Application of histone lactylation modification as target spot in preparation of medicine for preventing or treating chronic obstructive pulmonary disease

Abstract

The invention belongs to the technical field of biomedicine, and discloses application of histone lactic acid modification as a target spot in preparation of a medicine for preventing or treating chronic obstructive pulmonary diseases, in particular to lactic acid modification on 18th lysine residues of histone 3, and the expression level of ATM protein in airway epithelial cells is up-regulated through the histone lactic acid modification, so that the chronic obstructive pulmonary diseases can be prevented or treated. The expression of cell senescence key genes is promoted, so that the senescence phenotype of airway epithelial cells is aggravated, and the airway epithelial cells participate in the disease process of mediating the chronic obstructive pulmonary disease. The discovery provides a new therapeutic target for the chronic obstructive pulmonary disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to the application of histone lactic acid modification as a target in the preparation of drugs for preventing or treating chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a common respiratory disease characterized by persistent airflow limitation. Its main pathological changes are airway (bronchitis, bronchiolitis) and / or alveolar abnormalities (emphysema), which can lead to a variety of symptoms such as dyspnea, cough, and sputum. COPD is usually associated with exposure to harmful particles or gases, and exposure to cigarette smoke is its main risk factor. In addition, intrinsic factors such as genetic susceptibility, abnormal inflammatory response, and poor lung development are also involved in the pathogenesis of COPD. In severe cases, COPD can lead to cor pulmonale, respiratory failure, and various complications, significantly affecting the patient's quality of life and mortality. (Celli, Bartolome et al. "Definition and Nomenclature of Chronic Obstructive Pulmonary Disease: Time for Its Revision." American journal of respiratory and critical care medicine vol.206,11 (2022): 1317-1325.) Currently, the clinical treatment options for COPD are very limited. In addition to quitting smoking and avoiding environmental exposure, bronchodilators are mainly used during the stable period of the disease, and glucocorticoids are added during the acute exacerbation period. Existing drugs are difficult to safely and effectively reduce oxidative stress and inflammatory responses in the lungs, and the development of new drugs is still limited by the current understanding of the pathogenesis of COPD.

[0003] Lactylation, also known as lactylation, is a new post-translational modification that uses lactic acid, an important product of cellular metabolism, as raw material. It can regulate the expression of many genes by modifying histones in chromatin, such as the lactylation modification on the 18th lysine residue of histone 3 (H3K18la). It can also modify various non-histone proteins to directly affect the function of specific proteins (Zhang, Di et al. "Metabolic regulation of gene expression by histonelactylation." Nature vol. 574,7779 (2019): 575-580.). Since its discovery, lactylation has shown its potential role in tumors and various chronic diseases, including regulating the metabolism, inflammation, immunity, aging and other phenotypes of tissue structural cells (Li, Hongde et al. "Lactylation in cancer: Current understandingand challenges." Cancer cell vol. 42,11 (2024): 1803-1807.) (Hu, Xue-Ting et al. "Lactate-mediated lactylation in human health and diseases: Progress andremaining challenges." Journal of advanced research, S2090-1232(24)00529-0. 9Nov. 2024.), but it is still unclear what role lactylation plays in the pathogenesis of chronic obstructive pulmonary disease.

[0004] Studies have shown that lung aging and lung tissue cell aging play an important role in the pathogenesis of COPD. (Pace, E et al. “Carbocysteine ​​counteracts the effects of cigarette smoke on cell growth and on the SIRT1 / FoxO3 axis in bronchial epithelial cells.” Experimental gerontology vol. 81 (2016): 119-28.) Accelerated lung aging and airway inflammation promote each other, leading to the occurrence and development of the disease. The core feature of cell aging is the stagnation of division and proliferation function, which in turn causes the damage of the barrier protection function of structural cells and the dysfunction of non-structural cells. At the same time, senescent cells still maintain metabolic activity, and there is a phenotypic change in the secretion spectrum and an increase in the secretion of multiple inflammatory factors and chemokines. As the first line of defense for lung tissue to respond to external stimuli, the airway epithelium plays a key role in the pathogenesis of COPD. There is increasing evidence that airway epithelial cells show severe accelerated aging under the stimulation of cigarette smoke, which in turn participates in the destruction of lung tissue structure and the occurrence of airway inflammation in COPD (Schneider, Jaime L et al. "The aging lung: Physiology, disease, and immunity." Cell vol. 184,8 (2021): 1990-2019.) (Nyunoya, Toru et al. "Cigarette smoke induces cellular senescence." American journal of respiratory cell and molecular biology vol. 35,6 (2006): 681-8.). Despite this, the current understanding of the mechanism of airway epithelial cell aging caused by cigarette smoke stimulation is still relatively limited, and there is a lack of effective targets for clinical intervention.

[0005] Therefore, it is urgent to conduct in-depth research on the pathogenesis of COPD, find intervention targets for lung aging, and fully combine cutting-edge advances in molecular biology such as lactylation modification to provide theoretical support for the development of new and effective therapeutic drugs. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide the use of histone lactylation modification as a target in the preparation of drugs for preventing or treating chronic obstructive pulmonary disease, and to provide a new intervention target for the development of new and effective therapeutic drugs for chronic obstructive pulmonary disease.

[0007] 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 histone lactylation modification as a target in the preparation of a drug for preventing or treating chronic obstructive pulmonary disease.

[0008] In the above technical solution, the chronic obstructive pulmonary disease is chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

[0009] In the above technical solution, the histone lactic acid modification is the lactic acid modification on the 18th lysine residue of histone 3, namely H3K18la.

[0010] In the above technical scheme, histone lactylation modification promotes the expression of key genes of cell senescence by upregulating the expression level of ATM protein in airway epithelial cells, thereby aggravating the aging phenotype of airway epithelial cells and participating in mediating the disease progression of chronic obstructive pulmonary disease.

[0011] In a second aspect, the present invention provides a drug for preventing or treating chronic obstructive pulmonary disease, which contains a component that targets and interferes with histone lactylation modification.

[0012] In the above technical scheme, the drug includes one or more of sodium oxamate, a component that inhibits lactate synthesis, AZD3965, a component that inhibits lactate transporter MCT1, A-485, a component that inhibits lactylation modification enzyme P300, and Tartryl-CoA, a component that inhibits lactyl-CoA synthetase GTPSCS.

[0013] Among them, the CAS No. of sodium oxamate is 565-73-1, and the structural formula is as follows: The CAS No. of AZD3965 is 1448671-31-5, and the structural formula is as follows: The CAS No. of A-485 is 1889279-16-6, and its structural formula is as follows: The PubChem CID of Tartryl-CoA is 146675159, and the structural formula is as follows: In the above technical solution, the chronic obstructive pulmonary disease is chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

[0014] In the above technical solution, the histone lactic acid modification is the lactic acid modification on the 18th lysine residue of histone 3, namely H3K18la.

[0015] The beneficial effects of the present invention are as follows: the present invention has discovered for the first time the key role of a novel post-translational modification of protein lactylation, especially the lactylation modification of histone H3K18la in COPD and its downstream cellular phenotype and molecular mechanism, which can provide new molecular targets and theoretical basis for the clinical prevention and treatment of COPD. By reducing the level of lactylation modification in airway epithelial cells and the abnormal expression of its downstream molecule ATM protein, the excessive aging of airway epithelial cells can be effectively alleviated, thereby inhibiting the aging process of lung tissue, and providing a new treatment strategy for COPD. Sodium oxamate, as an inhibitor of lactate dehydrogenase in lactate metabolism, can effectively inhibit the lactylation modification of histones by interfering with lactate synthesis, thereby downregulating the expression of ATM protein. In addition, the monocarboxylate transporter MCT1 is a transporter protein that transports lactate into cells, and its inhibitor AZD3965 can affect lactylation modification by blocking lactate from entering cells. Protein P300, as a lactylation modification enzyme, directly participates in mediating the lactylation modification of histones, and its inhibitor A-485 can be used to inhibit the lactylation modification of histones. Lactyl-CoA, an important substrate for lactylation, was found to be produced by lactate catalyzed by the GTP-specific succinyl-CoA synthetase GTPSCS, and Tartryl-CoA's inhibitory effect on GTPSCS can be used to inhibit lactylation in cells. At the same time, as a bridge between cell metabolism and phenotype, a deeper understanding of lactylation can help further explore the relationship between metabolic disorders and disease phenotypes in COPD, thereby more comprehensively understanding the pathogenesis and mechanism of COPD, and helping to advance the early prevention and treatment of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the immunohistochemical staining results of lactylated modification H3K18la on the 18th lysine residue of histone 3 in natural control and COPD patient clinical lung tissue specimens. The enlarged close-up area is the airway epithelial cells; Figure 2 The figure shows the results of Western blot experiments of H3K18la and internal reference protein histone 3 (H3) after cigarette smoke extract (CSE) stimulated the human airway epithelial cell line HBE at different concentrations. , ; Figure 3 This is a functional enrichment of differentially expressed genes with upregulated H3K18la levels in the promoter region of HBE airway epithelial cells after CSE stimulation discovered by chromatin immunoprecipitation sequencing; Figure 3a is the GO function enrichment result diagram, Figure 3 b is the KEGG function enrichment result diagram; Figure 4 This is a gene association diagram of the above genes related to cell cycle, p53 signaling pathway and cell senescence; Figure 5 The figure is a schematic diagram and statistical diagram of the Western blot experimental results of H3K18la and the internal reference protein histone 3 (H3) after the lactate synthesis inhibitor sodium oxamate was stimulated at different concentrations in the human airway epithelial cell line HBE. ; Figure 6 The figure is a schematic diagram and statistical diagram of the Western blot experimental results of ATM protein and internal reference protein β-Tubulin in airway epithelial cells HBE after cigarette smoke extract and sodium oxamate stimulated them alone or in combination. , ; Figure 7 The figure shows the Western blot results of senescence-related proteins p53, p21 and internal reference protein GAPDH in HBE airway epithelial cells after cigarette smoke extract and ATM protein small interfering RNA or ATM protein inhibitor KU-60019 were stimulated alone or in combination. , , , ;in, Figure 7 a is a schematic diagram of the experimental results of cigarette smoke extract and small interfering RNA of ATM protein alone or in combination. Figure 7 b is Figure 7 A bar chart of Figure 7 c is a schematic diagram of the experimental results of cigarette smoke extract and KU-60019 alone or in combination. Figure 7 d is Figure 7 c's bar chart; Figure 8 Schematic diagram of the experimental results of β-galactosidase staining of airway epithelial cells HBE after stimulation with cigarette smoke extract and ATM protein inhibitor KU-60019 alone or in combination. DETAILED DESCRIPTION

[0017] 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.

[0018] The present invention demonstrates through the following examples that histone lactylation modification can promote the senescent phenotype of airway epithelial cells by upregulating ATM expression, and airway epithelial cell senescence plays an important role in the occurrence and development of COPD and is an important potential target for the clinical treatment of COPD.

[0019] In order to better explain the present invention, it is described in detail below in conjunction with specific embodiments.

[0020] Example 1 Changes in histone lactic acid modification levels in lung tissue specimens of COPD patients Experimental specimens and materials: 1. Collection of experimental specimens: According to the diagnostic criteria for COPD (dyspnea, chronic cough or sputum, history of recurrent lower respiratory tract infection and / or history of exposure to risk factors for the disease, and FEV1 / FVC < 0.70 after the use of bronchodilators, which confirmed the presence of persistent airflow limitation) and the study inclusion criteria (40-70 years old, clear smoking history, excluding patients with other severe chronic respiratory diseases, active tuberculosis or recent inhaled or oral steroid treatment), clinical lung tissue specimens of COPD patients and natural controls (non-smoking non-COPD patients) were collected from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Specifically, normal lung tissue (≥ 5 cm from the lesion) was collected from patients who underwent lobectomy or segmentectomy in the Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology due to "lung nodule occupying space to be investigated", and the baseline data and some key indicators (including age, gender, height, weight, smoking history and lung function) of the subjects were collected through the hospital medical record system. The specimens were promptly placed in 4% paraformaldehyde and isolated from air for 24 hours. After fixation, routine paraffin embedding and sectioning were performed for subsequent experiments; 2. Main materials for the experiment: 50X sodium citrate antigen retrieval solution was purchased from Solebao Biotechnology Co., Ltd.; donkey serum for blocking was purchased from Solebao Biotechnology Co., Ltd.; H3K18la rabbit monoclonal antibody was purchased from Hangzhou Jingjie Biotechnology Co., Ltd.; HRP-goat anti-rabbit polyclonal antibody was purchased from Wuhan Promoter Biological Co., Ltd.; DAB staining kit was purchased from Wuhan Sevier Biotechnology Co., Ltd.; and hematoxylin staining solution was purchased from Wuhan Sevier Biotechnology Co., Ltd.

[0021] 3. Experimental methods: Select well-preserved paraffin sections and bake them in a 60°C oven for 30 to 60 min until the paraffin melts and drips in a "sweat" state. Then dewax the sections by immersing them in xylene-anhydrous ethanol-85% ethanol solution-75% ethanol solution-running distilled water in this order.

[0022] Prepare about 300 mL of antigen repair solution and pour it into the antigen repair box, soak the tissue sections in it. Note that the antigen repair solution should be slightly excessive to avoid drying the slices; put it in a microwave oven, heat it on medium heat for 8 minutes, let it stand for 8 minutes, and then heat it on medium-low heat for 7 minutes; after heating, let it cool naturally to room temperature, which usually takes 1 hour.

[0023] Discard the antigen repair solution, wash with PBS on a shaker for 3 times, and prepare 3% hydrogen peroxide solution at the same time; then take out each slice in turn, shake off the PBS and carefully wipe the area around the tissue slice with a clean paper towel to dry it, immediately circle the tissue to be stained with a tissue pen, and be careful to avoid contaminating the tissue; immediately add a sufficient amount of hydrogen peroxide solution and incubate in the dark for 20 minutes to inactivate the endogenous peroxidase of the tissue.

[0024] Discard the hydrogen peroxide solution, wash with PBS three times, add an appropriate volume of blocking solution and incubate for 1 h for blocking. Be careful to avoid incubation for too long. After blocking, discard the blocking solution, do not wash, and then add H3K18la antibody working solution diluted 1:100 in PBS to cover the tissue to be stained. Place the slices in a humidified box and incubate overnight at 4°C.

[0025] The next day, take out the wet box in advance and rewarm for 1 hour, recover or discard the antibody working solution, and wash the sections with PBS 3 times; add secondary antibody incubation solution (HRP-goat anti-rabbit antibody diluted 1:200 in PBS) and incubate at room temperature for 1 hour; discard the secondary antibody liquid and wash 3 times with PBS; prepare DAB liquid and keep it away from light; add DAB liquid to the tissue slice for color development for 5-20 minutes. After the color development is completed, immediately rinse under running water for 5 minutes to stop the color development.

[0026] Add hematoxylin solution for staining for about 3 minutes, then quickly rinse under running water for 5 minutes; add ammonia water to reverse blue, and rinse under running water again for 5 minutes. Soak the slices in ethanol solutions of increasing concentrations for dehydration. After the alcohol on the surface of the slices is completely evaporated, add neutral gum for sealing. Be careful to avoid bubbles during the process; randomly select the field of view under the microscope to photograph the staining of the airway epithelium.

[0027] 4. Experimental results: H3K18la is currently the most studied lactylation modification site on chromatin histones. The results of H3K18la immunohistochemical staining of clinical lung tissues are as follows Figure 1 As shown. The brown-yellow coloring area is mainly located in the cell nucleus (blue area), which is consistent with the subcellular localization of H3K18la. Compared with natural controls, H3K18la in the lung tissue of COPD patients is mainly located in airway epithelial cells (pseudostratified columnar cells that surround the lumen), and the H3K18la level in airway epithelial cells is significantly increased (darker brown color).

[0028] Example 2 Effect of cigarette smoke extract on histone lactylation modification in airway epithelial cells In order to further evaluate the changes in the level of histone lactylation modification in airway epithelial cells under the stimulation of cigarette smoke, the present invention detected the level of H3K18la in the human airway epithelial cell line HBE by Western blot.

[0029] HBE cell line, full name 16 Human Bronchial Epithelial cell line, is derived from normal human bronchial epithelial cells and has been immortalized. It can be used to establish respiratory epithelial disease models. It is one of the most commonly used cell lines for in vitro studies of respiratory diseases such as COPD, with good representativeness and reliability. HBE cells are routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, and cell morphology and growth status are closely observed. Under normal conditions, HBE cells are cobblestone-like, adherent to the wall, proliferate rapidly, and have a doubling time of 24-36 hours.

[0030] At the same time, fresh cigarette smoke extract (CSE) was prepared. Cigarette smoke is the most important risk factor for the onset of COPD, and adding CSE to cultured cells is a classic method to simulate cigarette smoke stimulation for in vitro modeling. The whole process was kept away from light and wind, and standardized 3R4F cigarettes were used. After lighting, they were connected to the filter tip, and cigarette smoke was sucked into the culture medium by negative pressure suction. Two cigarettes were dissolved in every 20 ml of culture medium, and each cigarette was controlled to burn out in about 5 minutes. The stock solution was filtered and sterilized with a filter with a pore size of 0.22 microns, which defined the concentration of cigarette smoke extract as 100%. CSE was dispensed into brown light-proof EP tubes at 1 ml per tube, and then stored in a -20°C low-temperature refrigerator for use.

[0031] Stimulation should be performed when the cells are in good condition. After stimulating the cells with freshly thawed CSE at a concentration gradient for 48 hours, scrape the HBE cells on ice using RIPA lysis buffer (with protease inhibitors added in advance), collect them in a 1.5 ml centrifuge tube, break the cell membrane by ultrasound, and then centrifuge at 4°C with parameters of 12,000 rpm and 15 min. After centrifugation, aspirate the supernatant into the protein tube and add 1 / 4 volume of the liquid in the tube with 5X protein loading buffer. After mixing, place in a metal bath and heat at 100°C for 10 minutes. After cooling, you can start detecting the target protein by Western blot. Otherwise, place in a -80°C refrigerator for storage.

[0032] Prepare SDS-PAGE gel according to the instructions, take out the protein sample to be tested, put it on ice to melt, put it in the electrophoresis tank after the gel is completely solidified, add electrophoresis solution, and add protein samples and pre-stained protein markers to the gel wells in sequence according to the plan. After the sample is added, cover the lid according to the positive and negative poles, turn on the power of the electrophoresis instrument, adjust the voltage to 70 V first, and perform constant voltage electrophoresis until the bromophenol blue is evenly compressed and the protein markers are clearly separated. Then adjust the voltage to 120 V and continue electrophoresis until the corresponding positions of each target protein are fully separated. During this period, prepare the items required for transfer, including transfer clips, transfer solution, transfer tank, and ice water bath.

[0033] Take out the gel sheet, rinse off the foam under running water, and use the seesaw to slowly lift the short board to avoid tearing the gel. Use the seesaw to cut off the excess gel on the long board, and then carefully transfer the required gel to the black side of the transfer clip. Measure the side length of the gel, cut the PVDF membrane, and be careful to put on dry gloves to avoid wetting the PVDF membrane. Immerse the membrane in methanol for 3 to 5 minutes to fully activate it. Then slowly cover the membrane on the gel to avoid the appearance of bubbles, then cover the filter paper, sponge and the opposite side clip, fasten the transfer clip, pour the transfer solution into the transfer tank, and put the transfer clip in it, put in an ice pack, turn on the power after connecting the positive and negative poles, adjust the constant current to 230 mA, start the transfer, and transfer for 2 hours.

[0034] After the transfer is completed, turn off the power, remove the transfer clip, check the transfer, and place the successfully transferred PVDF membrane in 5% skim milk (25 grams of skim milk powder fully dissolved in 500 ml of TBST solution) for blocking, and incubate on a shaker at room temperature for 1-2 hours. After blocking, wash the PVDF membrane 3 times with TBST solution on a shaker, 10-15 minutes each time. Then use a ballpoint pen to clearly mark the molecular weight corresponding to each marker, and use a blade to cut according to the molecular weight of the target protein. Prepare the primary antibody solution (H3K18la antibody and H3 antibody are diluted in antibody diluent at 1:1000 each), pour it into the antibody incubation box, and then place the membrane in the corresponding antibody solution and incubate it on a shaker at 4 degrees overnight. After the primary antibody incubation is completed, properly recover the primary antibody solution and wash the PVDF membrane 3 times with TBST solution. Then prepare the secondary antibody solution of the corresponding species at a ratio of 1:10000, add it to the incubation box, and incubate it on a shaker at room temperature for 1.5-2 hours. After the secondary antibody incubation is completed, the PVDF membrane is washed 3 times with TBST solution. The exposure instrument is turned on in advance to preheat, and the developer (A solution: B solution = 1:1) is prepared. Then the PVDF strip is fully immersed in the developer and placed in the exposure instrument for development. The image and the white film are saved for subsequent analysis. Figure 2 shown.

[0035] Combination Figure 2It can be seen that direct stimulation of CSE can upregulate the lactylation modification level on the 18th lysine residue of histone 3 in airway epithelial cells, and the upregulation effect is most significant at 2.5% and 5% ( P <0.05 or P <0.01), nearly 1.5 times; the effect weakened at 10%, which is speculated to be related to excessive toxicity at high concentrations, severe cell damage and imminent death, which is consistent with the toxicological characteristics of CSE. This result further proves that cigarette smoke, as the most important risk factor in the pathogenesis of COPD, can directly promote lactic acid modification on chromatin histones of airway epithelial cells, and the latter's regulatory function on gene expression may be involved in mediating the phenotypic abnormalities of airway epithelial cells in the pathogenesis of COPD, such as inflammation, aging, metaplasia, etc.

[0036] Example 3 Genes and biological processes involved in the regulation of histone lactylation modification in airway epithelial cells under cigarette smoke stimulation Studies have shown that various modifications on chromatin histones, including acetylation and lactylation, regulate the expression of corresponding genes mainly by changing the open level of specific chromatin regions. In order to discover the specific genes regulated by H3K18la in airway epithelial cells under the stimulation of cigarette smoke, the present invention extracts DNA fragments regulated by H3K18la through chromatin immunoprecipitation (ChIP).

[0037] Each ChIP extraction requires about four million cells to ensure the extraction effect. After culturing the required amount of HBE cells, collect them and use 37% formaldehyde solution for cross-linking to strengthen the binding of DNA to the proteins on them. Break the membrane, extract the cell nucleus, and then add an appropriate amount of micrococcal nuclease (according to preliminary experiments, add about 0.25 microliters per HBE cell), and digest the DNA into fragments of about 150-900 bases in a 37°C water bath for 20 minutes. Ultrasonic treatment breaks the nuclear membrane, centrifuges to remove the nuclear fragments, and obtains the chromatin fragment sample.

[0038] After confirming the concentration and enzyme digestion of chromatin by Nanodrop quantitative measurement and DNA gel electrophoresis quality inspection, sufficient chromatin fragments were incubated with antibodies in a 4°C rotor overnight, and a positive control group (incubated with H3 antibody) and a negative control group (incubated with IgG antibody) were designed. ChIP-grade protein G magnetic beads were used to extract the antibody-protein-DNA complex, and eluted, cross-linked, and centrifuged to purify, and finally DNA fragments were obtained. The H3K18la rabbit monoclonal antibody in the above experiment was purchased from Hangzhou Jingjie Biotechnology Co., Ltd., and the chromatin immunoprecipitation kit was purchased from Cell Signaling Technology. The specific experimental process refers to the instruction manual.

[0039] For the DNA fragments obtained by ChIP, high-throughput sequencing was performed using the MGISEQ-T7 sequencer. After sequencing, the data was filtered through quality control to obtain high-quality sequencing data, which was then aligned to the reference genome and peak called for the entire genome to obtain the corresponding genes. The promoter of a gene is an important region for regulating its transcription, so for the obtained genes, those with upregulated H3K18la levels in the promoter-transcription start site (promoter-TSS) region under the stimulation of CSE were selected for bioinformatics analysis.

[0040] Functional enrichment analysis is to discover significantly enriched biological functions based on the artificial functional annotation information of the biological pathways in which the genes participate through statistical analysis. For the genes obtained by sequencing, functional enrichment is first performed based on the "Gene Ontology" (GO) database and the "Kyoto Encyclopedia of Genes and Genomes" (KEGG) database. The results are as follows Figure 3 As shown, the enrichment results suggest that the genes regulated by cigarette smoke via H3K18la are mainly enriched in phenotypes such as cell senescence and signaling pathways such as p53, and the latter is an important molecular pathway for cell senescence.

[0041] In order to further discover key genes, we conducted correlation analysis on genes included in "cell cycle", "cell senescence" and "p53 signaling pathway". The results are as follows Figure 4 As shown, it points to molecules such as ATM and CHEK2. Among them, ATM, the full name of AtaxiaTelangiectasia-Mutated gene, encodes ATM protein, which was first discovered in patients with ataxia telangiectasia and is involved in mediating DNA damage-related DNA repair and cell cycle regulation. As a key regulatory factor in multiple signal cascades, ATM protein has many downstream molecules, including cell cycle checkpoint kinases Chk1 and Chk2, tumor suppressors p53 and p21, and is closely related to the regulation of cell cycle and cell aging.

[0042] There is extensive DNA damage in the lung tissue structural cells of COPD patients. It is believed that DNA damage caused by oxidative stress in COPD leads to cell senescence and excessive release of aging-related inflammatory factors, which in turn forms a vicious cycle with the inflammatory response, causing the disease to persist. It can be speculated that excessive activation of ATM plays an important role in this process.

[0043] Example 4 Sodium oxamate interferes with histone lactic acid modification by inhibiting lactate metabolism Lactic acid, as an important substrate for lactic acid modification, can directly affect the level of histone lactic acid modification. Therefore, we inhibited lactic acid synthesis by adding the lactate dehydrogenase inhibitor sodium oxamate and detected its inhibitory effect on histone lactic acid modification by Western blot.

[0044] HBE cells were routinely cultured and stimulated with sodium oxamate in a concentration gradient. After 48 hours, the cells were harvested and proteins were extracted for Western blot detection. Figure 5 As shown, sodium oxamate can significantly inhibit the lactic acid modification level of HBE cells, specifically at a concentration of 10mM, it can inhibit nearly 50%, which indicates that sodium oxamate is an effective means to inhibit the lactic acid modification of H3K18la of histones in HBE cells.

[0045] Example 5 Cigarette smoke stimulation upregulates the expression of ATM protein in airway epithelial cells through histone lactylation modification In order to verify the expression change of ATM in airway epithelial cells, the present invention detected the level of ATM in the airway epithelial cell line HBE by Western blot.

[0046] On the basis of the above results, we further studied the mediating role of histone lactylation in the up-regulation of ATM expression in HBE cells stimulated by cigarette smoke. HBE cells were routinely cultured, and sodium oxamate was used to inhibit histone lactylation modification separately or superimposed on CSE stimulation. The proteins in the cells were extracted by RIPA lysis buffer, and the target proteins were detected by Western blot. The results are shown in Figure 2. Figure 6 shown.

[0047] Combination Figure 6 It can be seen that cigarette smoke stimulation can significantly upregulate the expression of ATM protein in airway epithelial cells ( P <0.01, while the up-regulation was significantly inhibited after the addition of sodium oxamate ( P <0.0001). This result confirms the above sequencing results, that is, cigarette smoke can promote the expression of ATM gene through histone lactylation modification. Combined with the role of ATM in various diseases, it can be speculated that it plays an important role in the aging of airway epithelial cells.

[0048] Example 6 ATM protein is involved in regulating airway epithelial cell senescence caused by cigarette smoke stimulation At present, many hallmark features of cell aging have been found. Evidence shows that p53 and p21 are marker molecules of cell aging process, which are involved in mediating cell cycle arrest and the expression of related genes; and β-galactosidase staining is a classic indicator for evaluating cell aging. The change of its content is closely related to the lysosomal stress response and can characterize the degree of cell aging. Therefore, in order to further evaluate the effect of ATM on the aging of airway epithelial cells stimulated by cigarette smoke, the present invention detected the protein levels of p53 and p21 in the human airway epithelial cell line HBE by Western blot, and detected the β-galactosidase content in HBE by β-galactosidase staining.

[0049] HBE cells were cultured and stimulated with cigarette smoke extracts. ATM expression or activity was inhibited by small interfering RNA of ATM molecules or KU-60019, an inhibitor of ATM, either separately or superimposedly. The small interfering RNA sequence of ATM molecules was 5'-GGGCCAUUCUUAAUGUAAUTT-3'. Proteins in cells were extracted by RIPA lysis buffer, and the target proteins were detected by Western blot. The results are shown in Figure 7 shown.

[0050] At the same time, use the β-galactosidase staining kit to stain HBE cells. Aspirate the cell culture medium, wash once with PBS, add 1 ml of β-galactosidase staining fixative, and fix at room temperature for 15 minutes. Aspirate the cell fixative, wash the cells 3 times with PBS, 3 minutes each time. Add 1 ml of staining working solution to each well and incubate at 37°C overnight. Observe and photograph under an ordinary optical microscope. The results are as follows Figure 8 As shown, the dark blue product is the cell senescence-related β-galactosidase.

[0051] Combination Figure 7 and Figure 8 It can be seen that the reduction of ATM expression or functional inhibition can significantly alleviate the senescent phenotype of airway epithelial cells, which is specifically manifested by the reduction of p53 and p21 protein expression ( P <0.0001 or P <0.001), as well as a decrease in β-galactosidase. This suggests that ATM protein is involved in the senescence of airway epithelial cells induced by cigarette smoke, and that inhibiting ATM can improve the disease phenotype and thus alleviate the lung aging process in COPD.

[0052] In summary, the above experimental data show that the upregulated histone lactylation modification H3K18la in COPD can directly promote the expression of ATM, aggravate the senescent phenotype of airway epithelial cells stimulated by cigarette smoke, and discover new effective targets for the development of clinical therapeutic drugs for COPD. At the same time, the changes and effects of lactylation modification in COPD suggest the importance of metabolism in abnormal cell phenotypes, which helps to have a more comprehensive understanding of the pathogenesis of COPD.

[0053] 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. Application of reagents that target and interfere with histone lactylation modification in the preparation of drugs for preventing or treating chronic obstructive pulmonary disease.

2. The application according to claim 1, characterized in that: The chronic obstructive pulmonary disease is chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

3. The application according to claim 1, characterized in that: The histone lactic acid modification is the lactic acid modification on the 18th lysine residue of histone 3, namely H3K18la.

4. The application according to claim 1, characterized in that: Histone lactylation modification promotes the expression of key genes of cell senescence by upregulating the expression level of ATM protein in airway epithelial cells, thereby aggravating the senescent phenotype of airway epithelial cells and participating in mediating the disease progression of chronic obstructive pulmonary disease.

5. Use of a drug in the preparation of a drug for preventing or treating chronic obstructive pulmonary disease, characterized in that: The drug contains a component that targets and interferes with histone lactylation modification; The drug comprises one or more of sodium oxamate, a component for inhibiting lactic acid synthesis, A-485, a component for inhibiting lactic acid modification enzyme P300, and Tartryl-CoA, a component for inhibiting lactyl-CoA synthetase GTPSCS.

6. The use according to claim 5, characterized in that: The chronic obstructive pulmonary disease is chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

7. The use according to claim 5, characterized in that: The histone lactic acid modification is the lactic acid modification on the 18th lysine residue of histone 3, namely H3K18la.

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