Application of kaempferol in preparation of medicine for treating chronic obstructive pulmonary disease

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of kaempferol in preparation of a medicine for treating chronic obstructive pulmonary disease. The invention provides application of kaempferol in preparation of a medicine for treating chronic obstructive pulmonary disease. The invention provides a medicine for treating chronic obstructive pulmonary disease. The medicine comprises kaempferol or pharmaceutically acceptable salt thereof as an active ingredient. The invention aims to provide the application of kaempferol in preparation of the medicine for treating the chronic obstructive pulmonary disease, and the chronic obstructive pulmonary disease is treated by adjusting an AMPK signal channel, a Hippo signal channel, an MAPK signal channel and a PI3K-AKT signal channel.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of kaempferol in preparing a medicine for treating chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by irreversible, progressive airflow limitation. Its morbidity and mortality rates remain high, and the World Health Organization predicts that COPD will be the third leading cause of death worldwide by 2030. The pathogenesis of COPD is complex, involving multiple factors, including abnormal oxidative stress, imbalance between proteases and antiproteases, and persistent activation of the inflammatory response. These factors collectively lead to lung tissue remodeling, chronic respiratory symptoms, and airflow limitation, posing a serious threat to human health. Currently, there is still an unmet clinical need for the treatment of COPD, and the exploration of new, effective therapeutic drugs is urgently needed.

[0003] Kaempferol is a natural flavonoid compound widely found in vegetables, fruits and medicinal plants. Existing studies have confirmed that kaempferol has multiple biological activities, including antioxidant, anti-inflammatory, anti-cancer, and regulation of blood lipids and blood sugar. In terms of anti-inflammatory, it can reduce the transcription of proinflammatory cytokines by inhibiting cyclooxygenase-2 (COX-2) activity, reducing prostaglandin E2 (PGE2) synthesis, or inhibiting nuclear factor κB (NF-κB) activation; in the field of anti-tumor, it can regulate Bcl-2 family proteins, activate mitochondrial apoptosis pathways or block the cell cycle to induce tumor cell apoptosis; in metabolic regulation, it can reduce cholesterol synthesis by inhibiting 3-hydroxy-3-methylvaleryl CoA reductase (HMGCR), or inhibit α-glucosidase to improve blood sugar levels. In addition, patents have reported the use of kaempferol in antiviral treatments such as new coronavirus, pseudorabies virus, treatment of autoimmune diseases, osteoarthritis, topical analgesics and hemostatics, and auxiliary compositions for interventional treatment of liver cancer.

[0004] However, no studies or reports have yet mentioned the use of kaempferol in the treatment of COPD, and this discovery of its potential use as a COPD medication is unprecedented. Given the high risk of COPD and the limitations of existing treatments, exploring new uses for kaempferol in the treatment of COPD has important clinical value and scientific significance. Summary of the Invention

[0005] The present invention aims to provide the use of kaempferol in the preparation of a drug for treating chronic obstructive pulmonary disease, regulating the AMPK signaling pathway, the Hippo signaling pathway, the MAPK signaling pathway and the PI3K-AKT signaling pathway, thereby treating chronic obstructive pulmonary disease.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] Application of kaempferol in preparing medicine for treating chronic obstructive pulmonary disease.

[0008] Preferably, the kaempferol is used to treat, prevent or improve chronic obstructive pulmonary disease.

[0009] Preferably, the kaempferol exerts its effect by regulating at least one of the AMPK signaling pathway, the Hippo signaling pathway, the MAPK signaling pathway, and the PI3K-AKT signaling pathway.

[0010] The present invention also provides a medicine for treating chronic obstructive pulmonary disease, comprising the kaempferol or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0012] Preferably, the pharmaceutically acceptable carrier is one or more of a solvent, a dispersant, a suspending aid, a surfactant, an isotonic agent, a thickener, a preservative, a solid binder or a lubricant.

[0013] Compared with the prior art, the present invention has the following advantages and technical effects:

[0014] This study demonstrates that kaempferol can treat chronic obstructive pulmonary disease (COPD). Kaempferol effectively increases cell survival, significantly inhibits apoptosis, and exerts anti-inflammatory effects by reducing the mRNA expression of pro-inflammatory factors such as IL-6 and TNF-α. It also improves oxidative stress by upregulating superoxide dismutase (SOD) levels and downregulating malondialdehyde (MDA) levels. Studies on COPD model mice have shown that kaempferol significantly improves lung function indicators, alleviates lung tissue pathological damage, improves alveolar structural damage, reduces inflammatory factor infiltration and collagen fiber deposition, and alleviates airway remodeling. It also enhances anti-inflammatory effects by reducing the levels of inflammatory factors such as TNF-α, IL-6, and IL-1β, inhibits cleaved caspease-3 expression to reduce apoptosis, and enhances antioxidant capacity by upregulating SOD and downregulating MDA.

[0015] Kaempferol also exerts its effects by regulating signaling pathways such as AMPK, Hippo, MAPK, and PI3K-AKT. Molecular docking and kinetic simulations confirm its stable binding to AKT1, providing a clear target for its action. Furthermore, this compound offers the advantages of low cost and minimal side effects, providing a new, effective and safe option for the treatment of COPD.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the structural formula of kaempferol;

[0018] Figure 2 This is a statistical graph showing the effect of kaempferol on the activity of BEAS-2B cells after stimulation with cigarette smoke extract (CSE) in Example 1;

[0019] Figure 3 The results of Example 2 show the effect of kaempferol on apoptosis of BEAS-2B cells after CSE stimulation;

[0020] Figure 4 The results of Example 3 show the effect of kaempferol on the expression of related cytokines in Beas-2b cells after CSE stimulation, wherein: Figure 4 A in the table is the effect on IL-6. Figure 4 B in the figure is the effect on TNF-α;

[0021] Figure 5 The changes in MDA and SOD levels of Beas-2b cells after kaempferol stimulation with CSE in Example 4 are shown. Figure 5 A in is the MDA change level, Figure 5 B in the figure is the change level of SOD;

[0022] Figure 6 The results of the analysis of the overall gene expression level of kaempferol on the survival rate of Beas-2b cells induced by CSE in Example 5;

[0023] Figure 7 The results of differential gene expression analysis of kaempferol on the CSE-induced Beas-2b cell survival rate in Example 5;

[0024] Figure 8 The cluster analysis results of differential gene expression analysis of kaempferol on CSE-induced Beas-2b cell survival rate in Example 5;

[0025] Figure 9 The GO functional analysis results of the differential gene expression of kaempferol on the CSE-induced Beas-2b cell survival rate in Example 5;

[0026] Figure 10 KEGG pathway analysis of the differential gene expression of kaempferol on CSE-induced Beas-2b cell survival rate in Example 5;

[0027] Figure 11 This is the molecular docking result of kaempferol and AKT1 in Example 5;

[0028] Figure 12 The molecular dynamics simulation results of kaempferol and AKT1 in Example 5;

[0029] Figure 13 is the lung function index of the COPD disease animal model after kaempferol treatment in Example 5, wherein, Figure 13 A in the figure is the FEV1 / FVC content of mice. Figure 13 B in the figure represents the dynamic lung compliance changes of mice. Figure 13 C in the figure represents the respiratory index change of mice;

[0030] Figure 14 HE staining results of lung tissue sections in Example 6;

[0031] Figure 15 The results of Masson staining of lung tissue sections in Example 6 are shown;

[0032] Figure 16 The results of the detection of TNF-α, IL-6, and IL-1β levels in the lung tissue of mice in Example 6 are shown in FIG. Figure 16 A in the figure is the result of TNF-α test. Figure 16 B in the figure is the IL-6 test result. Figure 16 C in the figure is the IL-1β detection result;

[0033] Figure 17 The results of the mRNA level detection of TNF-α, IL-6, and IL-1β in the lung tissue of mice in Example 6 are shown in FIG. Figure 17 A in the figure is the result of TNF-α test. Figure 17 B in the figure is the IL-6 test result. Figure 17 C in the figure is the IL-1β detection result;

[0034] Figure 18 This is the positive expression result of Cleaved caspease-3 in mouse lung tissue in Example 6;

[0035] Figure 19 The results of the detection of SOD and MDA levels in the lung tissue of mice in Example 6 are as follows: Figure 19 A in the figure is the SOD level test result. Figure 19 B in the figure is the MDA level test result. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0037] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0038] Source of test materials:

[0039] Zunyi cigarettes were purchased aseptically from the Zunyi Cigarette Factory of Guizhou Zhongyan Industrial Co., Ltd., China, and used to extract cigarette smoke extracts. Kaempferol was purchased from MCE, and CCK8 was purchased from Tongren (Japan). BEAS-2B cells were provided by the Department of Pharmacology, School of Basic Medical Sciences, Jilin University. H-DMEM medium was purchased from Gibco. Fetal bovine serum (FBS) was purchased from Clark Bioscience.

[0040] AnnexinV-FITC apoptosis detection kit was purchased from Beyotime Biotechnology; Agilent flow cytometry was purchased from Aglient;

[0041] Tizol was purchased from Thermo Fisher Scientific, chloroform was purchased from Beijing Chemical Industry, reverse transcription kit was purchased from Beijing Quanshijin, and primers were synthesized by Sangon Biotechnology;

[0042] The total SOD activity detection kit was purchased from Biyuntian Biotechnology, and the micro malondialdehyde (MDA) test kit was purchased from Nanjing Jiancheng Bioengineering Institute;

[0043] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0044] In the embodiment of the present invention, the structural formula of kaempferol is as follows Figure 1 shown.

[0045] Among them, the molecular weight of kaempferol is 286.23; the molecular formula is C 15 H 10 O6; Chemical name: 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one.

[0046] Example 1

[0047] The effect of kaempferol on the activity of BEAS-2B cells after stimulation with cigarette smoke extract CSE, the experimental method is as follows: monitor the cell status and density under a microscope, digest, centrifuge and resuspend the cells when the cell density is 70%-80%, and seed Beas-2b cells into 96-well plates at a density of 5000 per well. After the cells adhere to the wall, add H-DMEM complete culture medium containing 12% CSE and kaempferol at different concentrations of 1μM, 5μM, 10μM, 100μM, and 200μM to each well, and then incubate at 37°C for 24 hours. After that, add 10% CCK-8 solution to each well, and continue to incubate the 96-well plate at 37°C for 1 hour. Finally, use an enzyme reader to measure the absorbance at a wavelength of 450nm, and calculate the cell survival rate based on this. The results are as follows. Figure 2 .

[0048] Depend on Figure 2It can be seen that compared with 12% CSE stimulation, 100 μM and 200 μM kaempferol can improve cell survival rate.

[0049] Example 2

[0050] Flow cytometry was used to detect the effect of kaempferol on apoptosis of BEAS-2B cells after CSE stimulation. The experimental method is as follows:

[0051] The cell status and density were monitored under a microscope. When the cell density was 70%-80%, the cells were digested and resuspended to obtain a cell suspension. BEAS-2B cells were cultured at a density of 1×10 5 Cells were seeded at a density of 100 μg / mL in a 6-well plate and cultured in a constant-temperature incubator. After cell attachment, the cells were treated with the drug. Three groups of Beas-2b cells were set up: a control group (no intervention), a model group (stimulated with 12% CSE), and a treatment group (treated with both 12% CSE and 100 μM kaempferol). After 24 hours of incubation, the 6-well plate was removed for subsequent experiments. The supernatant was transferred to a suitable centrifuge tube, and the cells were washed once with PBS buffer. An appropriate amount of EDTA-free trypsin was added to digest the cells. The supernatant was incubated at room temperature until the adherent cells detached with gentle pipetting. The digestion was then terminated by adding the previously collected supernatant (to prevent over-digestion with trypsin). For the experiment, the suspension was first transferred to a centrifuge tube and the centrifuge was set to 1000 g for 5 minutes to pellet the cells. After centrifugation, the cells were gently resuspended in PBS buffer and centrifuged again at the same centrifugal force for 5 minutes to ensure complete cell collection. Finally, carefully pour off the supernatant and retain the precipitated cells for subsequent experiments. Add 195μL Annexin V-FITC binding solution, gently resuspend the cells, then add 5μL Annexin V-FITC and gently stir evenly. Incubate in the dark for 10-20 minutes at room temperature (20-25°C), then place the cells in an ice bath. Before testing on the machine, add 10μL propidium iodide staining solution, gently mix, and after preparation is completed, use Agilent flow cytometer for analysis. The results are as follows Figure 3 .

[0052] Depend on Figure 3As can be seen, quadrants Q2-3 represent viable cells, indicating normal cell function; quadrant Q2-2 indicates late apoptotic cells, indicating that cells are undergoing death; and quadrant Q2-4 represents early apoptotic cells, reflecting signs of apoptosis. In the control, model, and treatment groups of Beas-2b cells, the proportion of viable cells among total cells was 92.2%, 30%, and 76.1%, respectively; the proportion of early apoptotic cells among total cells was 1.74%, 1.16%, and 2.8%, respectively; and the proportion of late apoptotic cells among total cells was 5.21%, 60.7%, and 17.9%, respectively. As shown in the figure, Beas-2b cells underwent significant apoptosis after CSE stimulation, while the incidence of apoptosis was significantly reduced after kaempferol treatment, indicating that kaempferol can clearly inhibit CSE-induced apoptosis.

[0053] Example 3

[0054] The effect of kaempferol on the expression of related cytokines in Beas-2b cells after CSE stimulation was tested using the following method:

[0055] After extracting total RNA from cells, the reaction system was prepared as shown in Table 1 according to the instructions of the Quan's Gold reverse transcription kit, and mixed thoroughly to perform the reverse transcription reaction.

[0056] Table 1 Reaction system configuration

[0057]

[0058]

[0059] The primer sequences and reaction system information are detailed in Tables 2 and 3 below. Using GAPDH as the endogenous reference, the reaction system was added to 8 tube strips, and each sample was repeated 3 times. The amplification conditions were 95℃ pre-denaturation for 5 minutes, 95℃ denaturation for 20 seconds, 50℃ annealing for 20 seconds, and 72℃ extension for 20 seconds, for 40 cycles. The reaction tubes were placed in a real-time fluorescence quantitative PCR instrument and the program was started. Based on the Ct values ​​obtained for each gene amplification, 2 (-ΔΔCt) Law, 2 -△△Ct The expression level of the target gene was normalized with the reference gene as the reference. After completing at least 3 independent repeated experiments, statistical analysis was performed on the experimental data to calculate the relative expression level of the target gene. Figure 4 .

[0060] Table 2 Primer sequences

[0061]

[0062] Table 3 Reaction system

[0063] Components volume SYBR Green qPCR Mix 10 μL Upstream primer 0.8μL Downstream primer 0.8μL RT product (cDNA) 2μL Deionized water 6.4μL

[0064] Depend on Figure 4 CSE stimulation significantly increased the mRNA levels of IL-6 and TNF-α in cells, indicating that CSE can induce the production of inflammatory factors. In contrast, treatment with kaempferol significantly inhibited the expression of IL-6 and TNF-α, demonstrating its potential anti-inflammatory effects. This study suggests that kaempferol plays an important role in the treatment of COPD.

[0065] Example 4

[0066] The effects of kaempferol on the levels of SOD and MDA in Beas-2b cells stimulated by CSE were determined using the following method:

[0067] 1. Sample preparation: After cells are seeded in a 10 mm culture dish and cultured for 24 hours, aspirate the cell culture medium and wash once with PBS or physiological saline pre-cooled at 4°C or in an ice bath. Add 100-200 μL of the SOD sample preparation solution provided in this kit per 1 million cells. Pipette appropriately to fully lyse the cells. Centrifuge at approximately 12.000 g at 4°C for 3 minutes and collect the supernatant as the sample to be tested.

[0068] 2. Preparation of WST-8 / enzyme working solution: Prepare an appropriate amount of WST-8 / enzyme working solution for a 160 μL reaction volume. Evenly mix 151 μL SOD assay buffer, 8 μL WST-8, and 1 μL enzyme solution to make 160 μL of WST-8 / enzyme working solution.

[0069] 3. Preparation of reaction starting solution: Dissolve the reaction starting solution (40X) in the kit and mix thoroughly. Dilute it by adding 39μL SOD detection buffer to every 1μL reaction starting solution (40X). Mix thoroughly to obtain the reaction starting solution.

[0070] 4. Sample Assay: Refer to the instructions for setting up sample wells and various blank control wells in a 96-well plate. Add the sample to be tested and other solutions in sequence. Add the reaction starter solution and mix thoroughly.

[0071] 5. Calculation of total SOD activity:

[0072] Inhibition percentage = [(A blank control 1 - A blank control 2) - (A sample - A blank control 3)] / (A blank control 1 - A blank control 2) × 100%;

[0073] SOD enzyme activity units = SOD enzyme activity units in the test system = inhibition percentage / (1-inhibition percentage) units;

[0074] Malondialdehyde (MDA) test

[0075] After the cells were inoculated in a 10mm culture dish and cultured for 24 hours, they were washed twice with pre-cooled PBS at 4°C, and 100μL of RIPA lysis buffer was added to each well. After lysis, the cells were centrifuged at high speed at 4°C for 10 minutes (condition: 10000g), and the protein concentration was determined using a BCA protein concentration detection kit. TBA storage solution, MDA detection working solution, and standards with different concentration gradients were prepared. The detection reaction system was prepared by adding reagents according to the instructions, mixed well, heated at 100°C for 15 minutes, cooled to room temperature in a water bath, centrifuged at 1000g for 10 minutes, and 200μL of supernatant was added to a 96-well plate. The absorbance at 532nm was detected using an enzyme-labeled instrument. The results are as follows. Figure 5 .

[0076] Depend on Figure 5 It can be seen that after CSE stimulation, the expression level of SOD in cells decreased, but after treatment with kaempferol, SOD expression levels were upregulated. In contrast, after CSE stimulation, the expression level of MDA increased, but after kaempferol intervention, the expression level was reduced. This study results suggest that after CSE treatment, cells are in a state of oxidative stress, while kaempferol treatment improves oxidative stress, indicating that kaempferol has antioxidant effects in the treatment of COPD.

[0077] Example 5

[0078] The mechanism of action of kaempferol on CSE-induced Beas-2b cell survival was studied based on RNA-seq. The experimental method is as follows:

[0079] 1. Cell Treatment: Cell status and density were monitored under a microscope. When the cell density reached 70%-80%, cells were prepared into a cell suspension and plated. The cells were divided into the control group (Con), the model group (CSE stimulation), and the drug treatment group (CSE + kaempferol treatment). Beas-2b cells were treated with kaempferol for 24 hours. After treatment, the cells were harvested on ice and lysed with TRIZOL reagent for total RNA extraction. Each experiment was repeated three times, and the entire operation was completed on ice to ensure the integrity and stability of the RNA.

[0080] 2. Library construction and sequencing: The sequencing process includes: total RNA detection, mRNA enrichment, mRNA shearing, end modification with A and adapters, fragment selection and PCR enrichment, library quality control, and Illumina sequencing.

[0081] 3. Sequencing quality control: (1) Reads containing adapter sequences were removed; (2) low-quality reads were removed.

[0082] 4. Reference Gene Alignment: A specific genome is selected as a reference for sequence alignment and subsequent analysis. HISAT2 software is used to align processed clean reads with the reference genome, thereby obtaining information about the reads' location within the reference genome. StringTie software is used to assemble the aligned reads and reconstruct the transcriptome, laying the foundation for subsequent analysis.

[0083] 5. Library quality assessment: To ensure that the library quality meets the requirements, a comprehensive quality assessment of the sequencing library is required from multiple aspects, including randomness testing, fragment length distribution determination, and saturation detection.

[0084] 6. Gene Expression Analysis: Gene quantification effectively measures fluctuations in gene expression levels. Analysis of gene expression levels provides a visual representation of transcript abundance within a sample. Higher transcript abundance indicates higher gene expression levels. Gene expression exhibits tissue-specific and spatiotemporal specificity, resulting in variations in gene expression in samples under different physiological conditions or at different developmental stages. Analyzing gene expression levels facilitates in-depth exploration of gene expression fluctuations across different experimental samples.

[0085] 7. Analysis of differential gene expression: Differentially expressed genes were screened based on the total number of genes (Count value) in each sample using differential analysis software. For differential groupings with biological replicates, DESeq2 software was used to perform differential analysis; for differential groupings without biological replicates, edgeR software was used for analysis. In the process of identifying differentially expressed genes, the screening criteria were set as fold change (Fold Chang) ≥ 2 and false discovery rate (FDR) < 0.05. The fold change is used to characterize the ratio of the expression levels of the two groups of samples; the FDR is the value obtained after correcting the significant P value of the difference, so as to show the significance of the difference. In order to achieve more convenient comparison, the logarithm of the fold change is taken and expressed as log2FC.

[0086] 8. Differential gene enrichment analysis: Differential gene enrichment analysis was performed using the GO database and KEGG database.

[0087] 9. Molecular docking: The three-dimensional structure of AKT1 (7WM2) was downloaded from the Protein Data Bank (PDB) database. The protein molecule was processed by Pymol (version 2.5) to eliminate water molecules and ligands. The 2D structure of kaempferol was downloaded from the PubChem database and energy minimization and format conversion were performed using Chem3d (version 20.0). Molecular docking was performed using AutoDockTools software (version 1.5.7) and Pymol software (version 20.0) and Ligplot. + Visualize it.

[0088] 10. Molecular dynamics simulation: Based on the molecular docking results, we selected The Desmond part of the system was used to further explore the stability of the protein-complex and conduct molecular dynamics simulations. In order to analyze the stability and conformational behavior of the protein-complex within 100ns, the following process was simulated. First, the T1P3P system was selected for the construction of the system. For the neutralization stability of the solution system, Na + / Cl - The particles are then mixed to simulate the ion concentrations found in real biological environments. Next, energy minimization is performed to reduce atomic collisions. Finally, formal molecular dynamics simulations are performed, and corresponding simulation results are obtained, including RMSD, RMSF, and protein-ligand interaction calculations.

[0089] The results are as follows Figure 6-Figure 12 shown.

[0090] Depend on Figure 6 It can be seen that the expression trends of the three biological replicates are highly consistent. This experimental result fully proves that each group of samples has excellent performance in consistency.

[0091] Depend on Figure 7 As can be seen, the results intuitively demonstrate changes in the expression of relevant genes. Using a significance threshold of |log2foldchange|>1 and padj<0.05, a detailed comparison of the model and kaempferol-treated groups revealed 223 genes showing significant changes. Of these, 73 genes showed low expression in the treatment group, while 150 genes showed high expression. These significant changes in gene expression provide key insights into the mechanisms by which kaempferol affects CSE-induced Beas-2b cell survival and facilitate further exploration of its potential role in related physiological and pathological processes.

[0092] Depend on Figure 8As can be seen, the heat map exhibits a distinct block pattern, indicating significant differences in gene expression patterns between samples. This heat map clearly demonstrates the differences in gene expression between samples, providing an important data foundation for further research on gene function, sample classification, and exploration of underlying biological mechanisms.

[0093] Depend on Figure 9 It can be seen that in biological processes, differentially expressed genes are manifested in metabolic processes, cellular processes, and biological regulation; in cellular composition, they are mainly cellular anatomical entities, intracellular, and protein-containing complexes; and molecular functions are mainly concentrated in binding, catalytic activity, and molecular function regulator.

[0094] Depend on Figure 10 Through KEGG pathway enrichment analysis, significantly differentially expressed genes were enriched in the corresponding pathways, and their role in the pathways reflected whether the pathways were activated or inhibited. The differentially expressed genes were mainly enriched in the AMPK signaling pathway, the Hippo signaling pathway, the MAPK signaling pathway, and the PI3K-AKT signaling pathway.

[0095] Depend on Figure 11-12 It can be seen that the binding energy of kaempferol to AKT1 is -8.8kcal / mol (≤-5kcal / mol), indicating that kaempferol and AKT1 may spontaneously bind. In the molecular dynamics simulation experiment, the root mean square deviation (RMSD) showed that the fluctuation of protein and ligand was within an acceptable range. In particular, starting from about 65ns, the fluctuation range of protein Ca atoms and ligand weight factors was within 100 nm. Within. In terms of root mean square fluctuation (RMSF), the interaction between protein residues and ligand is less variable than other regions of the protein. Both aspects indicate that the complex is stable. The protein-ligand contact map shows that there are multiple interactions between the protein and the ligand, including hydrogen bonds, hydrophobic interactions, ionic interactions and water bridges. Residues such as TRP73, LYS193 and CYS196 interact with the ligand to form hydrogen bonds, and there are hydrophobic interactions between VAL69, LEU168, and VAL197 and the ligand. The small molecule compound binds stably to protein residues LYS193, CYS196 and VAL197.

[0096] Example 6

[0097] The therapeutic effect of kaempferol on COPD animal models is tested as follows:

[0098] Experimental Materials: This experiment used 60 SPF-grade female Balb / c mice weighing between 18 and 22 grams. The mice were acclimated for 7 days under a 12-hour light-dark cycle and had free access to food and water.

[0099] The experimental method is as follows: 60 female Balb / c mice were divided into 6 groups, with 12 mice in each group. The specific groups are as follows: blank group (labeled as Control), COPD smoke model group (abbreviated as Smoke, marked as S), low-dose treatment group (DL, dose of 50 mg kg -1 ·d -1 ), high-dose treatment group (DH, dose of 100 mg kg -1 ·d -1), and the positive drug treatment group (Y, the drug used was dexamethasone). Construction process and administration method of COPD disease animal model The COPD animal model was constructed by passive smoking, and the brand and batch of cigarettes used were consistent. Except for the Control group, the remaining mice were placed in a transparent plastic sealed box with a size of 90×70×40cm. To ensure normal ventilation of the mice and avoid suffocation, a small hole with a radius of 0.2cm was opened on each side of the plastic of the box. By connecting the air pump to the small hole on the top of the box, lighting the cigarette, and manually pressing the air pump, the smoke can be forced to enter the box at a uniform speed, forming a stable smoke environment for observation and research. The specific modeling operation is as follows: the mice inhale 6 cigarettes each time every day, each smoking lasts for 30 minutes, and the smoking operation is performed twice a day (6 hours apart). Smoking is performed 5 days a week, and the entire smoking modeling process lasts for 20 weeks. From the 16th week onwards, mice in the corresponding groups were treated with low-dose and high-dose kaempferol and dexamethasone by gavage every day, while the control group and the model group were simultaneously gavaged with normal saline (10 mg / kg). Figure 13 .

[0100] Depend on Figure 13 Compared to the control group, mice in the smoke group showed significant decreases in both FEV1 / FVC (%) and Cdyn, and a significant increase in RI, indicating the presence of airway obstruction. This indicates a successful smoke-induced COPD animal model. Different doses of kaempferol and dexamethasone increased FEV1 / FVC (%) and Cdyn, and decreased RI, in mice. The higher dose was more effective than the lower dose, indicating that kaempferol can improve lung function changes induced by smoke.

[0101] HE staining results of lung tissue sections Figure 14 As shown. Figure 14 It can be seen that the lung tissues of mice in the blank group and the model group changed. No obvious pathological changes were observed in the lung tissues of mice in the blank group, showing normal structure and function. However, after smoke modeling stimulation, the lung tissues of mice in the model group showed significant pathological changes, including infiltration of inflammatory factors, destruction of alveolar walls, and expansion of alveolar cavities. The fact shows that smoke has the ability to induce emphysema in mice, causing histological damage, indicating the success of the COPD animal disease model. After treatment with different doses of kaempferol and dexamethasone, there was a significant improvement compared with the smoke group, including a significant improvement in alveolar structure, infiltration of inflammatory factors and other pathological manifestations. Based on the results, kaempferol can alleviate the morphological changes in lung tissue caused by cigarette smoke stimulation.

[0102] In order to directly evaluate the effect of kaempferol on the deposition of collagen fibers in the lung tissue of COPD mice, Masson staining was performed. Figure 15 As shown. Figure 15 The blank group showed no significant collagen fiber deposition or fibrous foci in the lung tissue of rats. However, compared with the blank group, the model group showed significant collagen fiber deposition in the lung tissue of mice, confirming that long-term smoke exposure induced airway remodeling. After intervention with dexamethasone and varying doses of kaempferol, the collagen fiber content in the lung tissue of mice in all treatment groups decreased compared to the model group. These results suggest that kaempferol can alleviate airway remodeling induced by smoke exposure in mice.

[0103] ELISA was used to detect TNF-α, IL-6, and IL-1β in mouse lung tissue. Figure 16 shown.

[0104] Comparison revealed that after exposure to cigarette smoke, the inflammatory factors IL-6, TNF-α, and IL-1β increased significantly in the lung tissue of model mice, indicating that cigarette smoke can induce a significant inflammatory response. The model mice were treated with kaempferol and dexamethasone by oral gavage. Both drugs significantly reduced the levels of inflammatory factors in the lung tissue, with the inhibitory effect of kaempferol increasing with increasing dose. The changes in inflammatory factor levels detected by ELISA validated the potential application of kaempferol in alleviating cigarette smoke-induced COPD.

[0105] The mRNA levels of TNF-α, IL-6, and IL-1β in mouse lung tissue were detected by RT-qPCR. Figure 17 As shown. Figure 17 Compared with the blank group, the mRNA levels of TNF-α, IL-6, and IL-1β in the model group mice showed a significant increase. This result indicates that smoke stimulation can increase inflammatory factors in the lung tissue of mice. However, after intervention with kaempferol, the production of these inflammatory factors was significantly reduced, indicating that kaempferol has anti-inflammatory effects and can effectively alleviate smoke-induced lung inflammation.

[0106] IHC detected the positive expression of Cleaved caspease-3 in mouse lung tissue. Figure 18 As shown. Figure 18Compared to the blank control group, the model group mice showed a significant increase in the content of brown or tan particles in their lung tissue. This phenomenon suggests that prolonged exposure to cigarette smoke triggers apoptosis in the mice's lungs. Compared to the model group, oral administration of different doses of kaempferol and dexamethasone reduced the expression of cleaved caspease-3, an inflammation-associated enzyme, in the lungs of mice, indicating that kaempferol can inhibit apoptosis to a certain extent.

[0107] The antioxidant capacity of kaempferol in the lung tissue of COPD mice was evaluated by detecting the content of SOD and MDA. After establishing a COPD disease animal model and intervening with different doses of kaempferol and dexamethasone, the results of the measured SOD and MDA contents were as follows: Figure 19 shown.

[0108] Depend on Figure 19 It can be seen that compared with the blank group, the SOD content in the model group mice decreased and the MDA content increased. This result shows that smoke stimulation can induce oxidative stress in the lung tissue of mice. However, after kaempferol intervention, the SOD content increased and the MDA content decreased, indicating that kaempferol has an antioxidant effect and can effectively alleviate the oxidative stress damage to the lungs caused by smoke.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of kaempferol in the preparation of drugs for treating chronic obstructive pulmonary disease.

2. The application according to claim 1, characterized in that The kaempferol is used for treating, preventing or improving chronic obstructive pulmonary disease.

3. The application according to claim 1, characterized in that The kaempferol exerts its effect by regulating at least one of the AMPK signaling pathway, the Hippo signaling pathway, the MAPK signaling pathway and the PI3K-AKT signaling pathway.

4. A drug for treating chronic obstructive pulmonary disease, characterized in that: The invention comprises the kaempferol or a pharmaceutically acceptable salt thereof as claimed in claim 1 as an active ingredient.

5. The drug according to claim 4, characterized in that Also included are pharmaceutically acceptable carriers.

6. The medicine according to claim 5, characterized in that The pharmaceutically acceptable carrier is one or more of a solvent, a dispersant, a suspending aid, a surfactant, an isotonic agent, a thickener, a preservative, a solid binder or a lubricant.

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