Use of histone lactylation modification as a target in the preparation of drugs for preventing or treating chronic obstructive pulmonary disease

Abstract

The present invention belongs to the field of biomedical technology, and discloses the application of histone lactylation modification as a target in the preparation of drugs for preventing or treating chronic obstructive pulmonary disease. In particular, it is the lactylation modification on lysine residue 18 of histone 3. Histone lactylation modification up-regulates the expression level of ATM protein in airway epithelial cells, promotes the expression of key genes for cell senescence, and further aggravates the senescence phenotype of airway epithelial cells, participating in mediating the disease process of chronic obstructive pulmonary disease. And this discovery provides a new therapeutic target for chronic obstructive pulmonary disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of histone lactylation 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, with main pathological changes in the airways (bronchitis, bronchiolitis) and / or alveoli (emphysema), which can cause various symptoms such as dyspnea, cough, and expectoration. COPD is usually associated with exposure to harmful particles or gases, and cigarette smoke exposure is the most important risk factor. In addition, intrinsic factors such as genetic susceptibility, abnormal inflammatory responses, and abnormal lung development also participate in the pathogenesis of COPD. In severe cases, COPD can lead to cor pulmonale, respiratory failure, and various complications, significantly affecting the quality of life and mortality of patients. (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.)

[0003] Currently, the clinical treatment options for COPD are still very limited. In addition to smoking cessation and environmental exposure avoidance, bronchodilators are mainly used during the stable phase of the disease, and glucocorticoids are added during the acute exacerbation phase. 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.

[0004] Lactylation modification, also known as lactyl modification, is a novel post-translational modification using lactic acid, an important product of cell metabolism, as a raw material. It can regulate the expression of numerous genes by modifying histones in chromatin, such as lactylation modification on lysine residue 18 of histone 3 (H3K18la), and can also modify various non-histones to directly affect the functions of specific proteins (Zhang, Di et al. “Metabolic regulation of gene expression by histone lactylation.” Nature vol. 574,7779 (2019): 575-580.). Since its discovery, lactylation modification has shown its potential roles in tumors and various chronic diseases, including regulating phenotypes such as metabolism, inflammation, immunity, and aging of tissue structure cells (Li, Hongde et al. “Lactylation in cancer: Current understanding and challenges.” Cancer cell vol. 42,11 (2024): 1803-1807.) (Hu, Xue-Ting et al. “Lactate-mediated lactylation in human health and diseases: Progress and remaining challenges.” Journal of advanced research, S2090-1232(24)00529-0. 9 Nov. 2024.), but it is currently unclear what role lactylation modification plays in the pathogenesis of chronic obstructive pulmonary disease.

[0005] Studies have shown that lung aging and senescence of lung tissue cells play important roles 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 cellular senescence is the arrest of the function of division and proliferation, which in turn causes damage to the barrier protection function of structural cells and dysfunction of non-structural cells. At the same time, senescent cells still maintain metabolic activity, and there are phenotypic changes in the secretory profile and increased secretion of a variety of inflammatory factors and chemokines. As the first line of defense for lung tissue to respond to external stimuli, airway epithelium plays a key role in the pathogenesis of COPD. Moreover, more and more evidence indicates that airway epithelial cells show severe accelerated senescence under the stimulation of cigarette smoke, and then participate 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.). Nevertheless, the current understanding of the mechanism of airway epithelial cell senescence caused by cigarette smoke stimulation is still relatively limited, and there is a lack of effective targets for clinical intervention.

[0006] Therefore, it is urgent to deeply study the pathogenesis of COPD, find intervention targets for lung aging, and fully combine the latest progress in molecular biology such as lactylation modification to provide theoretical support for the development of new and effective therapeutic drugs. 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 histone lactylation modification as a target in the preparation of drugs for preventing or treating chronic obstructive pulmonary disease, so as to provide a new intervention target for the development of new and effective therapeutic drugs for chronic obstructive pulmonary disease.

[0008] To achieve the above object, the present application adopts the following technical solutions:

[0009] 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.

[0010] In the above technical solution, the chronic obstructive pulmonary disease is chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

[0011] In the above technical solution, the histone lactylation modification is the lactylation modification on the 18th lysine residue of histone 3, that is, H3K18la.

[0012] In the above technical solution, histone lactylation modification participates in mediating the disease process of chronic obstructive pulmonary disease by up-regulating the expression level of ATM protein in airway epithelial cells, promoting the expression of key genes of cell senescence, and further aggravating the senescence phenotype of airway epithelial cells.

[0013] 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.

[0014] In the above technical solution, the drug includes one or more of sodium oxamate, a component that inhibits the lactate transporter MCT1, AZD3965, a component that inhibits the lactylation-modifying enzyme P300, A-485, and a component that inhibits lactyl-CoA synthetase GTPSCS, Tartryl-CoA.

[0015] Among them, the CAS No. of sodium oxamate is 565-73-1, and the structural formula is as follows:

[0016]

[0017] The CAS No. of AZD3965 is 1448671-31-5, and the structural formula is as follows:

[0018]

[0019] The CAS No. of A-485 is 1889279-16-6, and the structural formula is as follows:

[0020]

[0021] The PubChem CID of Tartryl-CoA is 146675159, and the structural formula is as follows:

[0022]

[0023] In the above technical solution, the chronic obstructive pulmonary disease is a chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

[0024] In the above technical solution, the histone lactylation modification is the lactylation modification on the 18th lysine residue of histone 3, namely H3K18la.

[0025] The beneficial effects of the present invention are as follows: The present invention discovers for the first time a new type of post-translational modification of proteins, lactylation modification, especially the key role of histone lactylation modification H3K18la in COPD and its downstream cell phenotypes and molecular mechanisms, 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 senescence of airway epithelial cells can be effectively alleviated, and further the senescence process of lung tissue can be inhibited, providing a new treatment strategy for COPD. Sodium oxalate, as an inhibitor of lactate dehydrogenase in lactate metabolism, can effectively inhibit the lactylation modification of histones by interfering with lactate synthesis, and then down-regulate the expression of ATM protein. In addition, 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-modifying 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 modification, is found to be catalyzed by GTP-specific succinyl-CoA synthetase GTPSCS to produce lactate, and the inhibitory effect of Tartryl-CoA on GTPSCS can be used to inhibit lactylation modification in cells. At the same time, lactylation modification, as a bridge connecting cell metabolism and phenotype, the deepening of the understanding of it helps to further explore the association between metabolic disorders and disease phenotypes in COPD, so as to more comprehensively understand the pathogenesis and mechanism of COPD, and contribute to the early prevention of the disease and the advancement of the treatment threshold. Description of the Drawings

[0026] Figure 1 It is a schematic diagram of the results of immunohistochemical staining of lactylation modification H3K18la on the 18th lysine residue of histone 3 in clinical lung tissue specimens of natural controls and COPD patients. The magnified close-up area is airway epithelial cells;

[0027] Figure 2 It is a schematic diagram and statistical chart of the Western blot experimental results of H3K18la and the reference protein histone 3 (H3) after human airway epithelial cell line HBE is stimulated with cigarette smoke extract (CSE) at different concentrations. Among them, , ;

[0028] Figure 3 Functional enrichment was performed on the differential genes with up-regulated H3K18la levels in the promoter region after CSE stimulation in airway epithelial cells HBE discovered by chromatin immunoprecipitation sequencing; among them, Figure 3 a is the result graph of GO functional enrichment, Figure 3 b is the result graph of KEGG functional enrichment;

[0029] Figure 4 is the gene association graph related to the cell cycle, p53 signaling pathway and cell senescence among the above genes;

[0030] Figure 5 is the schematic diagram and statistical graph of the Western blot experiment of H3K18la and the reference protein histone 3 (H3) after stimulating the human airway epithelial cell line HBE with different concentrations of the lactate synthesis inhibitor oxamate; among them, ;

[0031] Figure 6 is the schematic diagram and statistical graph of the Western blot experiment of the ATM protein and the reference protein β-Tubulin in the cells after stimulating the airway epithelial cells HBE with cigarette smoke extract and oxamate alone or in combination; among them, , ;

[0032] Figure 7 is the schematic diagram and statistical graph of the Western blot experiment of the senescence-related proteins p53, p21 protein and the reference protein GAPDH in the cells after stimulating the airway epithelial cells HBE with cigarette smoke extract and the small interfering RNA of the ATM protein or the ATM protein inhibitor KU-60019 alone or in combination; among them, , , , ; among them, Figure 7 a is the schematic diagram of the experimental results of stimulating with cigarette smoke extract and the small interfering RNA of the ATM protein alone or in combination, Figure 7 b is Figure 7 the column statistical graph of a, Figure 7 c is the schematic diagram of the experimental results of stimulating with cigarette smoke extract and KU-60019 alone or in combination, Figure 7 d is Figure 7 the column statistical graph of c;

[0033] Figure 8 is the schematic diagram of the experimental results of β-galactosidase staining of the cells after stimulating the airway epithelial cells HBE with cigarette smoke extract and the ATM protein inhibitor KU-60019 alone or in combination. Detailed implementation method

[0034] To better illustrate the objectives, 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 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 be defined only by the claims.

[0035] The present invention confirms through the following embodiments that histone lactylation modification can promote the senescence phenotype of airway epithelial cells by upregulating the expression of ATM, and the senescence of airway epithelial cells plays an important role in the occurrence and development of COPD and is an important potential target for the clinical treatment of COPD.

[0036] To better explain the present invention, the following detailed description is provided in conjunction with specific embodiments.

[0037] Example 1 Changes in the level of histone lactylation modification in lung tissue specimens of COPD patients

[0038] Experimental specimens and materials:

[0039] 1. Collection of experimental specimens:

[0040] According to the COPD diagnostic criteria (history of dyspnea, chronic cough or expectoration, repeated lower respiratory tract infection history and / or exposure to risk factors of the disease, and the presence of persistent airflow limitation confirmed by FEV1 / FVC < 0.70 after using bronchodilators) and the inclusion and exclusion criteria for the study (aged 40 - 70 years, with a clear smoking history, excluding patients with other severe chronic respiratory diseases, active pulmonary tuberculosis, or those who have recently received inhaled or oral steroid treatment), clinical lung tissue specimens of COPD patients and natural controls (non-smoking and non-COPD patients) were collected from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Specifically, normal lung tissue (≥5 cm away from the lesion) of patients who underwent lobectomy or segmentectomy due to "pulmonary nodule occupancy to be investigated" in the Department of Thoracic Surgery of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology was collected, and baseline data and some key indicators of the subjects (including age, gender, height, weight, smoking history, and lung function) were collected through the hospital medical record system. The specimens were immediately placed in 4% paraformaldehyde and isolated from air, and fixed for 24 hours. After fixation, routine paraffin embedding and sectioning were performed for subsequent experiments;

[0041] 2. Main experimental materials:

[0042] The 50X sodium citrate antigen retrieval solution was purchased from Solarbio Science & Technology Co., Ltd.; the blocking donkey serum was purchased from Solarbio Science & Technology Co., Ltd.; the rabbit monoclonal antibody against H3K18la was purchased from Jingjie Biotech Co., Ltd. in Hangzhou; the HRP-goat anti-rabbit polyclonal antibody was purchased from Wuhan Promoter Biotechnology Co., Ltd.; the DAB staining kit was purchased from Wuhan Sevier Biotechnology Co., Ltd.; the hematoxylin staining solution was purchased from Wuhan Sevier Biotechnology Co., Ltd.

[0043] 3. Experimental methods:

[0044] Select well-preserved paraffin sections, place them in an oven at 60 °C and bake for 30 - 60 min until the paraffin melts and drips down in a "sweating" state. Then, immerse the sections in xylene - absolute ethanol - 85% ethanol solution - 75% ethanol solution - running distilled water in sequence for dewaxing.

[0045] Prepare about 300 mL of antigen retrieval solution and pour it into the antigen retrieval box. Immerse the tissue sections in it, noting that the antigen retrieval solution should be slightly excessive to avoid dry slices. Put it into the microwave oven, heat on medium fire for 8 min, let it stand for 8 min, and then heat on medium - low fire for 7 min. After heating, cool it naturally to room temperature, which generally takes 1 h.

[0046] Discard the antigen retrieval solution, wash 3 times with PBS on a shaker, and at the same time prepare 3% hydrogen peroxide solution. Then, take out each section in turn, shake off the PBS and carefully wipe it dry with a clean tissue paper to make the area around the tissue section dry. Immediately use a histochemical pen to circle the tissue to be stained, taking care not to contaminate the tissue. Immediately add a sufficient amount of hydrogen peroxide solution and incubate in the dark for 20 minutes to inactivate the endogenous peroxidase in the tissue.

[0047] Shake off the hydrogen peroxide solution, wash 3 times with PBS, add an appropriate volume of blocking solution and incubate for 1 h for blocking, taking care not to incubate for too long. After blocking, shake off the blocking solution without washing, and then immediately add the working solution of H3K18la antibody diluted 1:100 in PBS to cover the tissue to be stained. Place the section in a wet box and incubate at 4 °C overnight.

[0048] The next day, take out the wet box in advance and warm it up for 1 h. Recover or discard the antibody working solution, and wash the section 3 times with PBS. Add the secondary antibody incubation solution (HRP - goat anti - rabbit antibody diluted 1:200 in PBS) and incubate at room temperature for 1 h. Discard the secondary antibody solution, wash 3 times with PBS. Prepare the DAB solution, taking care to avoid light. Add the DAB solution to the tissue section for color development for 5 - 20 min. After color development is completed, immediately rinse under running water for 5 min to stop color development.

[0049] Dropwise add hematoxylin staining solution and stain for about 3 min, then quickly rinse under running water for 5 min; add ammonia water for bluing, and rinse again with running water for 5 min. Soak the sections successively in ethanol solutions with increasing concentrations for dehydration. After the alcohol on the surface of the sections has completely evaporated, dropwise add neutral balsam for mounting, and pay attention to avoid generating bubbles during the process; randomly select fields of view under the microscope to photograph the staining of airway epithelium.

[0050] 4. Experimental results:

[0051] H3K18la is the most studied lactylation modification site on chromatin histones at present. The immunohistochemical staining results of H3K18la in clinical lung tissues are as Figure 1 shown. The yellowish-brown staining area is mainly located in the cell nucleus (blue area), which is consistent with the subcellular localization of H3K18la. Compared with the natural control, H3K18la in the lung tissues of COPD patients is mainly located in airway epithelial cells (pseudostratified columnar cells that surround and form the lumen), and the level of H3K18la in airway epithelial cells is significantly increased (the yellowish-brown color is deeper).

[0052] Example 2 Effects of cigarette smoke extract on histone lactylation modification of airway epithelial cells

[0053] 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 of protein immunoblotting experiment.

[0054] The HBE cell line, with the full name of 16 Human Bronchial Epithelial cell line, is derived from normal human bronchial epithelial cells and has been immortalized. It can be used to establish a model of respiratory epithelial diseases and is one of the most commonly used cell lines for in vitro research on respiratory system 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 the cell morphology and growth status are closely observed. Under normal conditions, HBE cells are cobblestone-like, adherent growth, with a relatively fast proliferation rate and a doubling time of 24-36 hours.

[0055] Meanwhile, prepare fresh cigarette smoke extract (CSE). Cigarette smoke is the most important risk factor for the development of COPD, and culturing cells with added CSE is a classic method for in vitro modeling by simulating cigarette smoke stimulation. Keep away from light and wind throughout the process. Use standardized 3R4F cigarettes. After lighting, connect them to the filter tip, and aspirate the cigarette smoke into the culture medium by negative pressure. Dissolve two cigarettes in every 20 mL of culture medium, and control each cigarette to burn out in about 5 minutes. Filter and sterilize the stock solution with a filter with a pore size of 0.22 μm, and define the concentration of the cigarette smoke extract as 100%. Aliquot the CSE into brown light-proof EP tubes at 1 mL per tube, and then store them in a -20°C low-temperature refrigerator for future use.

[0056] The stimulation should be carried out when the cells are in good condition. After stimulating the cells with fresh thawed CSE at a concentration gradient for 48 hours, use RIPA lysis buffer (with protease inhibitor added in advance) to scrape HBE cells on ice, collect them into a 1.5 mL centrifuge tube, break the cell membrane by ultrasonic treatment, and then centrifuge at 4°C with parameters of 12,000 rpm and 15 min. After centrifugation, aspirate the supernatant into a protein tube and add 1 / 4 volume of 5X protein loading buffer to the liquid in the tube. Mix well and then heat in a metal bath at 100°C for 10 minutes. After cooling, start to detect the target protein by Western blot, otherwise store it in an -80°C refrigerator for future use.

[0057] Prepare the SDS-PAGE gel according to the instructions. At the same time, take out the protein samples to be detected and place them on ice to thaw. After the gel is completely solidified, place it in the electrophoresis tank, add the electrophoresis solution, and sequentially add the protein samples and the prestained protein marker into the gel wells according to the plan. After loading is completed, cover the lid according to the positive and negative poles, turn on the power of the electrophoresis instrument, first adjust the voltage to 70 V, and perform constant voltage electrophoresis until the bromophenol blue is aligned and the protein marker is clearly separated, then adjust the voltage to 120 V and continue electrophoresis until the corresponding positions of each target protein are fully separated and then stop. During this period, prepare the items required for membrane transfer, including the membrane transfer clip, membrane transfer solution, membrane transfer tank, and ice water bath, etc.

[0058] Take out the gel plate, wash off the foam under running water, use a spatula to slowly lift the short plate to avoid tearing the gel. Use a spatula to cut off the excess gel on the long plate, and then carefully transfer the required gel to the black side of the membrane transfer clip. Measure the side length of the gel and cut the PVDF membrane. Note to change into dry gloves to avoid wetting the PVDF membrane. Immerse the membrane in methanol for 3 - 5 minutes to fully activate it. Then slowly cover the membrane on the gel, avoiding the appearance of bubbles during this period. Then cover the filter paper, sponge, and the opposite clip, tighten the membrane transfer clip, pour the membrane transfer solution into the membrane transfer tank, place the membrane transfer clip into it, put an ice pack, connect it according to the positive and negative poles, turn on the power, adjust the constant current to 230 mA, start membrane transfer, and perform conventional membrane transfer for 2 h.

[0059] After the membrane transfer is completed, turn off the power supply, take out the membrane transfer clip, check the membrane transfer situation, put the successfully transferred PVDF membrane into 5% skim milk (25 g of skim milk powder is fully dissolved in 500 ml of TBST solution) for blocking, and incubate at room temperature on a shaker for 1 - 2 h. After blocking, wash the PVDF membrane 3 times with TBST solution on a shaker, 10 - 15 min each time. Then clearly mark the molecular weight size corresponding to each marker with a ballpoint pen, and cut it with a blade according to the molecular weight of the target protein. Prepare the primary antibody solution (H3K18la antibody and H3 antibody are each diluted 1:1000 in the antibody dilution solution), pour it into the antibody incubation box, then put the membrane into the corresponding antibody solution, and incubate overnight at 4 degrees on a shaker. After the primary antibody incubation is completed, properly recover the primary antibody solution, and wash the PVDF membrane 3 times with TBST solution. Subsequently, prepare the secondary antibody solution of the corresponding species at a ratio of 1:10000, add it to the incubation box, and incubate on a shaker at room temperature for 1.5 - 2 h. After the secondary antibody incubation is completed, wash the PVDF membrane 3 times with TBST solution. Preheat the exposure instrument in advance, and prepare the developing solution (Solution A: Solution B = 1:1). Then fully immerse the PVDF strip in the developing solution, put it into the exposure instrument for development, and save the picture and its blank film for subsequent analysis. The results are as Figure 2 shown.

[0060] Combined Figure 2 it can be seen that the direct stimulation of CSE can up - regulate the lactylation modification level of histone H3 lysine residue 18 in airway epithelial cells, and the up - regulation effect is most significant at 2.5% and 5% ( P <0.05 or P <0.01), nearly 1.5 - fold; the effect weakens at 10%, presumably related to excessive toxicity and severe cell damage and near - death at high concentrations, which is in line 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 the lactylation modification of chromatin histones in airway epithelial cells, and the latter's regulatory function on gene expression may be involved in mediating the phenotypic abnormalities of airway epithelial cells during the pathogenesis of COPD, such as inflammation, senescence, and metaplasia.

[0061] Example 3 Genes and biological processes regulated by histone lactylation modification in airway epithelial cells under cigarette smoke stimulation

[0062] Research shows that various modifications on chromatin histones, including acetylation modification and lactylation modification, mainly regulate the expression of corresponding genes by changing the open level of specific regions of chromatin. To explore the specific genes regulated by H3K18la in airway epithelial cells under cigarette smoke stimulation, the present invention extracts the DNA fragments regulated by H3K18la through chromatin immunoprecipitation technology (ChIP).

[0063] Each ChIP extraction requires approximately four million cells to ensure the extraction effect. After sufficient HBE cells are cultured, they are collected and cross-linked with 37% formaldehyde solution to strengthen the binding of DNA to the proteins on it. The cell membrane is disrupted, the cell nuclei are extracted, and then an appropriate amount of micrococcal nuclease is added (about 0.25 μL per HBE cell according to preliminary experiments), and the DNA is digested into fragments with a length of about 150 - 900 bases by water bath at 37°C for 20 minutes. The nuclear membrane is broken by sonication, and the nuclear debris is removed by centrifugation to obtain a chromatin fragment sample.

[0064] After confirming the concentration and digestion of chromatin by Nanodrop quantitative measurement and DNA gel electrophoresis quality inspection, an appropriate amount of chromatin fragments is taken and incubated with the antibody overnight on a rotor at 4°C. At the same time, a positive control group (incubating with H3 antibody) and a negative control group (incubating with IgG antibody) are designed. The antibody-protein-DNA complex is extracted using ChIP-grade protein G magnetic beads, and then eluted, de-cross-linked, and centrifuged for purification to finally obtain DNA fragments. The anti-H3K18la rabbit monoclonal antibody in the above experiment was purchased from Hangzhou Jingjie Biotech Co., Ltd., and the chromatin immunoprecipitation kit was purchased from Cell Signaling Technology. The specific experimental procedures refer to the instruction manual.

[0065] For the DNA fragments obtained by ChIP, high-throughput sequencing is performed using the MGISEQ-T7 sequencer. After the sequencing is completed, filtering is carried out through quality control to obtain high-quality sequencing data. The sequencing data is aligned to the reference genome for genome-wide peak calling to obtain the corresponding genes. The promoter of a gene is an important region for regulating its transcription. Therefore, for the obtained genes, those with an up-regulated H3K18la level in the promoter-transcription start site (promoter-TSS) region under the stimulation of CSE are selected for bioinformatics analysis.

[0066] Functional enrichment analysis is to discover significantly enriched biological functions through statistical analysis based on the manual functional annotation information of the biological pathways participated by genes. 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 Figure 3 shown. The enrichment results suggest that the genes regulated by cigarette smoke through H3K18la are mainly enriched in phenotypes such as cell senescence and signal pathways such as p53, and the latter is an important molecular pathway for cell senescence.

[0067] To further discover key genes, correlation analysis is performed on the genes included in "cell cycle", "cell senescence", and "p53 signaling pathway". The results are as Figure 4As shown, it points to molecules such as ATM and CHEK2. Among them, ATM, the full name is Ataxia Telangiectasia-Mutated gene, encodes the ATM protein, which was first discovered in patients with ataxia-telangiectasia, and is involved in mediating DNA repair related to DNA damage and cell cycle regulation. As a key regulatory factor of multiple signal cascades, the ATM protein has many downstream molecules, including cell cycle checkpoint kinases Chk1 and Chk2, tumor suppressors p53 and p21, which are closely related to the regulation of the cell cycle and cell senescence.

[0068] There is extensive DNA damage in the lung tissue cells of COPD patients. Some views hold that in COPD, the DNA damage caused by oxidative stress leads to cell senescence and the excessive release of senescence-related inflammatory factors, which in turn form a vicious cycle with the inflammatory response, resulting in the protracted course of the disease. It can be speculated that the over-activation of ATM plays an important role in this process.

[0069] Example 4 Sodium oxamate interferes with histone lactylation modification by inhibiting lactate metabolism

[0070] As an important substrate for lactylation modification, lactate can directly affect the level of histone lactylation modification. Therefore, here we inhibited lactate synthesis by adding the lactate dehydrogenase inhibitor sodium oxamate, and detected its inhibitory effect on histone lactylation modification by Western blot.

[0071] HBE cells were cultured routinely, stimulated with sodium oxamate at concentration gradients, harvested after 48 hours, and proteins were extracted for Western blot detection. The experimental results are as Figure 5 shown. Sodium oxamate can significantly inhibit the lactylation modification level of HBE cells. Specifically, it can inhibit nearly 50% at a concentration of 10 mM, indicating that sodium oxamate is an effective means to inhibit the histone lactylation modification H3K18la of HBE cells.

[0072] Example 5 Cigarette smoke stimulation upregulates the expression of ATM protein in airway epithelial cells through histone lactylation modification

[0073] To verify the change in the expression of ATM in airway epithelial cells, the present invention detected the level of ATM in the airway epithelial cell line HBE by Western blot.

[0074] On the basis of the above results, the research group further studied the mediating role of histone lactylation modification in the up-regulation of ATM expression in HBE cells stimulated by cigarette smoke. HBE cells were cultured routinely. While using CSE to stimulate, sodium oxamate was used alone or in combination to inhibit histone lactylation modification. Proteins in the cells were extracted with RIPA lysis buffer, and the target proteins were detected by Western blot. The results are as Figure 6 shown.

[0075] Combined Figure 6 it can be seen that cigarette smoke stimulation can significantly up-regulate the expression of ATM protein in airway epithelial cells ( P <0.01), and this up-regulation effect was significantly inhibited after adding sodium oxamate ( P <0.0001). This result proves the above sequencing result, that is, cigarette smoke can promote the expression of ATM gene through histone lactylation modification. Considering the role of ATM in various diseases, it can be speculated that it plays an important role in the senescence of airway epithelial cells.

[0076] Example 6 ATM protein is involved in regulating the senescence of airway epithelial cells caused by cigarette smoke stimulation

[0077] At present, multiple hallmark features of cellular senescence have been discovered. Evidence shows that p53 and p21 are marker molecules in the process of cellular senescence, participating in mediating cell cycle arrest and the expression of related genes; while β-galactosidase staining is a classical index for evaluating cellular senescence, and the change of its content is closely related to lysosomal stress response, which can characterize the degree of cellular senescence. Therefore, to further evaluate the effect of ATM on the senescence 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 content of β-galactosidase in HBE by β-galactosidase staining.

[0078] HBE cells were cultured. While using cigarette smoke extract to stimulate, small interfering RNA of ATM molecule or inhibitor KU-60019 of ATM was used alone or in combination to inhibit the expression or activity of ATM; among them, the small interfering RNA sequence of ATM molecule is 5’-GGGCCAUUCUUAAUGUAAUTT-3’. Proteins in the cells were extracted with RIPA lysis buffer, and the target proteins were detected by Western blot. The results are as Figure 7 shown.

[0079] Meanwhile, HBE cells were stained using a β-galactosidase staining kit. The cell culture medium was aspirated, and after washing once with PBS, 1 mL of β-galactosidase staining fixative was added and fixed at room temperature for 15 minutes. The cell fixative was aspirated, and the cells were washed 3 times with PBS for 3 minutes each time. 1 mL of staining working solution was added to each well and incubated overnight at 37 °C. Observation and photography were performed under an ordinary optical microscope. The results are as Figure 8 shown, where the dark blue product is the senescence-associated β-galactosidase in cells.

[0080] Combined Figure 7 with Figure 8 , it can be seen that the decrease in ATM expression or functional inhibition can significantly alleviate the senescence phenotype of airway epithelial cells, specifically manifested as a decrease in the expression levels of p53 and p21 proteins ( P <0.0001 or P <0.001), and a decrease in β-galactosidase. This indicates that the ATM protein is involved in the senescence of airway epithelial cells induced by cigarette smoke stimulation, and inhibiting ATM can improve the disease phenotype and thus alleviate the lung senescence process in COPD.

[0081] In summary, the above experimental data show that the histone lactylation modification H3K18la upregulated in COPD can exacerbate the senescence phenotype of airway epithelial cells under cigarette smoke stimulation by directly promoting the expression of ATM, identifying a new effective target for the development of clinical treatment drugs for COPD. At the same time, the changes and roles of lactylation modification in COPD suggest the significance of metabolism for abnormal cell phenotypes, contributing to a more comprehensive understanding of the pathogenesis of COPD.

[0082] Obviously, the above embodiments are merely 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 implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. Use of a reagent for targeted interference with histone lactylation modification in the preparation of a drug for preventing or treating chronic obstructive pulmonary disease; Histone lactylation modification up-regulates the expression level of ATM protein in airway epithelial cells, promotes the expression of key genes for cell senescence, and further exacerbates the senescence phenotype of airway epithelial cells, participating in mediating the disease process of chronic obstructive pulmonary disease; The reagent includes sodium oxamate, and the chronic obstructive pulmonary disease is chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

2. The application according to claim 1, characterized in that: The histone lactylation modification is lactylation modification at the 18th lysine residue of histone 3, i.e., H3K18la.

3. 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 for targeted interference with histone lactylation modification; The drug includes sodium oxamate, a component that inhibits lactate synthesis; The chronic obstructive pulmonary disease is chronic obstructive pulmonary disease induced by cigarette smoke or cigarette smoke extract.

4. The application according to claim 3, wherein: The histone lactylation modification is lactylation modification at the 18th lysine residue of histone 3, i.e., H3K18la.

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