, emphysema, impairment of pulmonary ventilation and resulting respiratory and critical illness.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003066047A1 (en) * | 2002-02-05 | 2003-08-14 | Astrazeneca Ab | Use of indole-3-acetic acids in the treatment of asthma, copd and other diseases |
| US20050165033A1 (en) * | 2002-02-05 | 2005-07-28 | Andrew Baxter | Use of indole-3-acetic acids in the treatment of asthma, copd and other diseases |
| US20060100425A1 (en) * | 2004-09-21 | 2006-05-11 | Athersys, Inc. | Indole acetic acids exhibiting CRTH2 receptor antagonism and uses thereof |
| WO2008012511A1 (en) * | 2006-07-22 | 2008-01-31 | Oxagen Limited | Compounds having crth2 antagonist activity |
| US20100330077A1 (en) * | 2005-03-11 | 2010-12-30 | Oxagen Limited | 1-acetic acid-indole derivatives with pgd2 antagonist activity |
| CN101970405A (en) * | 2007-12-14 | 2011-02-09 | 普尔马金医疗(哮喘)有限公司 | Indoles and their therapeutic use |
| CN108030786A (en) * | 2017-11-30 | 2018-05-15 | 吉林大学 | Application of the corymbose hedyotis herb B prime in the medicine for preparing treatment Chronic Obstructive Pulmonary Disease |
-
2021
- 2021-08-04 CN CN202110905544.2A patent/CN113425714B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003066047A1 (en) * | 2002-02-05 | 2003-08-14 | Astrazeneca Ab | Use of indole-3-acetic acids in the treatment of asthma, copd and other diseases |
| US20050101612A1 (en) * | 2002-02-05 | 2005-05-12 | Andrew Baxter | Use of indole-3-acetic acids in the treatment of asthma, copd and other diseases |
| US20050165033A1 (en) * | 2002-02-05 | 2005-07-28 | Andrew Baxter | Use of indole-3-acetic acids in the treatment of asthma, copd and other diseases |
| US20060100425A1 (en) * | 2004-09-21 | 2006-05-11 | Athersys, Inc. | Indole acetic acids exhibiting CRTH2 receptor antagonism and uses thereof |
| US20100330077A1 (en) * | 2005-03-11 | 2010-12-30 | Oxagen Limited | 1-acetic acid-indole derivatives with pgd2 antagonist activity |
| WO2008012511A1 (en) * | 2006-07-22 | 2008-01-31 | Oxagen Limited | Compounds having crth2 antagonist activity |
| CN101970405A (en) * | 2007-12-14 | 2011-02-09 | 普尔马金医疗(哮喘)有限公司 | Indoles and their therapeutic use |
| CN108030786A (en) * | 2017-11-30 | 2018-05-15 | 吉林大学 | Application of the corymbose hedyotis herb B prime in the medicine for preparing treatment Chronic Obstructive Pulmonary Disease |
Non-Patent Citations (2)
| Title |
|---|
| ASTRAZENECA AB,ET AL: ""he use of indole-3-acetic acids as CRTH2 receptor antagonists"", 《EXPERT OPINION ON THERAPEUTIC PATENTS》 * |
| RICHARD E. ARMER，ET AL: ""Indole-3-acetic Acid Antagonists of the Prostaglandin D2 Receptor CRTH2"", 《J. MED. CHEM.》 * |
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