Application of AH2QDS in preparation of medicine for improving chronic obstructive pulmonary disease

Abstract

The invention provides application of AH2QDS in preparation of medicines for improving chronic obstructive pulmonary disease, which comprises the following steps: establishing an SD rat COPD model and a human bronchial epithelial cell (HBE) cell model, and intervening and observing lung functions, lung histopathology, oxidative stress indexes, inflammatory factor levels and apoptosis by using AH2QDS; molecular docking is used for predicting expression of Keap1-Nrf2 protein, and immunoblotting verification of related protein is carried out. Results show that the AH2QDS can relieve lung tissue injury, improve lung functions (PEF and MMEF) of COPD rats, improve expression of antioxidant enzymes such as SOD, CAT and GSH-PX, and reduce levels, ROS and apoptosis of cell inflammatory factors TNF-alpha, IL-6, IL-1beta and IL-33 through Keap1-Nrf2. The AH2QDS activates a Keap1-Nrf2 pathway to improve the oxidative damage resistance of an organism and reduce the level of inflammatory cytokines, so that the lung function is improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of AH2QDS in the preparation of drugs to improve chronic obstructive pulmonary disease. Background Technology

[0002] COPD is a persistent respiratory disease characterized by airway inflammation and progressive obstruction of lung airflow, and it has become the third leading cause of death worldwide. The causes of COPD include smoking and long-term exposure to toxic gases and particulate matter, leading to apoptosis of airway epithelial cells, impairing cell integrity and function, and consequently causing chronic inflammation and impaired immune responses, ultimately resulting in respiratory disease. An imbalance between oxidation and antioxidation is a crucial factor in its development. Therefore, antioxidation, reducing oxygen free radicals and reactive oxygen species, and improving progressive lung function impairment are key measures for prevention and symptom improvement. However, there are currently no drugs with definitively proven efficacy, and finding novel compounds to improve lung function is an urgent problem to be solved.

[0003] AH2QDS (anthrahydroquinone-2,6-disulfonate) was previously discovered to be a compound with strong antioxidant activity. AH2QDS can activate the intracellular antioxidant system through the PI3K / AKT and Apelin / APJ pathways, increasing the expression level of capillary endothelial cell adhesion protein (ZO-1) and reducing pulmonary microvascular permeability. Simultaneously, it reduces oxidative stress levels in renal tissue, decreases endoplasmic reticulum stress and apoptosis, and enhances cellular antioxidant capacity. However, it remains unclear whether AH2QDS can alleviate oxidative damage, improve lung function, and reduce inflammation levels in COPD (chronic airway inflammation). This invention aims to establish a COPD animal model in SD rats by inducing LPS combined with CS, and to establish a cell model in HBE cells by intervening with LPS combined with cigarette extract (CSE). AH2QDS will then be applied as an intervention to observe its effects on oxidative stress indicators and lung function. Summary of the Invention

[0004] Therefore, this invention proposes the application of AH2QDS in the preparation of drugs to improve chronic obstructive pulmonary disease, thereby solving the above-mentioned problems.

[0005] The technical solution of the present invention is achieved as follows: the application of AH2QDS in the preparation of drugs to improve chronic obstructive pulmonary disease.

[0006] Furthermore, the active ingredient of the drug is AH2QDS.

[0007] Furthermore, the drug also includes pharmaceutically acceptable excipients selected from at least one of diluents, disintegrants, binders, and lubricants.

[0008] Furthermore, the dosage form of the drug is an oral preparation, an injection, or an inhalation preparation; the oral preparation includes tablets, capsules, or granules, and the inhalation preparation includes aerosols or powder inhalers.

[0009] Furthermore, the improvement of chronic obstructive pulmonary disease specifically refers to improving lung function in patients with chronic obstructive pulmonary disease.

[0010] Furthermore, the method for preparing the drug involves mixing the active ingredient AH2QDS with pharmaceutically acceptable excipients and preparing it into the target dosage form according to conventional formulation processes.

[0011] Furthermore, the drug works by activating the Keap1 / Nrf2 pathway to improve chronic obstructive pulmonary disease.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The drug of this invention uses AH2QDS as the active ingredient and is applied to improve chronic obstructive pulmonary disease (COPD). Experiments show that AH2QDS, by binding to Keap1, activates Nrf2 and translocates into the cell nucleus, regulates HO-1 and NQO1 proteins, inhibits oxidative stress, enhances the antioxidant capacity of tissue cells, reduces inflammation levels, improves LOS+CS-induced oxidative stress damage in the lung tissue of COPD rats, and improves lung function. This indicates that AH2QDS can not only enhance the body's antioxidant capacity by activating the Keap1 / Nrf2 pathway, but also reduce the level of inflammatory factors, reduce lung tissue damage, and ultimately improve the core lung function indicators of COPD patients. It can be used in the preparation of drugs to improve chronic obstructive pulmonary disease. Attached Figure Description

[0013] Figure 1 (A) Schematic diagram of LPS+CS-induced COPD model in rats; (B) HE staining of lung tissue from each group, namely Control group, LPS+CS 50-day group, and LPS+CS 100-day group; (C) Mean linear intercept (MLI) (µm) for each group; (D) Mean alveolar area (MAA) (µm) for each group. 2 (EG) Lung function MMEF, PEF, and FEV0.3 / FVC values. n≥3, ns had no statistical significance, *p<0.05, **p<0.01, ***p<0.001; Figure 2To determine the levels of antioxidants and inflammatory factors in plasma and bronchoalveolar lavage fluid of LPS+CS-induced COPD rats. (A) SOD level in serum of each group. (B) MDA level in serum of each group. (C) GSH-PX level in serum of each group. (D) CAT level in serum of each group. (E) IL-1β content in bronchoalveolar lavage fluid of each group. (F) IL-33 content in bronchoalveolar lavage fluid of each group. (G) IL-6 content in bronchoalveolar lavage fluid of each group. (H) TNF-α content in bronchoalveolar lavage fluid of each group. n≥3. ns were not statistically significant, *p<0.05**p<0.01***p<0.001; Figure 3 Effects of AH2QDS and erdosteine ​​on lung pathological damage and lung function in SD rats. (A) HE staining of lung tissue in each group (Bar=50μm). (B) Mean alveolar area (MAA) (µm) 2 (C) Mean linear intercept (MLI) measurement (µm). (D) Maximum mid-expiratory flow (MMEF) values ​​for each group. (E) Peak expiratory flow (PEF) values ​​for each group. n≥3, ns not statistically significant, *p<0.05**p<0.01***p<0.001****p<0.0001; Figure 4 To inhibit LPS+CS-induced oxidative stress and inflammation with AH2QDS. (AD) The levels of SOD, MDA, GSH-PX, and CAT in lung tissue homogenates of mice in each group were detected. (EG) Inflammatory cytokines, IL-1β, IL-33, IL-6, and TNF-α, in BAL supernatant were detected by ELISA. n≥3, ns no statistically significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 5 The effect of AH2QDS on intracellular oxidative stress damage in HBE cells. (A) Superoxide dismutase (SOD) content in each group. (B) Malondialdehyde (MDA) content in each group. (C) Glutathione peroxidase (GSH-PX) content in each group. (D) Catalase (CAT) content in each group. (E) Detection of reactive oxygen species (ROS) by fluorescence and flow cytometry, and quantitative map (Bar=50μm). (F) Mitochondrial membrane potential and quantitative map (Bar=100μm). n≥3, ns not statistically significant, *p<0.05, **p<0.01, ***p<0.001; Figure 6Effects of AH2QDS on LPS+CSE-induced HBE migration, apoptosis, and expression of inflammatory factors. (AB) Transwell plots and quantification of each group. (C) Apoptosis levels in each group. (DG) Levels of IL-6, IL-33, IL-1β, and TNF-α in each group. n≥3, ns not statistically significant, *p<0.05, **p<0.01, ***p<0.001; Figure 7 To investigate the effect of AH2QDS on Nrf2 protein in vitro. In HBE cells, nuclear lysates were extracted for NRF2 expression analysis, and total lysates were extracted for Keap1, HO-1, and NQO1 protein expression analysis. (A) RT-qRCR detection of Nrf2 mRNA expression in each group. (B) Confocal microscopy detection of Nrf2 immunofluorescence expression in each group (Bar=50μm). (C) RT-qRCR detection of Keap1 mRNA expression in each group. (D) Western blot detection of Nrf2 protein expression in the cell nucleus in each group. (E) Western blot detection of Keap1 protein expression in each group. (F) Western blot detection of HO-1 protein expression in each group. (G) Western blot detection of NQO1 protein expression in each group. n≥3, ns not statistically significant, *p<0.05, **p<0.01, ***p<0.001; Figure 8 The effect of AH2QDS on Nrf2 protein in COPD rats was investigated. Nuclear lysates were extracted from lung tissue cells for Nrf2 expression analysis, and total lysates were extracted for Keap1, HO-1, and NQO1 protein expression analysis. (A) Immunohistochemistry was used to assess Nrf2 and Keap1 protein expression in each group of rat lung tissue (Bar=100μm). (BC) mRNA expression of Nrf2 and Keap1 in each group of RT-qRCR rat lung tissue. (D) Western blot was used to detect Nrf2 protein expression in the nuclei of each group of rat lung tissue cells. (EG) Expression of Keap1, HO-1, and NQO1 proteins in each group of rat lung tissue. n≥3, ns showed no statistical significance, *p<0.05, **p<0.01, ***p<0.001; Figure 9ML385 eliminated the antioxidant effect of AH2QDS. HBE cells were treated with ML385, and Nrf2 expression was analyzed from nuclear lysates. HO-1 protein expression was also analyzed from total lysates. (A) Western blot analysis of Nrf2 protein expression in cell nuclei of each group. (B) Western blot analysis of HO-1 protein expression in cells of each group. (C) Superoxide dismutase (SOD) content in each group. (D) Malondialdehyde (MDA) content in each group. There was no statistically significant difference in ns compared to the LPS+CS group; *p < 0.05, **p < 0.01, ***p < 0.001. Figure 10 The effect of AH2QDS on Keap1. (A) Molecular formula of AH2QDS. (B) Interactions of amino acid residues TYR-334, ARG-380, ASN-382, and ASN-414 in AH2QDS and Keap1 protein. (C) Venn diagram of amino acids co-binding with AH2QDS, Nrf2, and Keap1. (D) Root mean square deviation curve (RSM). (D) Results graph; Figure 11 This study explores the mechanism by which AH2QDS alleviates LPS+CS-induced oxidative stress damage and inflammation. Detailed Implementation

[0014] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0015] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0016] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0017] This invention establishes a COPD animal model in SD rats by inducing COPD with LPS combined with CS, and establishes a cell model by intervening in HBE cells with a mixture of LPS and cigarette extract (CSE). AH2QDS is then used for intervention. Example 1 1. Methods and Materials 1.1 Reagents The lipopolysaccharide (LPS) material used in this embodiment of the invention was purchased from Sigma, Inc., USA, and was derived from Escherichia coli O111:B4. The antibodies used were as follows: Nrf2 polyclonal antibody (16396-1-AP), Keap1 polyclonal antibody (10503-2-AP), HO-1 polyclonal antibody (10701-1-AP), NQO1 polyclonal antibody (11451-1-AP), and PCNA polyclonal antibody (10205-2-AP) were purchased from Wuhan Sanying Biotechnology Co., Ltd., and GAPDH recombinant antibody (GB15004-100) was purchased from Wuhan Sewell Biotechnology Co., Ltd.

[0018] HRP-labeled goat anti-rabbit (BL052A) IgG (H+L) was purchased from Lanjieke Technology Co., Ltd. The enhanced ECL chemiluminescence assay kit (36222ES60) and BCA protein assay kit (20201ES76) were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0019] 1.2 Preparation of Cigarette Extract (CSE) Pour basal culture medium into a gas washing bottle. Light one cigarette (Huangshan: 10mg tar, 0.8mg nicotine, and 10mg carbon monoxide, 84mm length) for every 10ml of medium. The cigarette should burn for approximately 5 minutes. During this time, use a 20ml syringe to aspirate the smoke through the medium and dissolve it. Aspirate 5 times per minute. After the cigarette has burned out, let the gas washing bottle stand for 2 minutes. Measure the OD value of the obtained culture medium at 320nm using a microplate reader; the OD value should be within the range of 0.77±0.08. Then dissolve the cigarette extract in the culture medium and adjust the pH to 7.4. Filter using a 0.22µm filter. The prepared basal culture medium at this point is considered to have a CSE concentration of 100%.

[0020] 1.3 Cell culture, intervention, and cell viability assay Human airway epithelial cell line (HBE) was purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd. After co-culturing HBE cells with LPS+CSE for 24, 48, and 72 hours, cell viability was measured using a CCK8 assay kit (WhiteShark, China). Intervention with 100 μg / ml LPS and 10% CSE for 24 hours resulted in over 50% cell viability remaining, a better result than interventions at 48 and 72 hours. Pretreatment with 400 μM AH2QDS for 30 minutes showed better cell viability than pretreatment groups with 200 μM and 600 μM. Figure 2 AB).

[0021] Cell experiment groups: (1) control group; (2) LPS+CSE group; (3) LPS+CSE group after AH2QDS pretreatment for 30 min; (4) AH2QDS group.

[0022] 1.4 Flow cytometry and intracellular ROS detection The apoptosis rate of cells in each group was quantitatively determined using the Annexin V-FITC / PI apoptosis detection kit (40302, Yeasen, Shanghai, China). The apoptosis rate was detected using flow cytometry (BDAccuri™ C6Plus). Intracellular ROS levels were measured using a reactive oxygen species (ROS) detection kit (catalog number BL714A; Anhui Biosharp Technology Co., Ltd.). Cells were seeded at a density of 1 x 10⁵ / ml and incubated for 24 hours, followed by LPS+CSE intervention for 24 hours. Fluorescence values ​​were then detected using lasers at wavelengths of 488 nm and 515 nm. ImageJ and Flowjo software were used to analyze the fluorescence intensity of each group.

[0023] 1.5 ELISA detection, mitochondrial membrane potential level measurement and Transwell assay Following the instructions for the MDA, SOD, CAT, and GSH-PX detection kits, the relevant reagents were added to the reactions, and the absorbance (OD) values ​​were measured at 532 and 450 nm using enzyme-labeled markers, respectively. MMPs were measured using JC-1 staining according to the manufacturer's instructions. Bright red fluorescence indicates J-aggregates, and green fluorescence indicates J monomers. Fluorescence was observed using an inverted fluorescence microscope (Leica, Germany), and images were analyzed using ImageJ software. TNF-α, IL-1β, IL-33, and IL-6 in the samples were detected according to the ELISA kit provided by the manufacturer (Vancovel GmbH). Cell migration was assessed using 5µm Transwell plates. 10⁴ HBE cells / well were seeded into the upper migration chamber containing serum-free medium, and the lower chamber was placed in whole culture containing 20% ​​fetal bovine serum. After 24 hours of incubation, the cells on the back of the upper chamber were stained with 0.1% crystal violet (Biosharp Life Sciences) for 10 minutes at room temperature, and non-migrating cells were removed with a cotton swab. Cells were counted under an inverted optical microscope.

[0024] 1.6 Immunoblotting Proteins were extracted, and the concentration was determined using a BCA kit. Equal protein samples were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane under a constant current of 400 mA. The membrane was blocked for 1 hour at room temperature with 5% skim milk powder dissolved in TBST (0.05% Tween-20 in Tris buffer). The membrane was incubated overnight at 4°C with antibody I in a shaker, followed by washing three times with TBST and incubation with antibody II (1:10000 dilution) for 1 hour at room temperature. After washing, the membrane was developed using ECL reagent to observe the blot. Following development, the protein was removed with stripping buffer, and the blocking process was repeated. ImageJ software was used for analysis.

[0025] 1.7 Establishing an animal COPD model Thirty-six male SD rats (7-8 weeks old, average weight 130-150g) were purchased from Changsha Tianqin Co., Ltd. The animals were housed in a standard animal facility (Experimental Animal Center of Hainan Medical University) with a room temperature of 22-24˚C and a 12 / 12-hour light / dark cycle. After 7 days of acclimatization, the rats were randomly divided into four groups (n=6): (1) control group, (2) LPS+CS group, (3) LPS+CS+100mg / kg AH2QDS group, and (4) LPS+CS+30mg / kg Erdosteine ​​group. From the start of modeling, the rats were exposed to smoke daily (20 cigarettes for 20 minutes each time, twice a day). Lipopolysaccharide was instilled into the nasal cavity every 15 days. No cigarette smoke exposure was required on the day of lipopolysaccharide instillation. This treatment lasted for 100 days. From day 60, PBS, AH2QDS, and Erdosteine ​​were administered intraperitoneally twice a day. All animal experiments used were approved by the Ethics Committee of Hainan Medical University (No.: HYLL-2024-063).

[0026] 1.8 Bronchoalveolar lavage fluid, tissue collection, and lung tissue pathology 120 days after modeling, SD rats were euthanized by cervical dislocation after anesthesia. To obtain BALF (Bacillus Alveolar Fluid), the lungs were dissected, the left main bronchus was ligated, and 0.5 ml of ice-cold PBS was slowly injected into the left lung. BALF was then collected from the lungs for subsequent experiments. Right lung tissue was fixed with 4% paraformaldehyde, dehydrated in a gradient of alcohol, and then embedded in paraffin. The paraffin-embedded samples were cut into 4 μm sections and stained with hematoxylin and eosin (H&E). Tissue changes in the lungs were observed under an optical microscope. Mean linear intercept (MLI) and mean alveolar area (MAA) were measured according to reported methods to assess lung injury.

[0027] 1.9 Lung function test Anesthetize the SD rat model with 2% sodium pentobarbital, intubate the rats, place them in the cavity of a pulmonary function testing instrument, connect the instrument (Tawang, Shanghai, China), and measure MMEF and PEF.

[0028] 1.10RT-Qpcr Total RNA was extracted using the TAKARA kit, and DNA was obtained using a reverse transcription kit (Yisheng). Quantitative polymerase chain reaction (PCR) amplification was performed using a real-time quantitative PCR system (ThermoABI QuantStudio3). PCR primers were designed using GAPDH as an endogenous control. Data were analyzed using the 2-ΔΔCT method.

[0029] The specific primers are as follows: Nrf2 (rat forward:5′-CTGCCATTAGTCAGTCGCT-3′, reverse:5′-ACCGTGCCTTCAGTGTG-3′) (Cell forward:5′-CTGGGTTCAGTGACTCGGAAATGG-3′, reverse:5′-AATGTGCTGGCTGTGCTTTAGGG-3′) Keap1 (rat forward:5′-GGGTGGGTTGTTGCTGTC-3′,reverse:5′-GCTTGTTCTGCTGCCTCTT-3′) (Cell forward:5′-TTGGCTGTGTGGAGTTGC-3′,reverse:5′-CGCACGTTCAGGTCGTC-3′) GAPDH (rat forward:5′-CAAGAAGGTGGTGAAGCAG-3′,reverse:5′-CAAAGGTGGAAGAATGGG-3′) (Cell forward:5′-CCTTCCGTGTCCCCACT-3′,reverse:5′-GCCTGCTTCACCACCTTC-3′) 1.11 Molecular docking experiment Download the crystal structure of Keap1 (entry 7K2A) from the RCSB protein database (http: / / www.rcsb.org). Process the crystal structure using PYMOL software to remove water molecules, add hydrogen ions, and balance the charge. Draw the 3D structure file of AH2QDS using ChemDraw and Chem3D software. Perform molecular docking using AutoDockTools 1.5.6 software. Add hydrogen to the Keap1 protein, and perform hydrogen addition and torsion bond determination on smaller molecules. Set the molecular docking parameters using the Grid panel: Keap1: Center(X,Y,Z)=(-42.1,23.5,60.8), size(X×Y×Z)=(20.0×20.0×25.0). Set the docking mode to semi-flexible docking, the docking precision exhaustiveness=25, and the docking algorithm to Lamarck genetic algorithm. Run AutoDockVina 1.2.0 software for molecular docking.

[0030] 1.12 Immunohistochemistry and immunofluorescence The protein expression of Keap1 and Nrf2 in lung tissue was detected. Paraffin sections were dewaxed (using environmentally friendly dewaxing solution; catalog number G1128; Wuhan Kewei Biotechnology Co., Ltd.), and then rehydrated and restored using a series of alcohol-based solutions. The sections were blocked in 3% BSA at room temperature for 30 minutes, then blocked overnight at 4°C with Keap1 and Nrf2 antibodies, followed by incubation with HRP-labeled goat anti-rabbit IgG II antibody at room temperature for 1 hour. Finally, the sections were stained with diaminobenzidine, counterstained with hematoxylin (room temperature, 3 min), and observed under an optical microscope.

[0031] To evaluate the immunofluorescence of Nrf2 nuclear translocation, cells in each group were incubated with 4% paraformaldehyde for 10 min and 0.1% Triton X-100 (Beyotime) for 15 min. Cells were then incubated overnight at 4°C with antibody I (rabbit anti-Nrf2), followed by antibody II (goat anti-mouse IgG, Alexa Fluoride). TM The cells were incubated at 37°C for 1 hour (Plus 488 / 555), and the nuclei were labeled with DAPI. Images were obtained using a laser confocal microscope (Olympus, Tokyo).

[0032] Statistical analysis Data analysis was performed using Prism 10.0 software, and the values ​​are expressed as mean ± SEM. Two-tailed Student's t test was used for comparisons between two groups. Multiple group comparisons were conducted using both one-way and two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0033] 2. Results 2.1 LPS+CS to establish a COPD model in SD rats To establish an animal model of COPD and investigate the effects of AH2QDS on COPD. Since pathological morphological parameters of lung tissue are the most important parameters for evaluating the success of animal model establishment, this study will observe the pathological changes in lung tissue of the animal model. Rats will be exposed to CS for 100 days and LPS will be instilled intranasally once every 15 days. Figure 1 A). For example Figure 1 As shown in BD, compared with the normal group, during LPS+CS days 50 to 100, lung tissue pathology showed gradual widening of alveolar septa, alveolar cavity fusion and enlargement, and a gradual increase in mean linear intercept (MLI) and mean alveolar area (MAA) of the lung tissue. This indicates that the lung tissue pathology is consistent with the pathological changes of COPD. Pulmonary function tests are used to diagnose and grade the severity of chronic obstructive pulmonary disease; therefore, an assessment model based on peak expiratory flow (PEF), maximum mid-expiratory flow (MMEF), and FEV0.3 / FVC was established. Figure 1 As shown in the EG, compared with the control group, rats exposed to LPS+CS had significantly decreased peak expiratory flow (PEF), maximum mid-expiratory flow (MMEF), and FEV0.3 / FVC. Figure 1 (EG). This indicates that LPS+CS induced airway obstruction and a gradual decline in lung function in rats.

[0034] The oxidative stress status and lung inflammation in rats were assessed by detecting the levels of antioxidants and the expression of cellular inflammatory factors. For example... Figure 2 As shown in the AD diagram, compared with the control group, the expression levels of SOD, GSH-PX, and CAT decreased in rats induced by LPS+CS for 50 and 100 days, while the level of MDA increased. This indicates that oxidative stress persists and gradually intensifies during LPS+CS induction. Lung inflammation was detected by measuring inflammatory factors in BALF supernatant using ELISA, including IL-1β, IL-33, TNF-α, and IL-6. Figure 2 As shown in the EH, the expression levels of inflammatory factors were significantly higher in the control group than in the control group, and the expression levels of inflammatory factors gradually increased with the extension of LPS+CS exposure time. This indicates that airway inflammation persists and gradually worsens.

[0035] 2.2 AH2QDS alleviates lung tissue pathological damage and improves lung function in COPD rats like Figure 3 As shown in AC, lung tissue pathology revealed that AH2QDS treatment significantly reduced alveolar wall thickening, alveolar cavity fusion and expansion, and inflammatory cell infiltration. MLI and MAA also showed significant improvement. The AH2QDS treatment group was superior to erdosteine. Regarding lung function, such as... Figure 3As shown in the DE, the MMEF in the LPS+CS group was approximately 3.73 ml / s, and the PEF was approximately 5.75 ml / s. After AH2QDS treatment, the MMEF ranged from 6.35 to 8.99 ml / s, and the PEF ranged from 7.8 to 12.04 ml / s (Table 1). Both significantly improved PEF and MMEF, superior to the erdosteine ​​group. The results indicate that AH2QDS can improve lung function (PEF and MMEF) in COPD rats.

[0036] Table 1 Lung function parameters

[0037] Note: Data is displayed graphically. Figure 3 D, E. Measure lung function PEF and MMEF. 2.3AH2QDS reduced oxidative stress damage and inflammation levels in COPD rats. Evaluation of the expression of antioxidants SOD, GSH-PX, CAT, and oxidative damage product MDA. Figure 4 As shown in the AD diagram, the LPS+CS group exhibited significantly decreased levels of SOD, CAT, and GSH-PX, while MDA significantly increased. Both the AH2QDS and erdosteine ​​groups enhanced the levels of antioxidants SOD, CAT, and GSH-PX while reducing MDA expression. However, AH2QDS was superior to erdosteine ​​in increasing GSH-PX expression levels. These results indicate that AH2QDS can alleviate oxidative damage in the lungs and improve lung function, and is superior to erdosteine.

[0038] Small airway inflammation is a major characteristic of chronic obstructive pulmonary disease (COPD). This study assessed the regulatory role of the AH2QDS in airway inflammation by detecting the expression of inflammatory cytokines TNF-α, IL-6, IL-1β, and IL-33 in bronchoalveolar lavage fluid (BALF). Figure 4 As shown in the EH, the levels of inflammatory cytokines in the AH2QDS and erdosteine ​​groups were lower than those in the LPS+CS group, while AH2QDS showed a more significant inhibition of IL-33 expression than erdosteine. This indicates that AH2QDS can inhibit LPS+CS-induced small airway inflammation.

[0039] 2.4AH2QDS alleviates LPS+CSE-induced oxidative stress in HBE cells The ability of AH2QDS to reduce oxidative stress in vitro was evaluated by detecting SOD, CAT, GSH-PX, and MDA. Figure 5As shown in the AD diagram, the expression of SOD, CAT, and GSH-PX in the LPS+CSE group was significantly decreased compared to the Control group, while the MDA content was significantly increased. After treatment with AH2QDS, the expression levels of SOD, CAT, and GSH-PX were increased, while the expression of MDA was decreased. This indicates that AH2QDS can also alleviate LPS+CSE-induced oxidative stress damage in cells in vitro. Figure 3 As shown in Figure E, LPS+CSE-induced ROS production in HBE cells significantly decreased after AH2QDS treatment, while mitochondrial membrane potential significantly increased. This indicates that AH2QDS can alleviate LPS+CSE-induced intracellular ROS production and protect mitochondrial function.

[0040] Effects of 2.5AH2QDS on LPS+CSE-induced migration, apoptosis, and expression of inflammatory factors in HBE cells The effects of AH2QDS on LPS+CSE-induced HBE cell migration were validated using Transwell assays. Figure 6 As shown in Figure A, the number of cells migrating in the LPS+CSE group after AH2QDS intervention was significantly increased, promoting cell repair. Figure 6 As shown in Figure C, the apoptosis rate in the LPS+CSE group was approximately 26%, while the apoptosis rate in the LPS+CSE group treated with AH2QDS decreased to 16%. This indicates that AH2QDS reduces LPS+CSE-induced HBE cell apoptosis. Furthermore, the expression of IL-6, IL-33, IL-1β, and TNF-α in the AH2QDS-treated group was as follows... Figure 6 DG showed a significant decrease.

[0041] 2.6AH2QDS enhances antioxidant activity by activating the Keap1 / Nrf2 pathway. To further elucidate the potential mechanism by which AH2QDS improves lung function, it is hypothesized that AH2QDS alleviates oxidative stress damage through the Keap1-Nrf2 pathway. Figure 7 As shown in AB and D, RT-qPCR, immunofluorescence, and Western blot analysis revealed that AH2QDS treatment improved the decrease in Nrf2 induced by LPS+CSE in HBE cells and increased the nuclear translocation of Nrf2. Figure 7 As shown in C and E, LPS+CSE treatment increased KEAP1 expression, while the AH2QDS group reversed this change. AH2QDS also significantly increased the expression of HO-1 and NQO1. Figure 7 FG). Similarly, as Figure 8As shown in AG, the expression of Nrf2, HO-1, and NQO1 was decreased and Keap1 was significantly increased in in vitro lung tissue, while AH2QDS reversed these changes. This indicates that AH2QDS can participate in the regulation of the Keap1-Nrf2 pathway, releasing Nrf2 by binding to Keap1, allowing it to enter the cell nucleus and activate the intracellular antioxidant system.

[0042] To further confirm whether AH2QDS exerts its antioxidant effect by activating the Nrf2 pathway, HBE cells were pretreated with the Nrf2-specific inhibitor ML385. Following AH2QDS treatment, the following results were observed: Figure 10 As shown in the AD study, ML385 eliminated the effects of AH2QDS on LPS+CSE-induced increases in Nrf2 and HO-1 expression, as well as the increase in SOD and decrease in MDA. These results indicate that the antioxidant effect of AH2QDS is exerted through the Keap1-Nrf2 pathway.

[0043] 2.7AH2QDS promotes the dissociation of Nrf2 and Keap1 by competitively binding to Keap1 with Nrf2, thereby initiating cellular antioxidant damage mechanisms. The main pathway regulating Nrf2 activity is through its interaction with the Keap1 protein. When Keap1 binds to Nrf2, it inhibits Nrf2 activity; upon dissociation, Nrf2 enters the nucleus and initiates an antioxidant program. Previous studies have revealed multiple amino acid sites on Keap1 that bind to Nrf2. Using molecular docking analysis and computer models, the affinity between AH2QDS and Keap1 protein was predicted, such as... Figure 9 As shown in AB, the three-dimensional chemical structure of AH2QDS interacts with the amino acid residues TYR-334, ARG-380, ASN-382, and ASN-414 in the Keap1 protein through five hydrogen bonds (green dashed lines). Figure 9 As shown in Figure C, the amino acid binding sites of AH2QDS and Keap1 / Nrf2 in previous studies, including three amino acid sites (TYR-334, ARG-380, and ASN-382), are consistent in their involvement in Keap1 binding. The stability of the AH2QDS-Keap1 protein complex was assessed using 100 ns molecular dynamics simulations. Figure 10 As shown in DE, both the root mean square deviation curve (RSMD) and the root mean square fluctuation curve (RMSF) exhibit small fluctuations and good stability.

[0044] 3. Conclusion This study aimed to demonstrate that AH2QDS activates the Keapi-Nrf2 pathway to initiate intracellular antioxidant damage mechanisms, alleviating LPS+CS-induced small airway oxidative stress damage in COPD rats, reducing the expression of inflammatory factors in lung tissue, and improving lung function PEF and MMEF. AH2QDS, as a potent antioxidant, competitively binds to Keap1, releasing Nrf2 into the nucleus and activating Nrf2. This result was also confirmed in in vitro experiments on LPS+CS-stimulated HBE cells. Nrf2 expression was increased in the AH2QDS group. Furthermore, the AH2QDS group showed increased intracellular SOD, CAT, and GSH-PX expression levels, decreased MDA expression, and significantly reduced ROS levels; IL-6, IL-33, IL-1β, and TNF-α levels were also decreased to varying degrees. This indicates that AH2QDS activates the antioxidant system, increasing the levels of antioxidant factors and reducing the levels of inflammatory factors. Molecular docking analysis and immunoblotting results demonstrated that AH2QDS has a high affinity for Keap1, and through competitive binding with Keap1, it promotes the dissociation of Nrf2 from Keap1 and enhances the ability of Nrf2 to translocate.

[0045] An imbalance between oxidation and antioxidation is a crucial factor in the pathogenesis of COPD. Antioxidant damage can, to some extent, slow the onset and progression of COPD. Studies have reported that during the pathophysiology of COPD, lung tissue, under the influence of reactive oxygen species (ROS), experiences exacerbated lipid peroxidation, reduced antioxidant defense capacity, and stimulated the release of large amounts of inflammatory cytokines, leading to damage to small airway epithelial cells and causing airway obstruction. Superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and catalase (CAT) can protect small airways from damage by counteracting ROS. IL-6, TNF-α, and IL-1β can induce fibroblast proliferation, leading to thickening of the small airway walls, structural remodeling, further exacerbating airflow limitation, and ultimately causing a decrease in pulmonary arterial function (PEF) and partial pulmonary embolism response (MMEF), which is consistent with these findings. AH2QDS, as a potent antioxidant, has been shown to improve pulmonary vascular permeability and reduce lung injury. This study further demonstrates that AH2QDS effectively alleviates oxidative stress damage in lung tissue of a COPD rat model by increasing the expression of SOD, GSH-PX, and CAT, and improves PEF and MMEF. It also improves mitochondrial function, reduces apoptosis, lowers ROS and cytokine levels, and protects small airway epithelial cells. Furthermore, in vitro experiments have shown that AH2QDS can increase cell migration and proliferation, and enhance their repair function. Therefore, AH2QDS shows promise as a potential compound for the prevention and treatment of COPD.

[0046] The mechanism of action of AH2QDS involves activation of the Keap1-Nrf2 pathway. Keap1-Nrf2 is a classic antioxidant pathway that regulates the transcription of antioxidant genes and immune factors, playing a crucial role in oxidative stress and inhibiting disease progression. In this study, the application of the Nrf2 specific inhibitor ML385 by AH2QDS promoted increased HO-1 and SOD expression, and eliminated the reduction in MDA, further demonstrating that AH2QDS functions through the Keap1-Nrf2 pathway. Furthermore, molecular docking revealed that AH2QDS interacts with amino acid residues TYR-334, ARG-380, ASN-382, and ASN-414 in the Keap1 protein, also proving that Keap1-Nrf2 is the signaling pathway through which AH2QDS exerts its effects. LPS+ cigarette smoke induces an increase in Keap1 and a downregulation of nuclear Nrf2 and its downstream targets HO-1 and NQO1. AH2QDS alleviates airway oxidative stress damage by increasing Nrf2 expression in the cell nucleus. In vivo experiments have shown that AH2QDS is superior to erdosteine ​​in terms of antioxidant activity and reduction of pro-inflammatory cytokine levels.

[0047] Although preliminary findings suggest that AH2QDS affects the development and progression of COPD through Keap1-Nrf2, further research is needed to determine whether AH2QDS can also alter the activity of other pathways and thus influence the pathophysiological process of COPD. This is because COPD is a heterogeneous disease involving the regulation of multiple signaling networks, such as the RAGE / EGFR pathway. Furthermore, this study did not perform transcriptome sequencing or promoter sequencing on the AH2QDS treatment group, thus failing to fully understand the changes in related signaling pathways. This limits the scope of this study regarding the mechanism of action of AH2QDS on COPD.

[0048] In summary, AH2QDS, by binding to Keap1, activates Nrf2 and translocates to the nucleus, regulates HO-1 and NQO1 proteins, inhibits oxidative stress, enhances the antioxidant capacity of tissue cells, reduces inflammation levels, improves LOS+CS-induced oxidative stress damage in the lung tissue of COPD rats, and improves lung function (PEF, MMEF). Figure 11 ).

[0049] Therefore, AH2QDS improves lung function (PEF) and MMEF in COPD rats through Keap1 / Nrf2 pathway activation. This suggests that AH2QDS has the potential to become an important candidate compound for the treatment of COPD.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of AH2QDS in the preparation of drugs to improve chronic obstructive pulmonary disease.

2. The application as described in claim 1, characterized in that, The active ingredient of the drug is AH2QDS.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients selected from at least one of diluents, disintegrants, binders, and lubricants.

4. The application according to claim 1, characterized in that, The dosage form of the drug is an oral preparation, an injection, or an inhalation preparation; the oral preparation includes tablets, capsules, or granules, and the inhalation preparation includes aerosols or powder inhalers.

5. The application according to claim 1, characterized in that, The improvement of chronic obstructive pulmonary disease specifically refers to improving lung function in patients with chronic obstructive pulmonary disease.

6. The application of the drug according to claim 1, characterized in that, The method for preparing the drug is to mix the active ingredient AH2QDS with pharmaceutically acceptable excipients to prepare the target dosage form.

7. The application as described in claim 1, characterized in that, The drug works by activating the Keap1 / Nrf2 pathway to improve chronic obstructive pulmonary disease.

Images