Application of daminozide in preparation of medicine for preventing or treating COPD (chronic obstructive pulmonary disease)

Abstract

The invention belongs to the technical field of biological pharmacy, and discloses an application of daminozide in preparation of a medicine for preventing or treating COPD (chronic obstructive pulmonary disease). According to the application disclosed by the invention, the butyrohydrazide inhibits the expression of inflammatory factors IL-6 and IL-8 by recovering the function of airway epithelial cells in lung tissues, so that the COPD process is slowed down, and a new treatment strategy is provided for COPD.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to the application of butyrylhydrazide in preparing medicines for preventing or treating COPD. Background Art

[0002] Daminozide (chemical name: 1,2-naphthylene-1,2-dimethylamine-1-one, butyrylhydrazide) is a plant growth regulator. There is evidence that it can protect against osteoarthritis through inhibition of KDM2 / 7 histone demethylase, but the role of Daminozide in the pathogenesis of COPD is still unclear.

[0003] COPD (Chronic Obstructive Pulmonary Disease) is a group of chronic lung diseases characterized by airflow limitation, mainly including chronic bronchitis and emphysema. COPD is usually caused by airway inflammation and changes in lung structure caused by long-term exposure to harmful substances (especially smoking), which in turn causes long-term damage to the airways and airflow limitation.

[0004] The main characteristics of COPD are as follows: 1) Chronic airway inflammation: COPD often has long-term chronic inflammation in the airways, which leads to thickening of the airway walls and increased mucus secretion, thus affecting normal airflow. 2) Airflow limitation: Due to narrowing of the airways or thickening of the airway walls, it is difficult for air to enter the lungs smoothly, causing patients to have difficulty breathing, especially during physical activities. 3) Decreased lung function: As the disease progresses, the alveoli are damaged and oxygen exchange is reduced, causing patients to have long-term breathing difficulties. Symptoms include: cough, shortness of breath, wheezing, chest tightness, etc. The main pathological characteristics of COPD include airway inflammation, airway remodeling, emphysema, small airway lesions, changes in alveolar wall structure, pulmonary hypertension and oxygenation dysfunction. These pathological characteristics of COPD work together to cause airflow limitation, difficulty breathing, and gradual decline in lung function. The main causes of COPD are smoking, air pollutants, occupational exposure and genetic factors.

[0005] At present, the main treatment for COPD is drug therapy. However, existing drugs cannot reverse the course of COPD, and their clinical treatment effects and safety cannot meet the treatment needs. Studies have shown that in the chronic airway inflammation of COPD, airway epithelial cells play a key role in the pathological development process. Airway epithelial cells play a key role in the chronic airway inflammation of COPD. They are not only a barrier to the respiratory tract, but also can sense external harmful substances (such as smoke, pollutants, etc.) and activate immune responses. Damaged airway epithelial cells recruit inflammatory cells (such as neutrophils and macrophages) to the airways by secreting cytokines, chemokines and mucus, further promoting inflammatory responses and airway remodeling. Long-term inflammatory responses aggravate airway stenosis and airflow limitation, promoting the progression of COPD.

[0006] Therefore, there is an urgent need to conduct in-depth research on the pathogenesis of COPD, find new therapeutic targets, fully combine innovative medical development trends, and develop new drugs that can effectively treat COPD and are highly safe. Summary of the invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide a use of butyric acid hydrazide in the preparation of a drug for preventing or treating COPD, which has good clinical application value.

[0008] In order to achieve the above purpose, this application adopts the following technical solutions: In a first aspect, the present invention provides the use of butyric acid hydrazide in the preparation of a medicament for preventing or treating COPD.

[0009] In the above technical solution, diaminozidine significantly reduces the release of inflammatory factors in airway epithelial cells.

[0010] Daminozide restores the function of airway epithelial cells in lung tissue, inhibits the expression of inflammatory factors IL-6 and IL-8, and thus slows down the progression of COPD, providing a new treatment strategy for COPD.

[0011] In the second aspect, the present invention provides the use of butyric acid hydrazide in the preparation of a drug for restoring decreased lung function, alleviating emphysema, alleviating chronic lung inflammation, and alleviating the secretion of inflammatory factors in airway epithelial cells. The inventors of the present application found in their research that butyric acid hydrazide has a certain restorative effect on decreased lung function, can improve lung inflammatory cell infiltration, reduce emphysema, and can also reduce the secretion of inflammatory factors in airway epithelial cells. It can be used to prepare a drug for restoring decreased lung function, alleviating emphysema, alleviating chronic lung inflammation, and alleviating the secretion of inflammatory factors in airway epithelial cells.

[0012] Decreased lung function, emphysema, chronic lung inflammation, and secretion of inflammatory factors in airway epithelial cells are symptoms of COPD patients. In the new application of daminozide, especially for the decreased lung function, emphysema, chronic lung inflammation, and secretion of inflammatory factors in airway epithelial cells in COPD patients, daminozide has a good recovery and improvement effect on these symptoms caused by COPD.

[0013] In a third aspect, the present invention provides a drug for preventing or treating COPD, which is composed of diaminobenzidine at an effective dose, and can be a single component or a composition containing an effective dose of diaminobenzidine, and the composition can include a pharmaceutically acceptable carrier.

[0014] In the present invention, diaminozidine can be prepared together with a pharmaceutically acceptable carrier into a drug for preventing or treating COPD. The drug can be prepared into various dosage forms in the art, including but not limited to injections, tablets, capsules, and the like.

[0015] The beneficial effects of the present invention are as follows: the present invention discovers a new application of dihydrochloride in the prevention or treatment of COPD, dihydrochloride can partially restore decreased lung function, emphysema, chronic lung inflammation, and secretion of inflammatory factors in airway epithelial cells, and can be used to prevent or treat COPD, solving the problem that there is currently no small molecule metabolite treatment for COPD, and is expected to significantly reduce the mortality rate of COPD patients, which has important clinical prevention and treatment significance. Dihydrochloride can be combined with other active ingredients or excipients to prepare a drug for the prevention or treatment of COPD, providing a new treatment option for the treatment of COPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram showing the lung function results of mice lung tissue treated with intraperitoneal injection of Daminozide and saline after cigarette smoke exposure modeling. , ;in, Figure 1 a is the change of FEV0.05 index in lung function of mice in different treatment groups. Figure 1 b shows the changes in FEV0.05 / FVC index in lung function of mice in different treatment groups.

[0017] Figure 2 The MLI score and inflammatory infiltration diagram of emphysema index in the above mouse model are shown in Figure 1. , ;in, Figure 2 a is the representative picture and statistical bar chart of MLI. Figure 2 b is a representative picture and statistical bar graph of inflammatory infiltration.

[0018] Figure 3The figure is a schematic diagram of the inflammatory cell count results of BALF slides in the above mouse model and the detection of inflammatory factors IL-6, KC, and IL-1β in BALF. , , ;in, Figure 3 a is a schematic diagram of the inflammatory cell count results of BALF slides. Figure 3 b is a schematic diagram of IL-6 detection, Figure 3 c is a schematic diagram of IL-1β detection, Figure 3 d is a schematic diagram of KC detection; Figure 4 This is a schematic diagram of the results of detecting cell activity by treating human airway epithelial cells with different concentrations of Daminozide, where: ; Figure 5 To detect the changes in the transcriptional levels of intracellular inflammatory factors after pre-treating human airway epithelial cells with different concentrations of Daminozide for 1 hour and stimulating the cells with cigarette smoke extract for 24 hours. Figure 5 a is a bar graph of the relative expression of IL-6, Figure 5 b is a bar graph of the relative expression of IL-8, where , , .

[0019] Figure 6 This is a bar graph showing the expression of inflammatory factors in the cell supernatant after human airway epithelial cells were treated with Daminozide for 1 hour and then stimulated with cigarette smoke extract for 24 hours. Figure 6 a is the bar graph of IL-6, Figure 6 b is the bar graph of IL-8, where , . DETAILED DESCRIPTION

[0020] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0021] The "treatment" mentioned in the present invention refers to reducing the degree of chronic inflammation, or curing chronic inflammation to normalize it, or slowing down the progress of chronic inflammation.

[0022] The present invention verifies through the following examples that Daminozide (butyrylhydrazide) can significantly reduce the release of inflammatory factors in airway epithelial cells. The pro-inflammatory effect of airway epithelial cells plays an extremely important role in the pathogenesis of COPD.

[0023] In order to better explain the present invention, it is described in detail below in conjunction with specific embodiments.

[0024] Example 1 Effects of intraperitoneal injection of Daminozide on chronic obstructive pulmonary disease Experimental animals and materials: 1. Experimental animals: Source, strain, and line: Wild-type mice (WT, C57BL / 6) were ordered from the Hubei Branch of Bainte Experimental Animal Technology Co., Ltd.; Reproductive age: 8-10 weeks; 2. Experimental materials: Cigarettes: purchased from Hongjinlong Co., Ltd.; Physiological saline: purchased from Wuhan Promoter Biological Co., Ltd.; Daminozide: purchased from MedChemExpress.

[0025] 3. Experimental methods: 1) Order 15 wild-type C57 male mice and prepare the items required for modeling, including cigarette smoke exposure device, cigarettes, etc.; 2) Grouping: air control group, fumigation group and cigarette smoke exposure Daminozide administration group, 5 mice in each group; 3) Smoke modeling: The mice in the smoked group were placed in a cigarette smoke exposure device for 6 months of passive smoking modeling, 6 days a week, smoke exposure for about 3 hours a day, divided into 4 times, 10 to 12 cigarettes each time for 45 minutes. In the last month, the mice in the Daminozide treatment group were given intraperitoneal injections every other day at a dose of 10 mg / kg.

[0026] 4) After the model was established, the mice were weighed and anesthetized with 1% sodium pentobarbital injected intraperitoneally at a standard of 100 μL / 10 g; 5) After the mouse is anesthetized, tape is fixed to the foam board and the maxillary teeth are fixed with a rubber band to fully expose the neck; 6) Cut the skin on the neck and use forceps to bluntly separate the subcutaneous tissue and the soft tissue around the trachea. Place a suture of appropriate length under the trachea, use ophthalmic scissors to cut a small hole in the trachea horizontally, insert the ventilation tracheal intubation needle into the trachea, and fix the intubation needle to the trachea with sutures; 7) Turn on the pulmonary function ventilation mode, fix the mouse on the pulmonary function test board, and connect the endotracheal intubation needle to the instrument. Then, enter the mouse weight and number, click the corresponding test option, export the pulmonary function data and save it for later use.

[0027] 4. Experimental results: Specifically, the lung function results of mice are as follows Figure 1 As shown, combined Figure 1 It can be seen that after exposure to cigarette smoke, the downregulation of lung function in mice treated with intraperitoneal injection of Daminozide was significantly alleviated compared with the cigarette smoke exposure group.

[0028] Example 2 In order to further evaluate the degree of lung injury and inflammatory cell infiltration in each mouse after exposure to cigarette smoke, the present invention detected the level of lung injury and inflammatory cell infiltration in the lung tissue of each mouse by HE staining.

[0029] Specifically, the mouse lung tissues after the experiment in Example 1 were collected, fixed with paraformaldehyde for 48 hours, embedded in paraffin, and sliced ​​for HE staining.

[0030] The HE staining steps are as follows: The entire staining process includes five steps: dewaxing, staining, dehydration, transparency and sealing.

[0031] 1. Dewaxing: 1) Take out the dried slices from the incubator and immediately put them into xylene for dewaxing for 5-10 minutes (can be done in two bottles). The dewaxing time depends on whether the wax is completely dissolved. If the temperature is low, the time can be extended. If the temperature is high, the time can be appropriately shortened or the dewaxing can be accelerated in the incubator.

[0032] 2) Transfer into anhydrous alcohol (100%) (two bottles) for about 2 minutes.

[0033] 3) Transfer to 90% alcohol (two bottles) for about 2 minutes.

[0034] 4) Transfer to 80% alcohol (two bottles) for about 2 minutes.

[0035] 5) Transfer to 70% alcohol for about 2 minutes.

[0036] 6) Move into water and wash off the alcohol for about 2 to 3 minutes.

[0037] 7) Transfer to distilled water for about 2 minutes.

[0038] 2. Dyeing: 1) Transfer to hematoxylin and stain for 8 to 15 minutes. Generally, a slightly darker stain is appropriate.

[0039] 2) Transfer to water and wash off hematoxylin and floating color for about 1 to 2 minutes.

[0040] 3) Move to the differentiation solution (1% hydrochloric acid alcohol) and differentiate for a few seconds to 30 seconds until the slice fades to a light blue-red color. Differentiation can remove the blue color of the cytoplasm and make the nucleus clearer and more vivid. When the differentiation is insufficient, the cytoplasm is blue and the nucleus is overstained. When the differentiation is excessive, the nucleus is too light and difficult to identify. You can return to the hematoxylin staining solution and extend the staining time.

[0041] 4) Move into running water and wash for 30 to 60 minutes until the tissue turns bright blue or sky blue (bluish).

[0042] 5) Move into eosin solution and soak for 2-5 minutes. If dyeing is slow, add glacial acetic acid to the eosin solution (add 1-2 drops of glacial acetic acid to 100 ml of eosin solution) to aid dyeing.

[0043] 6) Move the slide into water, wash off the eosin suspension, and wipe off the excess dye on the slide with gauze.

[0044] 3. Dehydration: 1) After absorbing the water on the slide, immerse it in 80% alcohol (two bottles) for dehydration for about 1 to 2 minutes. If the slide fades quickly in alcohol, quickly move it to 90% alcohol or return it to eosin solution for re-staining.

[0045] 2) Transfer to 90% alcohol (two bottles) for dehydration for about 2 to 4 minutes.

[0046] 3) Transfer to anhydrous alcohol (100% alcohol) (two bottles) and dehydrate thoroughly for about 4 to 8 minutes.

[0047] 4. Transparency: 1) Transfer to xylene I and let it become transparent for 3 to 5 minutes.

[0048] 2) Transfer to xylene II and let it become transparent for 5 to 10 minutes.

[0049] 5. Sealing: To seal with gum, first take the slice out of xylene II, quickly wipe off the xylene on the outside of the tissue, then drop a drop of gum on the tissue slice, then take a clean cover glass, carefully add it to the sealing agent, and slowly flatten it so that the cover glass is in the right position. After the slice is sealed, place it in an incubator to dry, or place it flat to dry before packing it in a box.

[0050] 6. Shooting and evaluation: The slides were placed under an Olympus microscope and photographed at a magnification of 200x.

[0051] Combination Figure 1 and Figure 2It can be seen that compared with wild-type mice, the mice administered Daminozide by intraperitoneal injection had a lower emphysema level index MLI and a decreased inflammatory cell infiltration, which indicates that Daminozide can significantly reduce the degree of alveolar damage and chronic inflammation in mice.

[0052] Example 3 In order to further evaluate the degree of inflammatory cell infiltration in each mouse after exposure to cigarette smoke, the present invention counted the inflammatory cells in the BALF of the mice and detected the levels of IL-6, KC, and IL-1β in the BALF by ELISA.

[0053] Specifically, after the mouse completes the lung function test, the abdominal cavity is opened with scissors, the inferior vena cava is found and cut off; after the inferior vena cava is cut, the chest cavity is slowly opened, the mouse heart is fully exposed, and the right ventricle is found. Subsequently, a 10 mL syringe is used to draw about 4 mL of normal saline, which is slowly injected into the right ventricle to fully wash away the blood in the pulmonary circulation; a 1 mL syringe is used to draw normal saline and inject it into the endotracheal intubation needle for 1 min, and then back-drawn to collect bronchoalveolar lavage fluid. The specific method is to draw 700 μL of normal saline for the first time, 800 μL of normal saline for the second and third times, and the second and third recovered liquids are placed in the same EP tube; the above EP tube is centrifuged at 980 rpm for 8 min. Subsequently, the supernatant of the first tube is collected and stored at -80°C for subsequent ELISA testing. Then, discard the supernatant of the second tube, use red blood cell lysis buffer to lyse the red blood cells in the first and second tubes, centrifuge after termination with equal volumes of PBS, and add 100-200 μL of culture medium to resuspend; count the resuspended cells under a microscope, and adjust the cell suspension to an appropriate density based on the counting results. Then, fix the slide with a swinging clip, mark the slide with a number, add 80 μL of cell suspension, and use a swinging machine to swing the slide at a speed of 980 rpm for 5 min; Take out the A and B solutions of Liu's staining, use a rubber-tipped dropper to drip 3 drops of A solution for about 3-4 seconds and 10 drops of B solution for about 10 seconds on the swung slide, and quickly rinse under running water for 20-30 seconds, dry the liquid with absorbent paper, and then seal the slide with neutral resin; randomly collect at least 5 fields of view of the slide stained with Liu's staining solution under an ordinary optical microscope and save them. Subsequently, distinguish the macrophages, neutrophils and lymphocytes in the field of view, calculate their proportions, and combine them with the cell counting results to calculate the number of various types of cells in the mouse bronchoalveolar lavage fluid.

[0054] For the detection of inflammatory factors in BALF, after centrifuging BALF, the supernatant was taken and the expression of inflammatory factors was detected using the kits of IL-6, KC and IL-1β. According to the ELISA instructions of mouse IL-6, IL-8 and IL-1β, the standard, capture antibody and detection antibody dry powder reagents were dissolved and packaged in turn; one day before the experiment, the capture antibody of the three antibodies was diluted to the working solution concentration using coating buffer solution, and 100 μL of working solution was added to each well of the 96-well ELISA plate. After sealing the plate with a sealing film, the plate was centrifuged instantly and placed on a 37°C constant temperature shaker at 70 revolutions per minute for overnight incubation; before the experiment on the second day, the auxiliary kit was taken out in advance to restore to room temperature, and 1x washing solution and RD blocking solution were prepared. Take out the coated ELISA plate from the constant temperature shaker, discard the capture antibody solution in the well, and add 300 μL of washing solution in turn with a spray gun, gently tap the side wall for about 2 minutes to fully wash, and repeat the washing work 3 times. Then add 300 μL of blocking solution to each well, stick the sealing film, and incubate at room temperature for 1 h; take out the storage solution of the standard (concentration of 180 μg / mL) and return to room temperature, and prepare 8 1.5 mL EP tubes and 1x Assay Diluent diluent for multiple dilution; take out the supernatant sample to be tested in advance and dilute it appropriately. After the blocking is completed, discard the blocking solution and wash the plate wells 3 times with the washing solution, the method is the same as before. Then, add 100 μL of the standard and the sample to be tested to the ELISA plate in turn, cover the sealing film, and incubate at room temperature for 2 h; take out the detection antibody in advance to thaw and dilute it to the working solution concentration. After the primary antibody is bound, discard the liquid in the well and wash it 3 times with the washing solution, the method is the same as before. Then add 100 μL of the diluted detection antibody to each well, gently tap the side wall to mix, cover the sealing film, and incubate at room temperature for 2 h; then take out the 40x HRP liquid of the corresponding indicator and dilute it to the working solution concentration of 1x. After the incubation of the detection antibody is completed, the liquid is discarded and the plate is washed 3 times with the washing solution. Subsequently, 100 μL of HRP working solution is quickly added to the wells using a discharge gun, and incubated at room temperature in the dark for 20 minutes; the substrate liquid is prepared in a sufficient amount of substrate reaction solution at a ratio of A:B=1:1. After the HRP incubation is completed, the ELISA plate is washed 3 times with the washing solution, and then 100 μL of substrate reaction solution is quickly added to the wells using a discharge gun, and incubated at room temperature in the dark for 20 minutes; after the substrate reaction solution is incubated for 20 minutes, the color change of each well is observed, and the plate is read at a wavelength of 630 nm using an enzyme reader. When the optical density value (OD) of the first well of the standard is between 0.7 and 1.0, 50 μL of the stop solution is added to each well; After adding the stop solution, gently tap the side wall to mix thoroughly, and use a microplate reader to read the plate at a wavelength of 450 nm to obtain the OD value of each well. Draw a standard curve and curve function based on the OD value and corresponding concentration of the standard, and substitute the OD value of each well to calculate the concentration of each sample.

[0055] Figure 1 It can be seen that the number of macrophages and neutrophils in the bronchoalveolar lavage fluid BALF of mice treated with intraperitoneal injection of Daminozide was reduced compared with the group exposed to cigarette smoke alone. This result indicates that intraperitoneal injection of Daminozide can inhibit the infiltration of inflammatory cells in lung tissue. Figure 2-4 It can be seen that the expression of inflammatory factors IL-6, KC and IL-1β in the alveolar lavage fluid BALF of mice administered intraperitoneal injection of Daminozide was significantly decreased, which indicates that intraperitoneal injection of Daminozide can inhibit the secretion of inflammatory factors in the lungs of mice.

[0056] Example 4 Evidence shows that dysfunction of airway epithelial cells promotes the occurrence of chronic airway inflammation, which is necessary for maintaining COPD. Therefore, in order to further evaluate the effect of Daminozide on the secretion of inflammatory factors in airway epithelial cells stimulated by cigarette smoke extract, the present invention firstly uses the CCK8 method to find the optimal dosing concentration of Daminozide. The detailed steps of CCK8 are as follows: 1) Prepare cell suspension and count cells.

[0057] 2) Inoculate the cell suspension in a 96-well plate, about 100 μl per well. The same sample can be repeated 3 times. 3) Place the culture plate in an incubator for pre-incubation for a period of time (37°C, 5% CO2).

[0058] 4) Add different concentrations of Daminozide to each well of the culture plate.

[0059] 5) Place the culture plate in an incubator and incubate for 24 hours.

[0060] 6) Add 10 μl of CCK-8 solution to each well and gently shake the plate to help mix. Try not to create bubbles during the addition process to avoid affecting the OD value reading.

[0061] 7) Place the culture plate in the incubator and incubate for 1 hour.

[0062] 8) Measure the absorbance (OD) at 450nm using an ELISA reader. Calculate cell viability using the following formula: Viability calculation: Cell viability (%) = [A(drug added) - A(blank)] / [A(control) - A(blank)] × 100 The protein and mRNA levels of IL-6, KC and IL-1β in human airway epithelial cells were detected by ELISA and RT-PCR, respectively. The ELISA method has been described in detail in Example 3, and the RT-PCR method is described in detail below: 1) RNA extraction and reverse transcription Use TRZIOL to add to the cell culture plate, blow and then absorb, add to different EP tubes, mark them, pre-cool the chloroform in the 4℃ refrigerator in advance, and add chloroform at a ratio of chloroform: RNAiso Plus lysis solution = 1:5, then shake the EP tube vigorously for about 30 seconds to fully shake the reagent. Then place the EP tube on ice for 15 minutes, and three layers can be seen; pre-cool the low-temperature high-speed centrifuge, set the parameters to 4℃, 12,000 rpm, 15 min, centrifuge the above EP tube, and after the centrifugation is completed, three layers can be seen, and the top transparent layer is the required RNA liquid. Then, use a sterile pipette tip to absorb the upper layer of liquid into a new enzyme-free EP tube, add an equal volume of isopropanol, gently turn the EP tube upside down and mix it about 20 times, and then place it on ice for 30 minutes to fully precipitate; after sufficient precipitation, place the above EP tube in a centrifuge with parameters of 12,000 rpm and 15 minutes for centrifugation. At the same time, prepare a 75% ethanol solution, that is, 10 mL of DEPC water and 30 mL of anhydrous ethanol, mix them well, and screw the bottle cap to prevent evaporation. After the centrifugation is completed, white precipitates can be seen at the bottom of the tube. Pour out the liquid and tap dry on a clean paper towel. Then draw 1 mL of the prepared 75% ethanol solution into the tube, and turn the EP tube upside down to fully wash the RNA, based on the floating precipitate visible to the naked eye, and then let it stand on ice for 10 min. Repeat the ethanol washing step again and wait for the next step; After completing the washing step, use a low-temperature high-speed centrifuge to centrifuge at 4°C and 12,000 rpm for 15 min. Then, pour out the liquid, tap dry on a clean paper towel, and wipe the tube wall with a sterile cotton swab. Then place the EP tube on ice for 10-15 min to air dry naturally, and then add 40 μL of DEPC water to the tube to fully dissolve the RNA precipitate. Use clean absorbent paper to dry the probe, mix the RNA in each tube, and then aspirate 1 μL to measure the concentration, and enter the corresponding sample number in the computer.

[0063] According to the concentration of sample RNA, calculate the volume of the corresponding RNA, DEPC water and 5x Master Mix solution in the reverse transcription system, gently tap the tube wall to mix, and then centrifuge instantly. For example, in a 10 μL reverse transcription system, add the volume corresponding to 500 ng RNA, 2 μL 5x Master Mix solution, and use DEPC water to make up the total volume to 10 μL.

[0064] Place the reverse transcription system prepared in the previous step in a common PCR instrument for reverse transcription. The specific reverse transcription procedure is shown in the table below; Table 1 Reverse transcription procedure 2) Real-time fluorescence quantitative PCR Prepare the reaction system according to the ratio in the table below; Table 2 Amplification reaction system 3) Real-time fluorescence quantitative PCR Connect and turn on the CFX Connect PCR instrument, put in the prepared reaction system and proceed as follows. After the program is completed, calculate the relative expression of RNA in the sample based on the CT value.

[0065] Table 3 Fluorescence quantitative PCR reaction procedure Among them, the primers corresponding to each target gene are listed in the table below.

[0066] Table 4 List of primers corresponding to target genes After stimulating human airway epithelial cells with different concentrations of Daminozide for 24 hours, the results of CCK8 showed that administration at a concentration below 8uM would not have a significant effect on cell activity, such as Figure 4 shown.

[0067] Next, we pretreated the airway epithelial cells with different concentrations of Daminozide for 1 hour, and then stimulated them with cigarette smoke extract for 24 hours. The results of RT-PCR detection of IL-6 and IL-8 showed that after treatment with 2uM and 4uM concentrations of Daminozide, the transcription levels of IL-6 and IL-8 in airway epithelial cells were reduced. Figure 5 shown.

[0068] We further detected the expression of IL-6 and IL-8 in the cell supernatant after 1 hour of pretreatment of airway epithelial cells with 4uM Daminozide and 24 hours of stimulation with cigarette smoke extract. The results showed that the expression of IL-6 and IL-8 in the cell supernatant was reduced. Figure 6 shown.

[0069] In summary, the above experimental data indicate that Daminozide can slow down the progression of COPD by inhibiting the secretion of inflammatory factors in airway epithelial cells.

[0070] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of butyric acid hydrazide in the preparation of drugs for preventing or treating COPD.

2. Use of butyric acid hydrazide in the preparation of a drug for restoring decreased lung function, alleviating emphysema, or alleviating chronic lung inflammation.

3. Use of a drug in the preparation of a drug for preventing or treating COPD, characterized in that: The drug is composed of only an effective dose of diaminobenzidine or a composition containing an effective dose of diaminobenzidine, wherein the composition includes a pharmaceutically acceptable carrier.

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