Application of daminozide in the preparation of a medicament for preventing or treating COPD

Abstract

The present invention belongs to the technical field of biopharmaceuticals, and discloses the application of daminozide in the preparation of a drug for preventing or treating COPD. Daminozide slows down the progression of COPD by restoring the function of airway epithelial cells in lung tissue and inhibiting the expression of inflammatory factors IL-6 and IL-8, providing a new treatment strategy for COPD.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to the use of daminozide in the preparation of a drug for preventing or treating COPD. Background Art

[0002] Daminozide (chemical name: 1,2-naphthalene-1,2-dimethylamine-1-one, daminozide) is a plant growth regulator. There is evidence that it can protect against osteoarthritis through the inhibition of KDM2 / 7 histone demethylase, but it is not clear what role daminozide plays in the pathogenesis of COPD.

[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 resulting from long-term exposure to harmful substances (especially smoking), which in turn cause long-term damage to the airways and airflow limitation.

[0004] The main characteristics of COPD are as follows: 1) Chronic airway inflammation: There is often long-term chronic inflammation in the airways of COPD, leading to thickening of the airway wall and increased mucus secretion, thus affecting normal airflow. 2) Airflow limitation: Due to narrowing of the airways or thickening of the airway wall, air has difficulty entering the lungs smoothly, resulting in difficulty breathing for patients, especially during physical activity. 3) Decline in lung function: As the disease progresses, the alveoli are damaged and oxygen exchange decreases, leading to long-term difficulty breathing for patients. Symptoms include cough, shortness of breath, wheezing, chest tightness, etc. The main pathological features of COPD include airway inflammation, airway remodeling, emphysema, small airway lesions, changes in the structure of the alveolar wall, pulmonary hypertension, and oxygenation dysfunction. These pathological features of COPD act 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] Currently, 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 requirements. Research shows that in the chronic airway inflammation of COPD, airway epithelial cells play a key role in the pathological development process. They are not only the barrier of the respiratory tract but also can sense external harmful substances (such as smoke, pollutants, etc.) and activate the immune response. Damaged airway epithelial cells recruit inflammatory cells (such as neutrophils and macrophages) to the airway by secreting cytokines, chemokines, and mucus, further promoting the inflammatory response and airway remodeling. The long-term inflammatory response exacerbates airway stenosis and airflow limitation, driving the progression of COPD.

[0006] Therefore, it is urgent to deeply study the pathogenesis of COPD, find new treatment targets, fully combine the innovative pharmaceutical development trend, and develop new drugs that can effectively treat COPD and have high safety. Summary of the Invention

[0007] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide the application of daminozide in the preparation of drugs for preventing or treating COPD, which has good clinical application value.

[0008] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0009] In the first aspect, the present invention provides the application of daminozide in the preparation of drugs for preventing or treating COPD.

[0010] In the above technical solution, daminozide significantly reduces the release of inflammatory factors in airway epithelial cells.

[0011] Daminozide inhibits the expression of inflammatory factors IL-6 and IL-8 by restoring the function of airway epithelial cells in lung tissue, thereby slowing down the process of COPD and providing a new treatment strategy for COPD.

[0012] In the second aspect, the present invention provides the application of daminozide in the preparation of drugs for any one of restoring decreased lung function, reducing emphysema, reducing chronic lung inflammation, and reducing the secretion of inflammatory factors in airway epithelial cells. The inventors of the present application found in the research that daminozide has a certain effect on restoring decreased lung function, can improve the infiltration of inflammatory cells in the lungs, reduce emphysema, and can also reduce the secretion of inflammatory factors in airway epithelial cells, and can be used to prepare drugs for any one of restoring decreased lung function, reducing emphysema, reducing chronic lung inflammation, and reducing the secretion of inflammatory factors in airway epithelial cells.

[0013] Decline in lung function, emphysema, chronic pulmonary inflammation, and secretion of inflammatory factors by airway epithelial cells are symptoms of COPD patients. In the new application of daminozide, especially for the decline in lung function, emphysema, chronic pulmonary inflammation, and secretion of inflammatory factors by airway epithelial cells in COPD patients, daminozide has a good restorative and ameliorative effect on these symptoms caused by COPD.

[0014] In a third aspect, the present invention provides a drug for preventing or treating COPD, which consists of daminozide at an effective dose. It can be a single component or a composition containing an effective dose of daminozide, and the composition may include a pharmaceutically acceptable carrier.

[0015] In the present invention, daminozide can be jointly prepared 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, etc.

[0016] The beneficial effects of the present invention are as follows: The present invention discovers a new application of daminozide in preventing or treating COPD. Daminozide can partially restore the decline in lung function, emphysema, chronic pulmonary inflammation, and secretion of inflammatory factors by airway epithelial cells, and can be used to prevent or treat COPD, solving the problem that there is currently no small molecule metabolite treatment method for COPD, and is expected to significantly reduce the mortality rate of COPD patients, having important clinical preventive and therapeutic significance. Daminozide can be combined with other active ingredients or excipients, etc., to prepare a drug for preventing or treating COPD, providing a new treatment option for the treatment of COPD. Description of the Drawings

[0017] Figure 1 It is a graph of the lung function results of the lung tissues of mice after cigarette smoke exposure modeling, treated with intraperitoneal injection of daminozide and intraperitoneal injection of normal saline, where, , ; where, Figure 1 a shows the change in the FEV0.05 index in the lung function of mice in different treatment groups, Figure 1 b shows the change in the FEV0.05 / FVC index in the lung function of mice in different treatment groups.

[0018] Figure 2 It is a schematic diagram of the MLI score and inflammatory infiltration of the emphysema index in the above mouse model, where, , ; where, Figure 2 a is a representative picture and statistical bar chart of MLI, Figure 2 b is a representative picture and statistical bar chart of inflammatory infiltration.

[0019] Figure 3Schematic diagram of the inflammatory cell count in BALF smear and detection of inflammatory factors IL-6, KC, and IL-1β in BALF in the above mouse model. Among them, , , ; among them, Figure 3 a is the schematic diagram of the inflammatory cell count in BALF smear, Figure 3 b is the schematic diagram of IL-6 detection, Figure 3 c is the schematic diagram of IL-1β detection, Figure 3 d is the schematic diagram of KC detection;

[0020] Figure 4 Schematic diagram of the results of detecting cell viability after treating human airway epithelial cells with different concentrations of Daminozide. Among them, ;

[0021] Figure 5 After pre-treating human airway epithelial cells with different concentrations of Daminozide for 1 hour and then stimulating the cells with cigarette smoke extract for 24 hours, the change in the transcriptional level of intracellular inflammatory factors was detected. Figure 5 a is the bar chart of the relative expression level of IL-6, Figure 5 b is the bar chart of the relative expression level of IL-8. Among them, , , .

[0022] Figure 6 Bar chart of the expression of inflammatory factors in the cell supernatant after treating human airway epithelial cells with Daminozide for 1 hour and then stimulating the cells with cigarette smoke extract for 24 hours. Figure 6 a is the bar chart of IL-6, Figure 6 b is the bar chart of IL-8. Among them, , . Detailed implementation methods

[0023] To better illustrate the purpose, technical solution, 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 limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the present invention to those skilled in the art. The present invention will only be defined by the claims.

[0024] As used herein, "treatment" refers to reducing the degree of chronic inflammation, or curing chronic inflammation to make it normal, or slowing down the process of chronic inflammation.

[0025] The present invention has confirmed through the following examples that Daminozide 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.

[0026] To better explain the present invention, the following is a detailed description in combination with specific examples.

[0027] Example 1 Effects of intraperitoneal injection of Daminozide on chronic obstructive pulmonary disease

[0028] Experimental animals and materials:

[0029] 1. Experimental animals:

[0030] Source, strain, and line: Wild-type mice (WT, C57BL / 6) ordered from Hubei Branch of Bernt Experimental Animal Technology Co., Ltd.;

[0031] Reproductive age: 8 - 10 weeks old;

[0032] 2. Experimental materials:

[0033] Cigarettes: Purchased from Hongjinlong Co., Ltd.;

[0034] Normal saline: Purchased from Wuhan Promoter Biotechnology Co., Ltd.;

[0035] Daminozide: Purchased from MedChemExpress.

[0036] 3. Experimental methods:

[0037] 1) Order 15 wild-type C57 male mice and prepare the items required for modeling, including a cigarette smoke exposure device, cigarettes, etc.;

[0038] 2) Grouping: Air control group, smoking group, and cigarette smoke exposure Daminozide administration group, with 5 mice in each group;

[0039] 3) Smoking modeling: Place the mice in the smoking group in the cigarette smoke exposure device and conduct passive smoking modeling for 6 months, 6 days a week, with about 3 h of smoke exposure per day, divided into 4 times, with 10 - 12 cigarettes each time for 45 min of modeling. In the last month, the mice in the Daminozide treatment group were given intraperitoneal injection every other day, with a dose of 10 mg / kg.

[0040] 4) After the modeling is completed, after weighing the mice, according to the standard of 100 μL / 10 g, use 1% sodium pentobarbital to anesthetize the mice by intraperitoneal injection;

[0041] 5) After the mice were anesthetized, they were fixed on a foam board with adhesive tape, and the upper jaw teeth were fixed with a rubber band to fully expose the neck;

[0042] 6) Cut open the skin of 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 an ophthalmic scissors to make a small horizontal cut on the trachea, insert the tracheal intubation needle for ventilation into the trachea, and fix the intubation needle to the trachea with a suture;

[0043] 7) Turn on the lung function ventilation mode, fix the mice on the lung function detection board, and connect the tracheal intubation needle to the instrument. Subsequently, input the body weight and number of the mice, click the corresponding detection options, export the lung function data and save it for later use.

[0044] 4. Experimental results:

[0045] Specifically, the lung function results of the mice are as Figure 1 shown. Combining Figure 1 it can be seen that after exposure to cigarette smoke, compared with the cigarette smoke exposure group, the down-regulation of lung function in the mice treated with intraperitoneal injection of Daminozide was significantly alleviated.

[0046] Example 2

[0047] To further evaluate the lung injury and the degree of inflammatory cell infiltration in each mouse after exposure to cigarette smoke, the present invention detected the lung injury level and the inflammatory cell infiltration level in the lung tissues of each mouse by HE staining.

[0048] Specifically, the lung tissues of the mice after the experiment in Example 1 were collected, fixed with paraformaldehyde for 48 hours, embedded in paraffin after that, and then sliced and subjected to HE staining.

[0049] The steps of HE staining are as follows: The whole staining process includes five contents: dewaxing, staining, dehydration, clearing and mounting.

[0050] 1. Dewaxing:

[0051] 1) Take out the dried sections from the incubator and immediately immerse them in xylene for dewaxing for 5 - 10 min (this can be carried out 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 dewaxing can be accelerated in the incubator.

[0052] 2) Transfer them into absolute ethanol (100%) (two bottles) for about 2 min.

[0053] 3) Transfer them into 90% ethanol (two bottles) for about 2 min.

[0054] 4) Transfer them into 80% ethanol (two bottles) for about 2 min.

[0055] 5) Transfer it into 70% alcohol for about 2 minutes.

[0056] 6) Transfer it into water to wash away the alcohol for about 2 - 3 minutes.

[0057] 7) Transfer it into distilled water for about 2 minutes.

[0058] 2. Staining:

[0059] 1) Transfer it into hematoxylin and stain for 8 - 15 minutes. Generally, it is better to stain slightly darker.

[0060] 2) Transfer it into water to wash away the hematoxylin and floating color for about 1 - 2 minutes.

[0061] 3) Transfer it into the differentiating solution (1% hydrochloric acid alcohol) and differentiate for several seconds to 30 seconds until the section fades to light blue - red. The function of differentiation is to decolorize the cytoplasm and make the cell nucleus clearer and more vivid. When differentiation is insufficient, the cytoplasm is blue - stained and the cell nucleus is over - stained. When differentiation is excessive, the cell nucleus is too light to be recognized, and it can be returned to the hematoxylin staining solution for a certain period of time.

[0062] 4) Transfer it into running water and wash for 30 - 60 minutes to make the tissue show bright blue or sky - blue (blueing).

[0063] 5) Transfer it into eosin solution and stain for 2 - 5 minutes. If the staining is slow, glacial acetic acid can be added to the eosin solution (1 - 2 drops of glacial acetic acid are added to 100 ml of eosin solution) to assist staining.

[0064] 6) Transfer it into water to wash away the floating eosin solution and wipe off the excess dye on the glass slide with gauze.

[0065] 3. Dehydration:

[0066] 1) After sucking off the water on the glass slide, transfer it into 80% alcohol (two bottles) for dehydration for about 1 - 2 minutes. If the color fades quickly in alcohol, it can be quickly transferred into 90% alcohol or returned to the eosin solution for restaining.

[0067] 2) Transfer it into 90% alcohol (two bottles) for dehydration for about 2 - 4 minutes.

[0068] 3) Transfer it into absolute alcohol (100% alcohol) (two bottles) for thorough dehydration for about 4 - 8 minutes.

[0069] 4. Clearing:

[0070] 1) Transfer it into xylene I for clearing for 3 - 5 minutes.

[0071] 2) Transfer it into xylene II for clearing for 5 - 10 minutes.

[0072] 5. Mounting:

[0073] Seal with gum. First, take out the sections from xylene II, quickly wipe off the xylene around the tissue, drop a drop of gum on the tissue section, then take a clean cover glass and carefully place it on the mounting medium, gently flatten it to make the cover glass in the appropriate position. After the sections are sealed, bake them in an incubator or let them dry flat and then put them in a box.

[0074] 6. Photographing and evaluation:

[0075] Place the slides under an Olympus microscope and take pictures at a magnification of 200x. The pictures are

[0076] Combined with Figure 1 and Figure 2 It can be seen that compared with wild-type mice, in the mice administered with Daminozide by intraperitoneal injection, the index of emphysema level MLI decreases, and the infiltration of inflammatory cells decreases, indicating that Daminozide can significantly reduce the degree of alveolar damage and chronic inflammation in mice.

[0077] Example 3

[0078] To further evaluate the degree of infiltration of inflammatory cells in each mouse after exposure to cigarette smoke, the present invention counts the inflammatory cells in the BALF of mice and detects the levels of IL-6, KC, and IL-1β in the BALF by ELISA.

[0079] Specifically, after the mice complete the pulmonary function test, then use scissors to open the abdominal cavity, find the inferior vena cava and cut it off; after cutting the inferior vena cava, slowly open the thoracic cavity and fully expose the mouse heart to find the position of the right ventricle. Subsequently, use a 10 mL syringe to aspirate about 4 mL of normal saline and slowly inject it into the right ventricle to fully wash the blood in the pulmonary circulation; use a 1 mL syringe to aspirate normal saline and inject it into the tracheal intubation needle for 1 min, and then aspirate back to collect the bronchoalveolar lavage fluid. The specific method is to aspirate 700 μL of normal saline for the first time, aspirate 800 μL of normal saline for the second and third times, and place the liquid recovered in the second and third times in the same EP tube; centrifuge the above EP tube with parameters of 980 rpm for 8 min. Subsequently, collect the supernatant of the first tube and store it at -80 °C for subsequent ELISA detection. Subsequently, discard the supernatant of the second tube, lyse the red blood cells in the first and second tubes with red blood cell lysate, centrifuge after terminating with an equal volume of PBS, and resuspend with 100 - 200 μL of medium; count the resuspended cells using a microscope and adjust the cell suspension to an appropriate density according to the counting results. Subsequently, fix the glass slide with a spinner clip, label the number on the glass slide, add 80 μL of the cell suspension, and spin the slide at a speed of 980 rpm for 5 min using a spinner;

[0080] Take out Solution A and Solution B of Liu's stain. Use a dropper to sequentially add 3 drops of Solution A to the well-spun slide for staining for about 3 - 4 s and 10 drops of Solution B for staining for about 10 s, and quickly rinse under running water for 20 - 30 s. After blotting the liquid with absorbent paper, perform neutral resin mounting; Randomly collect at least 5 fields of view of the slide stained with Liu's stain under an ordinary optical microscope and save them. Subsequently, distinguish macrophages, neutrophils, and lymphocytes in the fields of view, calculate their proportions, and combine them with the cell count results to calculate the number of various types of cells in the bronchoalveolar lavage fluid of mice.

[0081] For the detection of inflammatory factors in BALF, after centrifuging the BALF, the supernatant was taken, and the expression levels of inflammatory factors in it were detected using kits for IL-6, KC, and IL-1β respectively. According to the ELISA instructions for murine IL-6, IL-8, and IL-1β, the standard products, capture antibodies, and detection antibody dry reagents were dissolved and aliquoted in sequence; one day before the experiment, the capture antibodies of the three antibodies were diluted to the working solution concentration using coating buffer solution, and 100 μL of the working solution was added to each well of a 96-well ELISA plate. After sealing the plate with a sealing film, it was centrifuged briefly and incubated overnight on a constant temperature shaker at 37°C at a rotation speed of 70 revolutions per minute; before the experiment the next day, the auxiliary kit was taken out in advance and restored to room temperature, and 1x washing solution and RD blocking solution were prepared. The coated ELISA plate was taken out from the constant temperature shaker, the capture antibody solution in the wells was discarded, and 300 μL of the washing solution was added to each well in sequence using a multi-channel pipette. The side walls were gently tapped for about 2 minutes for thorough washing, and the washing operation was repeated 3 times. Subsequently, 300 μL of the blocking solution was added to each well, the sealing film was attached, and it was incubated at room temperature for 1 h; the stock solution of the standard product (concentration 180 μg / mL) was taken out and restored to room temperature. At the same time, 8 1.5 mL EP tubes and 1x Assay Diluent were prepared for serial dilution; the supernatant samples to be detected were taken out in advance and appropriately diluted. After the blocking was completed, the blocking solution was discarded, and the plate wells were washed 3 times with the washing solution, using the same method as before. Subsequently, 100 μL of the standard product and the test samples were added to the ELISA plate in sequence, the sealing film was covered, and it was incubated at room temperature for 2 h; the detection antibody was taken out in advance and thawed and diluted to the working solution concentration. After the binding of the primary antibody was completed, the liquid in the wells was discarded, and it was washed 3 times with the washing solution, using the same method as before. Subsequently, 100 μL of the diluted detection antibody was added to each well. After gently tapping the side walls to mix evenly, the sealing film was covered, and it was incubated at room temperature for 2 h; then the 40x HRP solution for the corresponding index was taken out and diluted to 1x working solution concentration. After the incubation of the detection antibody was completed, the liquid was discarded, and it was washed 3 times with the washing solution. Subsequently, 100 μL of the HRP working solution was quickly added to the wells using a multi-channel pipette, and it was incubated at room temperature in the dark for 20 min; the substrate liquid was prepared into a sufficient amount of substrate reaction solution at a ratio of A:B = 1:1. After the incubation of HRP was completed, the ELISA plate was washed 3 times with the washing solution. Subsequently, 100 μL of the substrate reaction solution was quickly added to the wells using a multi-channel pipette, and it was incubated at room temperature in the dark for 20 min; after the substrate reaction solution was incubated for 20 min, the color change of each well was observed, and the plate was read using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 630 nm. When the optical density (OD) value of the first well of the standard product was between 0.7 and 1.0, 50 μL of the stop solution was added to each well;

[0082] After adding the stop solution, gently tap the side wall to mix well, and use a microplate reader to read the plate at a wavelength of 450 nm to obtain the OD values of each well. Draw a standard curve and a curve function based on the OD values and corresponding concentrations of the standards, and substitute the OD values of each well to calculate the concentrations of each sample.

[0083] Figure 1 It can be seen that in the mice administered with Daminozide by intraperitoneal injection, compared with the group exposed to simple cigarette smoke, the numbers of macrophages and neutrophils in the bronchoalveolar lavage fluid (BALF) were both decreased. This result indicates that intraperitoneal injection of Daminozide can inhibit the infiltration of inflammatory cells in the lung tissue. Figure 2 - 4 It can be seen that the expressions of inflammatory factors IL-6, KC and IL-1β in the bronchoalveolar lavage fluid (BALF) of the mice administered with Daminozide by intraperitoneal injection were significantly decreased, which indicates that intraperitoneal injection of Daminozide can inhibit the secretion of inflammatory factors in the lungs of mice.

[0084] Example 4

[0085] Evidence shows that the dysfunction of airway epithelial cells promotes the occurrence of airway chronic inflammation, which is essential for maintaining COPD. Therefore, 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 first explored the optimal administration concentration of Daminozide by the CCK8 method. The detailed steps of CCK8 are as follows:

[0086] 1) Prepare a cell suspension and count.

[0087] 2) Inoculate the cell suspension in a 96-well plate, about 100 μl per well, and the same sample can be replicated 3 times.

[0088] 3) Place the culture plate in an incubator for pre-incubation for a period of time (37 °C, 5% CO2).

[0089] 4) Add different concentrations of Daminozide to each well of the culture plate.

[0090] 5) Place the culture plate in an incubator and incubate for 24 h.

[0091] 6) Add 10 μl of CCK-8 solution to each well, and gently shake the culture plate to help mix. Try not to generate bubbles during the sample addition process to avoid affecting the OD value reading.

[0092] 7) Place the culture plate in an incubator and incubate for 1 h.

[0093] 8) Measure the absorbance (OD) at 450 nm with a microplate reader. Calculate the cell viability through the following formula: Viability calculation:

[0094] Cell viability (%) = [A (drug added) - A (blank)] / [A (control) - A (blank)] × 100

[0095] 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. The RT-PCR method is described in detail below:

[0096] 1) RNA extraction and reverse transcription

[0097] Add TRZIOL to the cell culture plate, pipette after blowing, transfer to different EP tubes, make marks. Pre-cool chloroform in a 4°C refrigerator and add chloroform at a ratio of chloroform:RNAiso Plus lysate = 1:5. Then shake the EP tube vigorously for about 30 s to fully mix the reagents. Then place the EP tube on ice for 15 min, and three distinct layers can be seen. Pre-cool a low-temperature high-speed centrifuge, set the parameters to 4°C, 12,000 rpm, 15 min, and centrifuge the above EP tubes. After centrifugation, three layers can be seen. The top transparent layer is the required RNA solution. Then, use a sterile pipette tip to transfer the upper liquid to a new enzyme-free EP tube, add an equal volume of isopropanol, gently invert the EP tube up and down about 20 times, and then place it on ice for 30 min to fully precipitate. After sufficient precipitation, centrifuge the above EP tubes in a centrifuge with parameters of 12,000 rpm, 15 min. Meanwhile, prepare a 75% ethanol solution by mixing 10 mL of DEPC water and 30 mL of absolute ethanol, and tighten the bottle cap to prevent volatilization. After centrifugation, a white precipitate can be seen at the bottom of the tube. Pour out the liquid and tap dry on a clean tissue. Then pipette 1 mL of the prepared 75% ethanol solution into the tube, invert the EP tube up and down to fully wash the RNA until the precipitate floats visibly, and then let it stand on ice for 10 min. Repeat the ethanol washing step for the next operation. After completing the washing step, centrifuge in a low-temperature high-speed centrifuge at 4°C, 12,000 rpm for 15 min. Then, pour out the liquid, tap dry on a clean tissue, wipe the tube wall with a sterile cotton swab, and then place the EP tube on ice for 10 - 15 min to air dry naturally. Then add 40 μL of DEPC water to the tube to fully dissolve the RNA precipitate. Wipe the probe dry with a clean absorbent paper, pipette 1 μL after mixing the RNA in each tube to measure the concentration, and enter the corresponding sample number into the computer.

[0098] Based on the concentration of the sample RNA, calculate the volumes of the corresponding RNA, DEPC water, and 5x Master Mix solution in the reverse transcription system. After gently flicking the tube wall to mix well, perform a brief centrifugation. For example, in a 10 μL reverse transcription system, add the volume corresponding to 500 ng RNA, 2 μL of 5x Master Mix solution, and use DEPC water to make up the total volume to 10 μL.

[0099] Place the prepared reverse transcription system from the previous step in a conventional PCR instrument for reverse transcription. The specific reverse transcription program is shown in the following table;

[0100] Table 1 Reverse Transcription Program

[0101]

[0102] 2) Real-time Fluorescent Quantitative PCR

[0103] Prepare the reaction system according to the ratios in the following table;

[0104] Table 2 Amplification Reaction System

[0105]

[0106] 3) Real-time Fluorescent Quantitative PCR

[0107] Connect and turn on the CFX Connect PCR instrument, place the prepared reaction system, and proceed according to the following program. After the program ends, calculate the relative expression of RNA in the sample based on the CT value.

[0108] Table 3 Fluorescent Quantitative PCR Reaction Program

[0109]

[0110] Among them, the primers corresponding to each target gene are listed in the following table.

[0111] Table 4 Primer List Corresponding to Target Genes

[0112]

[0113] After stimulating human airway epithelial cells with different concentrations of Daminozide for 24 hours, the results of CCK8 showed that administration at concentrations below 8 μM did not have a significant effect on cell viability, as Figure 4 shown.

[0114] Subsequently, we pretreated 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 2 μM and 4 μM concentrations of Daminozide, the transcriptional levels of IL-6 and IL-8 in airway epithelial cells were both decreased. The results are as Figure 5 shown.

[0115] We further detected the expression of IL-6 and IL-8 in the cell supernatant after stimulating airway epithelial cells pretreated with 4 μM Daminozide for 1 hour with cigarette smoke extract for 24 hours. The results indicated that the expression of IL-6 and IL-8 in the cell supernatant was both decreased. As Figure 6 shown.

[0116] 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.

[0117] Obviously, the above embodiments are only examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill 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 enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

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

2. 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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