Use of a compound specifically targeting oncostatin m in the preparation of a medicament for the treatment of pseudomonas aeruginosa infection-induced lung injury

Abstract

The application discloses application of a compound specifically targeting onimstatin M in preparation of a medicine for resisting Pseudomonas aeruginosa infectious lung injury, relates to the technical field of biological medicine, and the compound plays a role in resisting Pseudomonas aeruginosa infectious lung injury through specific binding of onimstatin M protein; the compound can be specifically and stably combined at a key function site of the onimstatin M protein; the compound is used for reducing the content of TNF-alpha, IL6 and IL1beta in serum of an individual with lung injury caused by Pseudomonas aeruginosa infection; a medicine composition is provided, and the medicine composition comprises an effective amount of the compound and a pharmaceutically acceptable carrier; based on the new target of onimstatin M, an active medicine capable of effectively reducing lung injury is developed, and in particular, a compound overcoming the limitations of high cost and injection administration of an antibody medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a compound that specifically targets tumor suppressor M in the preparation of a drug for treating Pseudomonas aeruginosa-induced lung injury. Background Technology

[0002] Pseudomonas aeruginosa (PA) is a common Gram-negative opportunistic pathogen causing lower respiratory tract infections and ventilator-associated pneumonia. Infections with this bacterium are common in immunocompromised patients, those with structural lung disease, or those undergoing invasive procedures, and are often severe with a high mortality rate. Currently, clinical treatment of PA infections primarily relies on antibiotics. However, with the widespread use of broad-spectrum antibiotics such as carbapenems, the prevalence of carbapenem-resistant and "refractory" drug-resistant Pseudomonas aeruginosa is becoming increasingly serious. According to data from the 2025 China Antimicrobial Resistance Surveillance (https: / / www.chinets.com), PA exhibits persistently high resistance rates to imipenem (resistance rate 22.6%) and meropenem (resistance rate 18.8%), posing a significant challenge to clinical treatment. Although novel enzyme inhibitor combination therapies (such as ceftazidime-avibactam) are effective against some drug-resistant bacteria (resistance rate 6.7%), their application remains limited by availability, cost, and the potential risk of resistance evolution. Current treatment strategies primarily focus on directly killing or inhibiting bacteria, lacking effective interventions for the excessive host inflammatory response and subsequent lung tissue damage caused by infection. PA, through its virulence factors (such as type III secretory system toxins), can directly damage alveolar epithelium and vascular endothelium, triggering a strong host immune response, leading to acute lung injury and even acute respiratory distress syndrome. This tissue damage is a key factor affecting patient prognosis.

[0003] In recent years, controlling infection and reducing tissue damage by modulating the host's immune response has become a new approach in anti-infective therapy. Cytokines are core regulators of the immune response. Oncosamine M (OSM), a member of the interleukin-6 (IL-6) family, is mainly secreted by activated immune cells such as macrophages, T cells, and granulocytes. Recent research has found that in respiratory viral infection models caused by influenza A virus, macrophage-derived OSM is crucial for repairing the damaged lung epithelial barrier and mitigating type I interferon-mediated immunopathological damage. This study indicates that OSM promotes the proliferation of type II alveolar epithelial cells and is a key mediator for post-infectious lung tissue repair. However, current research on OSM focuses on viral infection or chronic inflammatory disease models (such as pulmonary fibrosis), and its role in bacterial pneumonia, particularly lung injury caused by infection with the important Gram-negative bacterium *Pseudomonas aeruginosa*, has not been reported. OSM plays a protective role in Pseudomonas aeruginosa infection and participates in the pathological damage process. Current treatments for Pseudomonas aeruginosa infection primarily rely on antibiotics, but these face increasingly serious challenges due to bacterial resistance. Furthermore, this strategy cannot effectively intervene in lung tissue damage caused by excessive host immune responses during infection, severely impacting patient prognosis. While host-guided therapy offers new insights into anti-infective treatment, a clear and effective host target is still lacking in the field of Pseudomonas aeruginosa infection. Although recent studies have shown that oncostatin M plays a crucial role in the repair of viral lung injury, the expression patterns and functional roles of OSM in lung injury caused by bacteria such as Pseudomonas aeruginosa are completely unknown, making it impossible to determine whether it can serve as a therapeutic target. Moreover, no drugs (especially compounds) targeting this target have been developed for the treatment of bacterial pneumonia. Therefore, this paper aims to provide a compound that specifically targets oncostatin M for the application in the preparation of drugs against Pseudomonas aeruginosa-induced lung injury. Summary of the Invention

[0004] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide an application of a compound that specifically targets OSM in the preparation of a drug for treating Pseudomonas aeruginosa-induced lung injury. This invention clarifies whether OSM plays a key role in Pseudomonas aeruginosa-induced lung injury and verifies its feasibility as a drug target. Based on this new target, an active drug that can effectively alleviate lung injury is developed, especially a compound that overcomes the limitations of antibody drugs, such as high cost and the need for injection administration.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The application of a compound that specifically targets tumor suppressor M in the preparation of a drug for treating Pseudomonas aeruginosa-induced lung injury, wherein the compound has the following structural formula: ; The compound exerts its anti-pulmonary injury effect against Pseudomonas aeruginosa infection by specifically binding to the tumor suppressor M protein.

[0006] As a preferred embodiment, the compound is able to specifically and stably bind to key functional sites of the tumor suppressor M protein.

[0007] As a preferred embodiment, the compound is used to reduce the levels of TNF-α, IL6, and IL1β in the serum of individuals with lung injury caused by Pseudomonas aeruginosa infection.

[0008] As a preferred embodiment, the screening method for the compounds includes the following steps: S1. Provide a three-dimensional structural model of the tumor suppressor M protein; S2. Perform molecular docking simulation between the compounds to be screened and the tumor suppressor M protein, and calculate the binding free energy; S3. Select compounds that interact with the active site of the tumor suppressor M protein as candidate compounds.

[0009] As a preferred embodiment, the interactions include hydrogen bonds, salt bridges, π-π stacking, or van der Waals forces.

[0010] As a preferred embodiment, the candidate compound is capable of specifically binding to the tumor suppressor M protein and blocking its binding to the receptor.

[0011] A pharmaceutical composition comprising an effective amount of a compound and a pharmaceutically acceptable carrier.

[0012] As a preferred embodiment, the pharmaceutical composition is applied to treat Pseudomonas aeruginosa-induced lung injury, wherein the compounds in the pharmaceutical composition are used to alleviate the effects of Pseudomonas aeruginosa-induced lung injury and inhibit excessive inflammation.

[0013] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, as can be seen from the above technical solution: First, this invention demonstrates that OSM is a key host factor leading to acute lung injury in a specific pathogenic model of Pseudomonas aeruginosa infection, thus establishing OSM as a novel target for anti-infective therapy. Second, in a lethal animal infection model, this invention demonstrates that targeting and inhibiting OSM with this compound can significantly reduce pathological damage to lung tissue and substantially improve the survival rate of infected animals. Finally, this invention targets fatal acute lung injury caused by drug-resistant bacterial infections, providing a novel therapeutic approach that improves prognosis by modulating the host response (anti-injury) rather than directly killing bacteria, which has significant potential value in addressing the problem of antibiotic resistance.

[0014] Second, in PA infection, this application shows that the compound reduces pathological damage to lung tissue, inhibits excessive inflammation, and significantly improves survival; it mimics secondary lung injury caused by PA infection, a specific clinical etiology, in which OSM is a key mediator of the host response after infection; it addresses the problem of drugs for OSM-mediated fatal pneumonia caused by specific bacterial infections; and it provides a novel host-guided therapeutic drug for drug-resistant bacterial pneumonia.

[0015] Third, this compound was placed in a complex bacterial infection disease that is closer to clinical reality, demonstrating that it can intervene in the complex network of "infection → host immune response → tissue damage" by targeting OSM. In lung injury caused by Pseudomonas aeruginosa infection, the expression of the host factor OSM was significantly upregulated, and it was confirmed that inhibiting OSM (through neutralizing antibodies) can effectively reduce lung injury and improve survival rate. This establishes OSM as a novel therapeutic target for antibacterial infection.

[0016] Fourth, a novel OSM-targeting compound capable of achieving the aforementioned therapeutic uses was provided. Through high-throughput screening and molecular docking technology, a compound with a well-defined chemical structure (3-[(2H-1,3-benzodioxane-5-yl)methylene]-5-(4-methoxyphenyl)-2(3H)-furanone) was verified. This compound can specifically target OSM, and its efficacy in alleviating lung injury caused by PA infection was verified in animal models, similar to that of OSM-neutralizing antibodies.

[0017] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the expression of OSM protein in a lung injury model caused by Pseudomonas aeruginosa infection according to the present invention. Figure 2 This is a schematic diagram illustrating the effect of the OSM neutralizing antibody of the present invention on lung injury caused by PA infection; Figure 3 This is a schematic diagram of the binding conformation of the compound of the present invention with the OSM structure; Figure 4 This is a schematic diagram illustrating the effect of the compound of the present invention on lung injury caused by PA infection.

[0019] Explanation of reference numerals in the attached diagram: Figure 1A. HE staining images of different mouse lung tissue pathological sections, scale bar: 20μm. B. Bar chart showing lung injury scores of different mouse lung tissue sections, n=4, ****: P<0.0001. C. OSM immunofluorescence staining of lung tissue sections from PA and PBS groups observed by confocal microscopy, OSM is labeled in red, and DAPI staining of cell nuclei is labeled in blue, scale bar: 20μm. D. Bar chart showing the level of cytokine OSM in bronchoalveolar lavage fluid (BALF) of different mice detected by ELISA, n=4, **: P<0.01.

[0020] Figure 2 A. Representative images of HE-stained pathological sections of different mouse lung tissues observed under a microscope, scale bar: 20 μm. B. Bar chart showing lung injury scores of different mouse lung tissues, n=4, ****: P<0.0001. C. Survival curves showing the survival of the two groups of mice within 7 days after PA infection.

[0021] Figure 3 A. Overall view of the compound's binding position in the OSM protein structure. Carbon atoms are shown in cyan, oxygen atoms in red, nitrogen atoms in blue, and sulfur atoms in yellow. The OSM protein backbone is gray. B. Partial view of the compound's position in the OSM structure. Carbon atoms are shown in cyan, oxygen atoms in red, and nitrogen atoms in blue. Numbers such as 3.6, 3.5, and 3.3 (unit: Å) indicate the predicted distance between specific atoms on the compound and specific atoms on the protein side chain.

[0022] Figure 4 A. Representative images of HE staining of different mouse lung tissue pathological sections observed under a microscope, scale bar: 20 μm. B. Bar chart showing the levels of cytokines (IL1β, IL6, and TNF-α) in the serum of different mice as detected by ELISA, n=4, *: P<0.05, **: P<0.01, ****: P<0.0001. C. Survival curves showing the survival of the two groups of mice within 7 days after PA infection. Detailed Implementation

[0023] The present invention is as follows Figure 1 As shown in Figure 4, a compound that specifically targets tumor suppressor M is used in the preparation of a drug for treating Pseudomonas aeruginosa-induced lung injury. The structural formula of this compound is: ; This compound exerts its anti-pulmonary injury effect against Pseudomonas aeruginosa infection by specifically binding to the tumor suppressor M protein.

[0024] This compound can specifically and stably bind to key functional sites of the tumor suppressor M protein.

[0025] This compound is used to reduce the levels of TNF-α, IL6, and IL1β in the serum of individuals with lung injury caused by Pseudomonas aeruginosa infection.

[0026] The screening method for this compound includes the following steps: S1. Provide a three-dimensional structural model of the tumor suppressor M protein; S2. Perform molecular docking simulation between the compounds to be screened and the tumor suppressor M protein, and calculate the binding free energy; S3. Select compounds that interact with the active site of the tumor suppressor M protein as candidate compounds.

[0027] This interaction includes hydrogen bonds, salt bridges, π-π stacking, or van der Waals forces.

[0028] This candidate compound can specifically bind to the tumor suppressor M protein and block its binding to the receptor.

[0029] A pharmaceutical composition comprising an effective amount of a compound and a pharmaceutically acceptable carrier.

[0030] This pharmaceutical composition is used to treat Pseudomonas aeruginosa-induced lung injury. The compounds in this composition are used to reduce the effects of Pseudomonas aeruginosa-induced lung injury and inhibit excessive inflammation.

[0031] Example: Application of a compound that specifically targets tumor suppressor M in the preparation of a drug for treating Pseudomonas aeruginosa-induced lung injury. Experimental materials: OSM antibody (NBP3-16686, Novus, USA), mouse OSM neutralizing antibody (AF-495-NA, R&D Systems, USA), mouse (G3A1) IgG antibody (5415, CST, USA), goat anti-rabbit Alexa Fluor™ 594 antibody (Invitrogen, USA). DAPI (4′6-diamidinyl-2-phenylindole, Thermo Fisher Scientific, USA). FV3000 laser confocal microscope (OLYMPUS, Japan). Agilent Cary 60 UV spectrophotometer (Agilent Technologies, USA), Innova® 40 / 40R benchtop rocker (New Brunswick, USA), Leica CM1860 cryostat (Leica Biosystems, Germany). PAO1 bacterial strain was provided and preserved by the Chengdu Institute of Respiratory Health. Six- to eight-week-old male C57BL / 6J mice were purchased from Chongqing Tengxin Biotechnology Co., Ltd. All animal experiments were conducted in accordance with the guidelines of the Animal Protection and Utilization Committee of Southwest Jiaotong University. The target compound was synthesized by ChemDiv (product number: 5639-0112) and purchased from Shanghai TargetMol (50mg specification).

[0032] The common name of this compound is: 3-[(2H-1,3-benzodioxane-5-yl)methylene]-5-(4-methoxyphenyl)-2(3H)-furanone.

[0033] Molecular formula: C19H14O5.

[0034] SMILES: COc1ccc(cc1)C1=C / C(=C / c2ccc3c(c2)OCO3)C(=O)O1.

[0035] The structural formula of the compound is: .

[0036] I. Detection of OSM protein expression levels in a mouse lung injury model. Experimental methods: The resuscitation and subculturing of PAO1 bacteria: PAO1 cryopreserved strains were inoculated into LB broth and cultured at 37°C with shaking (200 rpm) for 12-16 hours to restore activity, until the medium became turbid (OD600 value 1.0-1.2). Every 24 hours, 1% of the inoculum was transferred to fresh medium and cultured at 37°C with shaking to maintain the strain in the logarithmic growth phase. All operations were performed in a biosafety cabinet under strict aseptic conditions.

[0037] Establishment of a mouse lung injury model: C57BJ / 6J mice were randomly divided into two groups: the PAO1-treated infection group (PA group) and the PBS-treated control group (PBS group). After one week of acclimatization, mice in the PA group were anesthetized by inhalation of isoflurane, and each mouse was given 30 μL of PAO1 (1 × 10^7 CFU) via the nose to induce lung infection. The PBS group was given an equal volume of PBS. Twenty-four hours later, the mice were euthanized by cervical dislocation under CO2 anesthesia, and lung tissue was collected for subsequent experiments.

[0038] Hematoxylin-eosin (HE) staining and lung injury scoring: Mouse lung tissue was soaked in 4% paraformaldehyde for 48 hours, then dewaxed with gradient ethanol, cleared with xylene, embedded in paraffin, and cut into 5µm thick sections. The sections underwent dewaxing with xylene → gradient hydration → hematoxylin for 5 minutes → differentiation with 1% hydrochloric acid and ethanol → eosin for 30 seconds → dehydration and clearing → mounting with neutral resin, and then observed and scanned using a whole-section scanning system (VS200, Olympus). A semi-quantitative histopathological scoring method was then used to score the lung tissue damage. The main steps were: blind evaluation of the HE-stained sections; random selection of multiple fields of view under a microscope; scoring of each field of view according to severity (0-4 points) based on lung injury indicators such as inflammatory cell infiltration, edema, and hemorrhage; calculation of the average score for each field of view; and the total score of all indicators as the quantitative value of the damage degree of the sample.

[0039] Enzyme-linked immunosorbent assay (ELISA) was used to detect OSM content in mouse bronchoalveolar lavage fluid (BALF). All procedures were strictly followed according to the manufacturer's instructions, as briefly described below: Collected mouse BALF and standards were sequentially added to 96-well plates pre-coated with anti-OSM monoclonal antibody and incubated at room temperature. After washing, biotin-labeled detection antibody was added, followed by incubation and washing again. Horseradish peroxidase-labeled streptavidin was then added, followed by incubation and washing, and TMB substrate was added for a light-protected colorimetric reaction. The reaction was terminated with dilute sulfuric acid, and the optical density (OD) of each well was immediately measured at 450 nm using a microplate reader. The absolute concentration of OSM in each sample was calculated using a standard curve plotted based on the standard concentration and its corresponding OD value. Final data are expressed as picograms of OSM per milligram of total protein (pg / mL).

[0040] Immunofluorescence staining of lung tissue: Fresh left lung tissue from mice was soaked in 4% paraformaldehyde for 48 hours, then dehydrated by soaking in 30% sucrose solution for 48 hours. After being embedded in OCT and rapidly frozen for 24 hours, the lung tissue was sectioned using a cryostat to a thickness of 5 µm. Subsequently, the frozen tissue sections were permeabilized with 0.05% Tween 20 for 10 minutes, incubated with 10% goat serum at room temperature for 1 hour to block non-specific antibodies, then incubated overnight at 4˚C with OSM mouse monoclonal antibody (1:200 dilution), and then incubated with goat anti-mouse Alexa Fluor™ 594 antibody (1:1000 dilution) at room temperature for 2 hours to bind primary antibody. The sections were washed three times with PBS to remove unbound secondary antibody. Cell nuclei were stained with DAPI, and the sections were mounted with an anti-fluorescence quencher. The sections were then observed and photographed using a laser confocal microscope.

[0041] Experimental results: In a lung injury model induced by Pseudomonas aeruginosa infection, OSM protein expression was increased. HE staining results are as follows Figure 1 As shown in Figure A, the alveolar structure of the lung tissue in the PBS group mice was clear and intact, with no interstitial thickening and very few inflammatory cells; the alveolar walls of the PA group were significantly thickened, with a large number of inflammatory cells infiltrating, and fluid exudation and hemorrhage were visible in the alveolar cavities, exhibiting typical pathological changes of acute lung injury. The lung injury of the mice in both groups was scored using the classic semi-quantitative overall lung injury scoring method, and the results are as follows: Figure 1 As shown in Figure B, compared with the PBS group, the lung injury score of mice in the PA group was significantly increased, and the difference was statistically significant. Immunofluorescence results are shown below. Figure 1 As shown in Figure C, the red fluorescence of OSM-labeled molecules in the lung tissue of mice in the PA group was significantly increased compared to the PBS group. ELISA results (as shown in Figure C) Figure 1 (D) Analysis showed that OSM expression in the BALF of the PA group was significantly higher than that in the PBS group, and the difference was statistically significant. In conclusion, OSM expression was increased in the lung tissue of mice with lung injury after PA infection.

[0042] II. OSM neutralizing antibodies can reduce lung injury caused by PA infection. Experimental methods: Animal model establishment: C57BJ / 6J mice were randomly divided into three groups: an experimental group treated with anti-OSM neutralizing antibody (anti-OSM+PA group), an infection group treated with IgG isotype control antibody (IgG+PA group), and a control group (Ctrl group). Mice in the anti-OSM+PA group were injected intraperitoneally with 200 µL of 2 μg / μL (400 μg / mouse) OSM neutralizing antibody, while mice in the IgG+PA and Ctrl groups were injected intraperitoneally with an equal amount of IgG isotype control antibody. Subsequently, the IgG+PA and anti-OSM+PA groups were anesthetized by inhalation of isoflurane, and then 30 μL of PAO1 (1×10^7 CFU) was instilled nasally in each group. Twenty-four hours later, the mice in each group were euthanized by cervical dislocation under CO2 anesthesia, and lung tissue was collected for frozen sections.

[0043] Survival analysis of mice after infection: Mice in the anti-OSM+PA group and the IgG+PA group were observed for 7 consecutive days after PA infection, and the time of death for each mouse was accurately recorded. Surviving mice were recorded as "surviving". Mice still alive at the end of the experiment had their survival time recorded as 7 days and were marked as "censored data" in the analysis. Survival curves were plotted using GraphPad Prism software.

[0044] The methods for HE staining and lung injury scoring are the same as described above.

[0045] Experimental results: HE staining results of lung tissue ( Figure 2 A) shows that the IgG+PA group mice exhibited extensive inflammatory cell infiltration around the trachea and blood vessels in their lung tissue, along with significant thickening of the alveolar walls and marked exudation within the alveolar cavities; however, no such changes were observed in the Ctrl group. Compared to the IgG+PA group, the Anti-osm+PA group mice showed significantly reduced inflammatory cell infiltration around the trachea and blood vessels in their lung tissue, lessened alveolar structural damage, and improved edema and hemorrhage. Figure 2 As shown in Figure B, the lung injury score in the IgG+PA group was significantly higher than that in the Ctrl group, with a statistically significant difference; while the lung injury score in the Anti-osm+PA group was significantly lower than that in the IgG+PA group, with a statistically significant difference. Survival analysis ( Figure 2 C) shows that the survival rate of mice in the IgG+PA group decreased sharply after infection, with deaths starting on day 2, and the survival rate reaching only 20% by the end of the observation period on day 7. This indicates that PA infection established a successful fatal pneumonia model. The decline in survival rate of mice in the anti-OSM+PA group was significantly slowed, and the survival rate remained significantly higher than that of the IgG+PA group throughout the 7-day observation period, maintaining 80% survival on day 7. Compared with the IgG+PA group, the risk of death in the anti-OSM+PA group was reduced by 47% (HR=0.53).

[0046] III. Compound screening methods and visualization of binding to OSM proteins Experimental methods: Virtual screening and molecular docking were performed using the Alphafold predicted structure of human OSM protein downloaded from the uniprot website (https: / / www.uniprot.org / ). The OSM protein structure was optimized using the Protein Preparation Wizard module in the Schrödinger software, removing irrelevant ligands and redundant parts, adding hydrogen atoms, and performing an energy minimization optimization step. Compound docking was performed on each compound in the Taoshu Biotechnology's marketed drug library (L1000) using Schrödinger software. Compounds with the optimal binding free energy (ΔG < -30 kcal / mol) and binding modes indicating the formation of at least three key hydrogen bonds and stable hydrophobic stacking with the OSM active site were selected as primary candidate molecules. The molecular docking results were visualized using PyMOL 2.6.0 software. The theoretical binding mode diagram shows that specific functional groups of this compound form spatial and electronic complementarity with key residues in the OSM active site.

[0047] Experimental Results: High-throughput screening of the commercially available compound library provided by Taoshu Biotechnology Co., Ltd. was performed using Schrödinger software, successfully identifying compounds capable of binding to OSM proteins. These compounds can form stable three-dimensional bindings to the target proteins, such as... Figure 3 As shown in Figure A, the target compound and the OSM protein have a binding energy of -8.0 kcal / mol, indicating stable binding. ARG-91 (arginine) and GLU-165 (glutamate) of the OSM protein form salt bridges or strong hydrogen bonds with the compound atoms. The benzene ring of PHE-169 (phenylalanine) interacts with the aromatic ring or hydrophobic fragments in the target compound through face-to-face π-π stacking or edge-to-face T-type interactions, helping to stabilize the target compound within the protein's hydrophobic pocket; the hydrophobic residue ALA-55 (alanine) binds to the compound via van der Waals forces. Meanwhile, Figure 3 B also shows interactions with residues such as ARG-84 (distance 3.3 Å), indicating that the compound does not bind to a single residue, but rather to a complex network of interactions consisting of ARG-91, GLU-165, PHE-169, ARG-84, ALA-55, etc.

[0048] Therefore, the above results indicate that the compounds screened in this invention can specifically and stably bind to key functional sites of OSM proteins.

[0049] IV. The compound can alleviate lung damage caused by PA infection. Experimental methods: Animal model establishment: C57BJ / 6J mice were randomly divided into three groups: the experimental group treated with the compound (compound + PA group), the PA infection group (PA group), and the control group (Ctrl group). Mice in the compound + PA group were injected intraperitoneally with 10 mg / kg of the compound, while mice in the PA and Ctrl groups were injected intraperitoneally with an equal volume of DMSO control solvent. Simultaneously, mice in the PA group and the compound + PA group were anesthetized by inhalation of isoflurane, and then each group received 30 μL of PAO1 (1 × 10^7 CFU) via nasal instillation. Twenty-four hours later, the mice in each group were euthanized by cervical dislocation under CO2 anesthesia, and lung tissue was collected for frozen sections.

[0050] The methods for HE staining and enzyme-linked immunosorbent assay (ELISA) for detecting cytokines are the same as above.

[0051] Experimental results: Results of HE staining of lung tissue ( Figure 4 A) shows that in the PA-infected mice, there was extensive inflammatory cell infiltration around the trachea and blood vessels in the lung tissue, alveolar wall thickening, and a large number of erythrocytes exuded in the alveoli. Compared with the PA group, the compound + PA group showed significantly reduced inflammatory cell infiltration around the airways and blood vessels, reduced alveolar wall thickening, and significantly reduced erythrocyte exudation in the alveoli. Serum cytokines were detected by ELISA, and the results are as follows. Figure 4 As shown in BD, compared with the Ctrl group, the serum levels of TNF-α, IL-6, and IL-1β in the PA group mice were significantly increased, and the differences were statistically significant; compared with the PA group, the serum levels of TNF-α, IL-6, and IL-1β in the compound + PA group mice were significantly decreased, and the differences were statistically significant. Survival analysis ( Figure 4 C) showed that the decline in survival rate in the compound + PA group was significantly slower than that in the PA group. Throughout the 7-day observation period, the survival rate remained significantly higher in the compound + PA group, and remained at 50% on day 7. Compared with the PA group, the risk of death in the compound + PA group was reduced by 50% (HR=0.5). These results indicate that the compound alleviated lung injury caused by PA infection.

[0052] The key design focus of this invention is: First, this invention demonstrates that OSM is a key host factor leading to acute lung injury in a specific pathogenic model of Pseudomonas aeruginosa infection, thus establishing OSM as a novel target for anti-infective therapy. Second, in a lethal animal infection model, this invention demonstrates that targeting and inhibiting OSM with this compound can significantly reduce pathological damage to lung tissue and substantially improve the survival rate of infected animals. Finally, this invention targets fatal acute lung injury caused by drug-resistant bacterial infections, providing a novel therapeutic approach that improves prognosis by modulating the host response (anti-injury) rather than directly killing bacteria, which has significant potential value in addressing the problem of antibiotic resistance.

[0053] Second, in PA infection, this application shows that the compound reduces pathological damage to lung tissue, inhibits excessive inflammation, and significantly improves survival; it mimics secondary lung injury caused by PA infection, a specific clinical etiology, in which OSM is a key mediator of the host response after infection; it addresses the problem of drugs for OSM-mediated fatal pneumonia caused by specific bacterial infections; and it provides a novel host-guided therapeutic drug for drug-resistant bacterial pneumonia.

[0054] Third, this compound was placed in a complex bacterial infection disease that is closer to clinical reality, demonstrating that it can intervene in the complex network of "infection → host immune response → tissue damage" by targeting OSM. In lung injury caused by Pseudomonas aeruginosa infection, the expression of the host factor OSM was significantly upregulated, and it was confirmed that inhibiting OSM (through neutralizing antibodies) can effectively reduce lung injury and improve survival rate. This establishes OSM as a novel therapeutic target for antibacterial infection.

[0055] Fourth, a novel OSM-targeting compound capable of achieving the aforementioned therapeutic uses was provided. Through high-throughput screening and molecular docking technology, a compound with a well-defined chemical structure (3-[(2H-1,3-benzodioxane-5-yl)methylene]-5-(4-methoxyphenyl)-2(3H)-furanone) was verified. This compound can specifically target OSM, and its efficacy in alleviating lung injury caused by PA infection was verified in animal models, similar to that of OSM-neutralizing antibodies.

[0056] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The application of a compound that specifically targets tumor suppressor M in the preparation of a drug for treating Pseudomonas aeruginosa-induced lung injury, characterized in that: The structural formula of the compound is: ； The compound exerts its anti-pulmonary injury effect against Pseudomonas aeruginosa infection by specifically binding to the tumor suppressor M protein.

2. The application according to claim 1, characterized in that: The compound can specifically and stably bind to key functional sites of the tumor suppressor M protein.

3. The application according to claim 1, characterized in that: The compound is used to reduce the levels of TNF-α, IL6, and IL1β in the serum of individuals with lung injury caused by Pseudomonas aeruginosa infection.

4. The application according to claim 1, characterized in that: The method for screening the compounds includes the following steps: S1. Provide a three-dimensional structural model of the tumor suppressor M protein; S2. Perform molecular docking simulation between the compounds to be screened and the tumor suppressor M protein, and calculate the binding free energy; S3. Select compounds that interact with the active site of the tumor suppressor M protein as candidate compounds.

5. The application according to claim 4, characterized in that: The interactions include hydrogen bonds, salt bridges, π-π stacking, or van der Waals forces.

6. The application according to claim 4, characterized in that: The candidate compound is able to specifically bind to the tumor suppressor M protein and block its binding to the receptor.

7. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises an effective amount of the compound used in any one of claims 1-6 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition is used to treat Pseudomonas aeruginosa-induced lung injury, and the compounds in the pharmaceutical composition are used to reduce the effects of Pseudomonas aeruginosa-induced lung injury and inhibit excessive inflammation.

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