Application of glutathione peroxidase 3 as therapeutic target in preparation of pulmonary fibrosis drugs

Abstract

The invention discloses an application of glutathione peroxidase 3 as a therapeutic target in preparation of a medicine for treating pulmonary fibrosis, and an application of a compound Ebselen in preparation of a medicine for treating pulmonary fibrosis. Through single cell and space transcriptomics analysis, it is found for the first time that expression of GPX3 in pulmonary fibrosis patients and model animal lung tissues is significantly reduced. In-vivo and in-vitro experiments prove that up-regulation of GPX3 expression can effectively inhibit fibroblast activation, migration, proliferation and oxidative stress and block a TGF-beta1 / Smad signal channel, so that collagen deposition and pulmonary fibrosis pathological change are relieved. Furthermore, a GPX3 simulant Ebselen is given by adopting an aerosol inhalation mode, is preferably loaded in liposome, and shows a remarkable anti-fibrosis effect in two mouse pulmonary fibrosis models induced by bleomycin and silicon dioxide. The invention provides a brand-new therapeutic target and a medicine scheme with clinical transformation potential for pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and molecular biology, specifically relating to the novel use of glutathione peroxidase 3 (GPX3) as a therapeutic target in pulmonary fibrosis, and the application of compound Ebselen in the preparation of drugs for the treatment of pulmonary fibrosis. Background Technology

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal interstitial lung disease characterized by abnormal activation of fibroblasts, their transformation into myofibroblasts, and excessive deposition of the extracellular matrix (ECM), ultimately leading to lung structural destruction and functional failure. Current treatment options for IPF are limited and ineffective, resulting in extremely poor patient prognosis.

[0003] Oxidative stress plays a crucial role in the pathogenesis of interstitial fibrosis (IPF). Elevated levels of reactive oxygen species (ROS) not only directly cause cell damage but also activate key pro-fibrotic signaling pathways such as transforming growth factor-β1 (TGF-β1), creating a vicious cycle that jointly drives the fibrotic process. Therefore, targeting oxidative stress pathways is a promising strategy for treating IPF.

[0004] Glutathione peroxidase 3 (GPX3) is an important extracellular antioxidant enzyme responsible for scavenging peroxides and maintaining tissue oxidative homeostasis. However, the specific expression changes, biological functions, and clinical significance of GPX3 in the development and progression of IPF remain unclear. Elucidating the role of GPX3 in IPF is of great significance for the development of new targeted therapies. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a novel target for the treatment of pulmonary fibrosis and a treatment strategy based on this target. Specifically, this invention demonstrates for the first time the role of GPX3 as a key protective factor in pulmonary fibrosis and provides an effective method for treating pulmonary fibrosis using the GPX3 agonist Ebselen.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] This invention discloses the application of glutathione peroxidase 3 as a therapeutic target in the preparation of drugs for pulmonary fibrosis.

[0008] The present invention also discloses the use of a GPX3 agonist in the preparation of a medicament for the prevention and / or treatment of pulmonary fibrosis.

[0009] The GPX3 agonist is ebselen.

[0010] The present invention also discloses a pharmaceutical composition for the prevention and / or treatment of pulmonary fibrosis, characterized in that it comprises a therapeutically effective amount of a GPX3 agonist and a pharmaceutically acceptable carrier.

[0011] The GPX3 agonist is ebselen.

[0012] The pharmaceutical composition is prepared into a dosage form suitable for pulmonary administration.

[0013] The dosage form suitable for pulmonary administration is an inhalation preparation, preferably a nebulized inhalation liquid, a dry powder inhaler, or an aerosol.

[0014] The pharmaceutical composition comprises liposomes loaded with the GPX3 agonist.

[0015] The present invention also discloses the use of any of the above-described pharmaceutical compositions in the preparation of a medicament for the prevention and / or treatment of pulmonary fibrosis.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] For the first time, GPX3 has been systematically revealed and validated as a key negative regulator of pulmonary fibrosis, providing a novel biological target with a clearly defined mechanism of action for the diagnosis and treatment of pulmonary fibrosis.

[0018] This study is the first to directly link Ebselen to the GPX3 target and confirm its efficacy in treating pulmonary fibrosis, providing solid experimental evidence and a clear direction for the repurposing of existing drugs or the development of new GPX3 agonists.

[0019] The treatment strategy provided by this invention directly targets one of the core pathological aspects of pulmonary fibrosis, oxidative stress, and intervenes in the disease process from the etiology. Compared with existing symptomatic treatments, it may have better efficacy and radical cure potential.

[0020] Experiments have demonstrated that Ebselen itself possesses therapeutic activity, providing a simple and feasible approach for the clinical application of this strategy, such as direct administration via nebulized inhalation, which reduces the complexity of drug development. Attached Figure Description

[0021] Figure 1Single-cell transcriptome analysis of IPF lung tissue reveals the regulatory role of GPX3 in fibroblast activation; (A) UMAP plot showing five major cell populations, four stromal cell subpopulations, and two fibroblast subpopulations from lung tissues of 32 IPF patients and 28 normal controls; (B) Volcano plot of differentially expressed genes (DEGs) in fibroblasts of IPF and normal lung tissues; (C) Feature plot of GPX3 expression distribution in fibroblasts; (D) Dot plot of GPX3 expression distribution in different cell types; (E) Violin plot of GPX3 expression levels in common fibroblasts (Fib) and myofibroblasts (MyoFib) of IPF patients; (F) Pseudo-time series analysis showing the dynamic changes in GPX3 and fibrosis-related genes during fibroblast differentiation into myofibroblasts.

[0022] Figure 2 1. Validation of the regulatory role of GPX3 in fibroblast activation using independent single-cell datasets; (A) UMAP plot showing four major cell subpopulations in the IPF single-cell dataset GSE135893; (B) UMAP and dot plots showing the enriched expression of GPX3 in stromal cells; (C) Volcano plot showing that GPX3 is the most significantly downregulated gene in fibroblasts; (D) UMAP plot of fibroblast subpopulation re-clustering, identifying four subpopulations, including an IPF-specific HAS1-high-expressing subpopulation; (E) Violin plot comparing GPX3 expression levels in the four fibroblast subpopulations; (F) Pseudo-time series analysis showing the dynamic expression changes of GPX3 and pro-fibrotic genes during fibroblast differentiation into the HAS1-high-expressing subpopulation; (G) Violin plot based on batch RNA-seq data showing that GPX3 expression in lung tissue of IPF patients was significantly lower than that in normal controls;

[0023] Figure 3 Spatial transcriptome analysis revealed the loss of GPX3 expression in fibrotic regions and its negative correlation with pro-fibrotic signals; (A) H&E staining, microenvironment annotation, and spatial distribution map of GPX3 and COL1A1 expression in lung tissue sections; (B) Heatmap analysis of GPX3 and fibrosis-related gene expression levels in different ecological niches.

[0024] Figure 4Bleomycin (BLM)-induced mouse pulmonary fibrosis model: GPX3 downregulation accompanied by enhanced oxidative stress and TGF-β1 activation; (A) HE staining, Ashcroft score, Masson trichrome staining and quantitative results (Student ttest, n=5, scale bar: 50 μm); (B) Immunohistochemical staining results of α-SMA and 4-HNE (Student ttest, n=5, scale bar: 50 μm); (C) Hydroxyproline content in lung tissue (Student ttest, n=5); (D, E) MDA and GSH levels in lung tissue homogenate (n=5); (F, G) qRT-PCR detection of GPX3 and α-SMAm RNA expression levels (Student ttest, n=3); (H) Western blot detection of GPX3 and α-SMA protein expression levels (Student ttest, n=3). (I, J) ELISA detection of GPX3 protein content in lung tissue and bronchoalveolar lavage fluid (BALF) (Student ttest, n=5); (K, L) ELISA detection of active TGF-β1 level in lung tissue and BALF (Student ttest, n=5); (M) Western blot detection of phosphorylation level of SMAD2 protein (Student ttest, n=5); (N) Schematic diagram of the mechanism by which GPX3 deficiency promotes pulmonary fibrosis by activating the ROS-TGF-β1 axis;

[0025] Figure 5 GPX3 overexpression inhibited TGF-β1-induced activation, proliferation, migration, and oxidative stress in MRC-5 cells; (A) EdU cell proliferation assay (Student t test, n=5, scale bar: 50 μm); (B) Transwell cell migration assay (Student t test, n=5, scale bar: 50 μm); (C) Immunofluorescence staining for α-SMA expression and distribution (Student t test, n=5, scale bar: 25 μm); (D) Detection of ROS, MDA, and GSH levels (Student t test, n=5, scale bar: 50 μm); (E, F) qRT-PCR detection of GPX3 and α-SMA RNA expression levels (Student t test, n=3); (G) Western blot detection of GPX3 and α-SMA protein expression levels (Student t test, n=3); (H) Western blot detection of SMAD2 protein phosphorylation levels (Student t test, n=3). test, n = 3);

[0026] Figure 6AAV-mediated GPX3 overexpression alleviates bleomycin-induced pulmonary fibrosis in mice; (A) Schematic diagram of animal intervention treatment; (B) Verification of AAV transfection efficiency; (C) HE staining, Ashcroft score, and Masson trichrome staining of lung tissue morphology (Student t test, n=5, scale bar: 50 μm); (D) Immunohistochemical staining of α-SMA and 4-HNE (Student t test, n=5, scale bar: 50 μm); (E) Hydroxyproline content determination (Student t test, n=5); (F, G) MDA and GSH levels in lung tissue homogenate (Student t test, n=5); (H, I) ELISA detection of active TGF-β1 levels in lung tissue and BALF (Student t test, n=5); (J) Western blot detection of GPX3 and α-SMA protein expression in lung tissue (Student t test, n=5). (test, n=3); (K, L) ELISA detection of GPX3 protein levels in lung tissue and BALF (Student ttest, n=5); (M) Western blot detection of SMAD2 protein phosphorylation levels (Student ttest, n=3);

[0027] Figure 7Antifibrotic effect of nebulized Ebselen liposomes in a bleomycin- and silica-induced pulmonary fibrosis model; (A) Schematic diagram of animal liposome treatment; (B) HE staining, Masson trichrome staining, and α-SMA immunohistochemical results of lung tissues in each group (Student t test, n=5, scale bar: 50 μm); (C) Ashcroft score results (Student t test, n=5); (D) Masson staining collagen quantification results (Student t test, n=5); (E) α-SMA immunohistochemical quantification results (Student t test, n=5); (F) Hydroxyproline content determination (Student t test, n=5); (G, H) MDA and GSH levels in lung tissue homogenate (Student t test, n=5); (I) Schematic diagram of animal liposome treatment; (J) HE staining, Masson trichrome staining, and α-SMA immunohistochemical results of lung tissues in each group (Student t test, n=5). (ttest, n=5, scale bar: 50μm); (K) Ashcroft score results (Student t test, n=5); (L) Masson staining collagen quantification results (Student t test, n=5); (M) α-SMA immunohistochemical quantification results (Student t test, n=5); (N) Hydroxyproline content determination (Student t test, n=5); (O, P) MDA and GSH levels in lung tissue homogenate (Student t test, n=5);

[0028] Figure 8 The antibodies used in this study. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0030] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0031] I. Reagents and Instruments

[0032] 1. Main Reagents: Bleomycin (Selleck); Silica particles (SiO2, Sigma-S5631); Sterile PBS; Hematoxylin-eosin staining reagent (Solepro); Masson's trichrome staining reagent (Maisin); Malondialdehyde (MDA) assay kit (Nanjing Jiancheng Biotechnology Institute); Glutathione (GSH) assay kit (Nanjing Jiancheng Biotechnology Institute); GPX3 ELISA kit (Jianglai Biotechnology); Activated TGF-β1 ELISA kit (Biolegend); TRIzol reagent (Accurate Biotechnology); Reverse transcription kit (AG11711, Accurate Biotechnology); qPCR kit (AG11701, Accurate Biotechnology). Biotechnology); RIPA lysis buffer (Beyotime); protease inhibitor cocktail (Selleck); BCA protein concentration assay kit (Beyotime); PMSF (Beyotime); SDS-PAGE related reagents (Yamei); PVDF membrane (Millipore); ECL luminescence solution (Yamei); Human embryonic lung fibroblasts MRC-5 (Chinese Academy of Sciences Cell Bank); MEM medium (Gibco); DMEM high glucose medium (Gibco); Fetal bovine serum (FBS, Gibco); Recombinant human TGF-β1 Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific); GPX3 overexpression plasmid (Gikai Gene); EdU cell proliferation assay kit (Raybro Biotech); DAPI staining solution (Solepro); DCFH-DA reactive oxygen species detection probe (Beyotime, S0033); Crystal violet staining solution (Solepro); Transwell chamber (Corning, #3422); Adeno-associated virus type 6 (AAV6-GPX3 and AAV6-NC, Shanghai Hanheng Biotechnology).

[0033] 2. Main Instruments: Biological microscope (Zeiss GmbH, Germany); Vortex mixer (Wuhan Sewell Biotechnology Co., Ltd.); Multifunctional microplate reader (TECAN, USA); Forced-air drying oven (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.); Inverted fluorescence microscope (Zeiss GmbH, Germany); Mini high-speed refrigerated centrifuge (SIGMA, USA); Constant temperature incubator shaker (Shanghai Yiheng Technology Co., Ltd.); Magnetic stirrer (Guangzhou Instrument & Experiment Technology Co., Ltd.); Mini vertical electrophoresis tank (BIO-RAD, USA); Electric heating constant temperature water bath (Shanghai Yiheng Technology Co., Ltd.); Multifunctional gel imaging system (Micro, Israel). Chemi); Paraffin microtome (Leica, Germany); Slice spreader (Leica, Germany); CO2 incubator (Current Instruments, USA); Rotary automatic tissue dehydrator (Leica, Germany); Shaker (Heidolph, Germany); High-speed tissue homogenizer (Wuhan Sewell Biotechnology Co., Ltd.); Pure water system (MILLIPORE, USA); Analytical balance (Sartorius Scientific Instruments Co., Ltd., Beijing); Paraffin embedding machine (Leica, Germany); Electric thermostatic shaking water bath (Shanghai Senxin Experimental Instruments Co., Ltd.); Autoclave (Toshiba, Japan); Real-time quantitative PCR instrument 480II (Roche)

[0034] II. Methods.

[0035] 1. Bioinformatics Analysis. Human lung tissue single-cell RNA sequencing data (GSE136831, GSE135893) underwent quality control, standardization, and cell segmentation using the Seurat standard workflow. Further differential expression analysis was performed on fibroblast subpopulations, and pseudo-timeline differentiation trajectories were constructed using Monocle3. Spatial transcriptome data were processed using Space Ranger (v2.0.0) and visualized using SpatialFeaturePlot; gene expression heatmaps were generated based on Z-score standardization and using the pheatmap R package. In addition, based on transcriptome data from IPF and normal lung tissue obtained from the GEO database, we performed batch RNA sequencing differential analysis to screen for disease-related differentially expressed genes.

[0036] 2. Animal Experiments. This study used 6–8 week old male C57BL / 6 mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.). All mice were housed in a specific pathogen-free environment. Animal experimental procedures strictly adhered to international guidelines for the use and care of laboratory animals and were approved by the Animal Ethics Committee of China Medical University.

[0037] 3. Silica-induced mouse silicosis model. SiO2 particles (5 μm, Sigma#S5631) were mechanically ground for 2 h to disperse agglomerates, followed by dry heat sterilization at 200℃ for 2 h to remove endotoxins and microbial contamination. The sterilized particles were uniformly suspended in sterile PBS to prepare a 600 mg / mL suspension. Mice were anesthetized with isoflurane inhalation and received a single intratracheal infusion of 20 μL of SiO2 suspension, while the control group received an equal volume of PBS. Six weeks after model establishment, the animals were sacrificed, and lung tissue was collected for histological and molecular biological analysis.

[0038] 4. Bleomycin-induced mouse pulmonary fibrosis model. A pulmonary fibrosis model was established by intratracheal infusion of a single dose of bleomycin (3 mg / kg), while the control group received an equal volume of PBS. To systematically observe the fibrosis process and the effect of drug intervention, all animals were sacrificed on day 21 after modeling, and lung tissue was collected for histopathological and biochemical analysis.

[0039] AAV6-mediated GPX3 overexpression model

[0040] To investigate the functional role of GPX3 in pulmonary fibrosis, adeno-associated virus type 6 (AAV6, Shanghai Hanheng Biotechnology) was used as a gene delivery vector to construct a mouse model of lung tissue-specific overexpression of GPX3. Specifically, 7 days before bleomycin induction, mice anesthetized with isoflurane were administered AAV6-GPX3 (experimental group) or AAV6-NC (empty virus control group) via intratracheal infusion to achieve specific overexpression of GPX3 in lung tissue, laying the foundation for subsequent phenotypic analysis.

[0041] 5. Timing of liposome intervention. In the bleomycin-induced pulmonary fibrosis model, the optimal timing of intervention was determined by reference (PMID:32165401). Nebulized inhalation therapy was started on the 7th day after modeling, 3 times a week. Each time, the mice were placed in a closed administration chamber and inhaled the drug-containing liposome nebulized suspension.

[0042] In a silica-induced silicosis model, the intervention starting point was set according to a relevant study (PMID:35551173). Nebulized treatment was started in the second week after SiO2 exposure, with the same dosing frequency of 3 times a week, to systematically evaluate the inhibitory effect of liposomes on the pathological process of silicosis.

[0043] 6. Histological analysis. Fresh left lung tissue was fixed in 10% formalin for 24 h, dehydrated, and embedded in paraffin. Sections (5 μm) were stained with hematoxylin-eosin (H&E) and Masson trichrome. The area of ​​fibrosis (%) was quantified using the Ashcroft score (H&E) and ImagePro Plus 6.0 software.

[0044] 7. Hydroxyproline Determination. Hydroxyproline content was measured using a hydroxyproline assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. Briefly, lung tissue was homogenized, hydrolyzed, and centrifuged. The supernatant was incubated with the assay reagent, and the absorbance was measured at 560 nm.

[0045] 8. MDA and GSH assays. The levels of malondialdehyde (MDA) and glutathione (GSH) in lung tissue homogenates and cultured cells were quantitatively determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol.

[0046] 9. Immunohistochemistry (IHC). Paraffin sections were dewaxed, hydrated, and subjected to heat-induced epitope retrieval in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 3% H2O2. Sections were incubated overnight with primary antibody at 4°C, followed by incubation with HRP-labeled secondary antibody. DAB staining was performed, followed by hematoxylin counterstaining. The percentage of positive areas was quantified.

[0047] 9. ELISA. Measure the expression levels of GPX3 and activated TGF-β1 in lung homogenates and BALF using an ELISA kit according to the manufacturer's instructions.

[0048] 10. Cell Culture and Treatment. Human embryonic lung fibroblasts (MRC-5) were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in MEM medium (Gibco) containing 10% fetal bovine serum (Gibco), 1 mM GlutaMAX, 1× non-essential amino acids, 1 mM sodium pyruvate, 100 U / mL penicillin, and 100 U / mL streptomycin. Cells were cultured routinely at 37°C and 5% CO2. All MRC-5 cells were identified by short tandem repeat sequence analysis and confirmed to be free of mycoplasma contamination. To simulate the fibrotic microenvironment, cells were treated with 10 ng / mL TGF-β1 for 24 h and then collected for subsequent experimental analysis.

[0049] 11. Cell Transfection. The plasmids and siRNA used in the experiment were purchased from Jikai Gene (Shanghai). Plasmid transfection was performed using Lipofectamine 3000 transfection reagent, strictly following the manufacturer's recommended procedures. Cells were cultured for 48 hours after transfection, and then collected for subsequent experimental analysis.

[0050] 12. Quantitative Real-Time PCR (qRT-PCR). Total RNA was extracted using TRIzol reagent (Accurate Biotechnology). RNA concentration and purity were measured. cDNA was synthesized from 1 μg of total RNA using a reverse transcription kit (AG11711, Accurate Biotechnology, Hunan). Quantitative PCR was performed using a qPCR kit (AG11701, Accurate Biotechnology). Gene expression was analyzed using Gapdh as an internal control and the 2-ΔΔCt method. Primers used in this study are listed in Table 1.

[0051] 13. Western Blot. Total protein was extracted from frozen lung tissue or MRC-5 cells using RIPA lysis buffer (Beyotime) supplemented with 1 mM PMSF and a protease inhibitor cocktail (Selleck). Protein concentration was determined using a BCA assay kit (Beyotime). Equal volumes of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). After blocking with TBST in 5% skim milk for 1 h at room temperature, the membrane was incubated overnight with primary antibody at 4°C. After washing three times with TBST, the membrane was incubated with HRP-labeled secondary antibody for 1 h at room temperature. Protein bands were detected using an ECL detection system, and quantification was performed using ImageJ software. Protein expression levels were normalized using GAPDH as an internal control. Details of the antibodies used in this study can be found in [link to antibody description]. Figure 8 .

[0052] 14. EdU Cell Proliferation Assay. The EdU reagent kit (Raybro, China) was used. Cells were seeded in 24-well plates and incubated with EdU for 2 hours after treatment. Cells were fixed, stained with Apollo dye, and counterstained with DAPI. Proliferation rate was calculated based on fluorescence microscopy images.

[0053] 15. Transwell migration assay. A Transwell chamber (Corning, #3422) was used. Serum-starved cells were seeded into serum-free medium in the upper chamber. Complete medium was added to the lower chamber. After 24 hours, unmigrated cells were removed, and migrating cells were fixed, stained with crystal violet, and counted under a microscope.

[0054] 16. Immunofluorescence staining. Fix, permeabilize, and block cells, then incubate with primary antibody overnight at 4°C. After incubation with fluorescent secondary antibody, stain the nuclei with DAPI. Acquire images using a fluorescence microscope.

[0055] 17. Reactive oxygen species (ROS) staining. Cells were incubated with DCFH-DA (Beyotime, S0033) probe, washed, and analyzed by fluorescence microscopy.

[0056] 18. Liposome Synthesis and Characterization. Liposomes were prepared using a thin-film evaporation-hydration-extrusion method. The composition included:

[0057] Lip@E: DPPC, cholesterol, DSPE-PEG, selenium epoxide

[0058] Liposomes were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements.

[0059] 19. Statistical Analysis. Statistical analysis was performed using GraphPad Prism 10 and R4.3.2 (https: / / www.r-project.org / ). Data are expressed as mean ± standard deviation. Differences between groups were analyzed using the Student's t-test, and statistical significance was defined as a two-tailed p-value < 0.05.

[0060] 20. GPX3 Expression Identification. Human lung tissue single-cell RNA sequencing data (GSE136831, GSE135893) underwent quality control, standardization, and cell segmentation using the Seurat standard workflow. Further differential expression analysis was performed on fibroblast subpopulations, and their pseudo-temporal differentiation trajectories were constructed using Monocle3. Spatial transcriptome data were processed using Space Ranger (v2.0.0) and visualized using SpatialFeaturePlot; gene expression heatmaps were generated based on Z-score standardization and using the pheatmap R package. In addition, based on transcriptome data from IPF and normal lung tissue obtained from the GEO database, we performed batch RNA sequencing differential analysis to screen for disease-related differentially expressed genes.

[0061] 21. In vitro functional experiments.

[0062] (1) Cell model: Human embryonic lung fibroblasts (MRC-5) were used.

[0063] (2) GPX3 overexpression: GPX3 was overexpressed in MRC-5 cells by transfecting the GPX3 plasmid.

[0064] (3) Functional testing.

[0065] a. Proliferation: Cell proliferation was detected using the EdU method.

[0066] b. Migration: Cell migration ability was detected using Transwell chambers.

[0067] c. Activation markers: α-SMA expression was detected by qPCR and Western Blot.

[0068] d. Oxidative stress: ROS levels were detected using fluorescent probes, and MDA and GSH levels were detected using a kit.

[0069] 22. Animal in vivo therapeutic experiments.

[0070] (1) Model establishment: A mouse pulmonary fibrosis model was established by intratracheal infusion of bleomycin or silica.

[0071] (2) Treatment intervention:

[0072] Gene therapy group: Adeno-associated virus (AAV-GPX3) carrying the GPX3 gene was administered via intratracheal infusion.

[0073] Drug treatment group: Ebselen solution was administered via nebulized inhalation.

[0074] (3) Effect evaluation.

[0075] a. Histopathology: Lung tissue was taken for H&E staining and Masson trichrome staining to assess inflammatory infiltration and collagen deposition, and Ashcroft scores and quantification of fibrosis area were performed.

[0076] b. Biochemical indicators: The levels of hydroxyproline, MDA, and GSH in lung tissue homogenate were detected.

[0077] c. Molecular level: The expression of molecules such as GPX3 and α-SMA was detected by qPCR, Western Blot or ELISA.

[0078] III. Results

[0079] 1. Multi-omics and multi-cohort data analysis identified GPX3 as a key regulator of fibroblast activation and IPF progression.

[0080] To systematically elucidate the key mechanisms of fibroblast activation in IPF, we first analyzed the largest current IPF single-cell RNA sequencing dataset, GSE136831 (PMID: 32832599), which contains lung tissue samples from 32 IPF patients and 28 healthy individuals. Cluster analysis identified five major cell populations, among which mesenchymal cells could be further divided into four subpopulations (…). Figure 1 A). Given the central role of fibroblast activation in the development and progression of IPF, this study focused on its in-depth characterization. By comparing differential gene expression between fibroblasts and myofibroblasts, we found that GPX3 expression was most significantly downregulated in lung fibroblasts of IPF patients. Figure 1 B). Further observation of its expression distribution revealed that GPX3 was mainly confined to stromal cells, exhibiting high cell type specificity. Figure 1(C and D). Notably, in IPF, the GPX3 expression level in myofibroblasts was significantly lower than that in normal fibroblasts (C and D). Figure 1 E). Pseudo-temporal trajectory analysis showed that GPX3 expression gradually decreased during the transformation of fibroblasts into myofibroblasts, while fibrosis marker genes (COL1A1, COL1A2, COL3A1, COL5A1, and COL5A2) showed a gradual upward trend. Crucially, the downregulation of GPX3 occurred before the upregulation of pro-fibrosis genes, suggesting that it may play a regulatory role in the early stages of fibroblast activation. Figure 1 F).

[0081] To validate these findings, we analyzed another independent dataset, GSE135893 (PMID: 32832599). The results showed that GPX3 was also primarily expressed in mesenchymal cells in this dataset. Figure 2 B), and the downregulation was most significant in IPF fibroblasts. Figure 2 C). This dataset further subdivides fibroblasts into four subpopulations, among which the HAS1-high expression subtype is specific to IPF and is mainly distributed in the subpleural region—the typical lesion area of ​​IPF—suggesting that this subpopulation plays a key role in disease development. Figure 2 D). Notably, GPX3 expression levels were lowest in fibroblasts with high HAS1 expression. Figure 2 E). Pseudo-time series analysis further showed that during the differentiation of fibroblasts into the HAS1-overexpressing subtype, GPX3 expression continuously decreased, showing an opposite trend to the expression changes of pro-fibrotic genes, and its downregulation also occurred earlier than the upregulation of pro-fibrotic genes. Figure 2 F).

[0082] Furthermore, batch transcriptomic analyses of multiple lung tissue cohorts consistently confirmed that GPX3 expression was significantly lower in IPF patients than in the control group. Figure 2 G). Spatial transcriptome analysis further revealed that GPX3 expression was significantly downregulated in fibroblast-rich niches and regions of active extracellular matrix (ECM) remodeling, while fibrosis-related genes were significantly upregulated, further supporting a negative correlation between GPX3 and pro-fibrosis gene expression. Figure 3 A, B).

[0083] In summary, by integrating multi-omics and multi-cohort data, this study suggests that GPX3 may play a key role in fibroblast activation and the pathogenesis of IPF.

[0084] 2. GPX3 expression was downregulated in a bleomycin-induced mouse pulmonary fibrosis model.

[0085] To investigate the pathogenesis of IPF, we used a bleomycin (BLM)-induced mouse pulmonary fibrosis model. This model can well simulate the key pathological features of human IPF and is widely used in research on this disease (PMID: 40473311). First, we assessed the degree of pulmonary fibrosis through histological analysis. HE staining showed that, compared with the control group, the BLM-treated mice had significant inflammatory cell infiltration in the lung tissue and exhibited typical fibrotic lesions; Ashcroft scores further confirmed a significantly increased severity of fibrosis. Masson trichrome staining results indicated a significant increase in collagen deposition in the lung tissue of BLM-treated mice. Figure 4 A). Immunohistochemical analysis showed that the expression of the myofibroblast marker α-SMA (α-smooth muscle actin) was upregulated in the BLM group, indicating increased myofibroblast activation; at the same time, the expression of the oxidative stress marker 4-HNE was enhanced, indicating an increased level of tissue oxidative stress. Figure 4 B). To further validate the above findings, we examined indicators related to collagen deposition and oxidative stress. Hydroxyproline content measurements showed a significant increase in collagen content in the BLM group (B). Figure 4 C). Elevated levels of the oxidative stress marker MDA and decreased levels of GSH suggest an exacerbated oxidative stress response. Figure 4 D, E). Regarding the molecular mechanism, both qPCR and Western blotting results consistently showed that GPX3 was significantly downregulated at both the mRNA and protein levels, and its expression was negatively correlated with α-SMA. Figure 4 FH). Since GPX3 is a secreted protein, we further detected its content in lung tissue and bronchoalveolar lavage fluid (BALF) using ELISA, and found that the GPX3 protein level was significantly reduced in both. Figure 4 I, J). Previous studies have suggested that ROS can activate potential TGF-β1, thereby inducing fibroblast activation and fibrosis (PMID: 38514615). Consistent with this, the levels of activated TGF-β1 in BLM-treated fibrotic lung tissue and BALF were significantly higher than those in the control group in this study. Figure 4 Meanwhile, the phosphorylation level of Smad2, a downstream signaling molecule of TGF-β1, was significantly enhanced, indicating that this pathway was activated.

[0086] In summary, the results of this study suggest that in a bleomycin-induced pulmonary fibrosis model, downregulation of GPX3 expression may lead to increased oxidative stress levels, thereby activating the TGF-β1 / Smad2 signaling pathway, promoting fibroblast activation and collagen deposition, and ultimately driving the progression of pulmonary fibrosis. Figure 4 M).

[0087] 3. GPX3 inhibits TGF-β1-induced fibroblast activation in vitro.

[0088] To elucidate the specific role of GPX3 in fibroblast activation, we conducted a systematic in vitro functional validation in human embryonic lung fibroblasts (MRC-5). First, EdU assays revealed that TGF-β1 significantly promoted MRC-5 cell proliferation, while GPX3 overexpression effectively inhibited this proliferative effect. Figure 5 A). Transwell experiments further demonstrated that TGF-β1 enhances cell migration, a phenomenon that can also be reversed by GPX3 overexpression. Figure 5 B). Regarding the cell activation phenotype, immunofluorescence results showed that TGF-β1 treatment significantly upregulated the expression of the myofibroblast marker α-SMA, while GPX3 overexpression significantly inhibited this change. Figure 5 C).

[0089] At the level of oxidative stress, TGF-β1 caused an increase in ROS and MDA levels, accompanied by a decrease in GSH content, indicating a disruption of redox balance; while GPX3 overexpression could effectively reverse these changes in oxidative stress-related indicators. Figure 5 D). To further clarify its molecular mechanism, we used qPCR and Western blot analysis to find that TGF-β1 not only downregulated GPX3 expression but also upregulated α-SMA and enhanced Smad2 phosphorylation; while GPX3 overexpression significantly inhibited TGF-β1-induced upregulation of α-SMA expression and Smad2 phosphorylation. Figure 5 E–H).

[0090] In summary, these results collectively demonstrate that TGF-β1 promotes the proliferation, migration, and activation of MRC-5 cells, accompanied by increased oxidative stress levels; while GPX3 overexpression effectively reverses the aforementioned fibrosis-related phenotypes. This part of the experiment confirms from an in vitro perspective that GPX3 can inhibit the TGF-β1-induced fibroblast activation process, further supporting its protective role in the development and progression of pulmonary fibrosis.

[0091] 4. Overexpression of GPX3 alleviates bleomycin-induced pulmonary fibrosis in mice by reducing oxidative stress and inhibiting TGF-β1 activation.

[0092] To verify the therapeutic potential of GPX3 overexpression for pulmonary fibrosis, this study used adeno-associated virus (AAV) as a gene delivery vector to achieve specific overexpression of GPX3 in lung tissue via intratracheal infusion. The experimental design is as follows: Figure 6 As shown in A and 6B: One week after AAV infusion, bleomycin (BLM) was administered via the trachea to establish a pulmonary fibrosis model, and samples were taken for analysis three weeks later.

[0093] Histological analysis showed that AAV-mediated GPX3 overexpression significantly improved BLM-induced lung pathological changes. HE staining indicated that GPX3 overexpression reduced inflammatory cell infiltration and fibrotic lesions, and Ashcroft score further confirmed a significant reduction in the degree of fibrosis. Figure 6 C). Masson staining results showed that collagen deposition in the lung tissue of mice in the AAV-GPX3 group was significantly reduced ( Figure 6 C). Immunohistochemical analysis revealed decreased expression of the myofibroblast marker α-SMA in the GPX3 overexpression group, along with a significant decrease in the expression of the oxidative stress marker 4-HNE, suggesting that fibrotic activity and oxidative stress levels were effectively alleviated. Figure 6 D).

[0094] At the molecular level, hydroxyproline content assays showed reduced collagen deposition in the GPX3 overexpression group. Figure 6 E). Oxidative stress index detection showed that AAV-GPX3 treatment could reduce MDA content and restore GSH levels (E). Figure 6 F,G), confirming that GPX3 overexpression effectively improved the redox imbalance. ELISA analysis showed that GPX3 overexpression significantly reduced the content of activated TGF-β1 in lung tissue and bronchoalveolar lavage fluid (BALF). Figure 6 Western blot results further showed that GPX3 overexpression downregulated α-SMA protein levels and inhibited Smad2 phosphorylation (H,I). Figure 6 J–M).

[0095] 5. Antifibrotic effect of nebulized Ebselen liposomes in a bleomycin- and silica-induced pulmonary fibrosis model.

[0096] Based on previous research, GPX3 has been confirmed as a key factor regulating fibroblast activation and the progression of pulmonary fibrosis. As a selenium-dependent antioxidant enzyme, GPX3 is mainly secreted by various tissues and widely distributed in extracellular fluid (such as blood). Its core function is to scavenge peroxides, thereby reducing oxidative stress damage to cells (PMID: 34031399).

[0097] Ebselen, the first synthesized organoselenium compound, can effectively mimic the activity of glutathione peroxidase (such as GPX3) and has multiple pharmacological functions such as anti-inflammatory and oxidative stress regulation (PMID: 34031399). Therefore, it can theoretically be used as a functional substitute for GPX3 to intervene in the process of pulmonary fibrosis.

[0098] To achieve efficient targeted delivery of Ebselen to lung tissue, this study employed nebulized inhalation as the administration route. This strategy allows the drug to directly accumulate in the respiratory tract and lung lesions, demonstrating unique advantages in the treatment of respiratory diseases (PMID: 40185752). To further enhance drug stability and lung retention, we selected liposomes as the delivery carrier. Liposomes are nanoscale vesicles composed of a phospholipid bilayer, exhibiting good biocompatibility and degradability, which can effectively improve drug distribution and release behavior in the lungs (PMID: 36768966).

[0099] Against this background, this study constructed liposomes loaded with Ebselen (Lip@E) and systematically evaluated its anti-fibrotic effect after nebulized inhalation using a mouse pulmonary fibrosis model induced by bleomycin (BLM) and silica (SiO2), aiming to comprehensively verify its therapeutic potential from both the perspectives of drug delivery and mechanism regulation.

[0100] In a BLM-induced pulmonary fibrosis model, we initiated Lip@E nebulized inhalation therapy in mice at the previously established key intervention time point (day 7 post-modeling, PMID: 32165401). Analysis of samples taken on day 21 revealed that, compared to the model group, Lip@E significantly reduced collagen deposition in lung tissue, inhibited fibroblast activation, and effectively decreased oxidative stress levels. Figure 7 A–H).

[0101] In a SiO2-induced pulmonary fibrosis model (PMID: 32622305), Lip@E also demonstrated significant therapeutic effects. Experimental results showed that after Lip@E intervention, SiO2-induced collagen deposition, fibroblast activation, and oxidative stress levels in lung tissue were significantly improved. Figure 7 I–P).

[0102] In summary, this study confirms that in two different mechanism-induced pulmonary fibrosis models, nebulized Ebselen liposomes significantly reduced pulmonary fibrosis pathological changes and collagen deposition by alleviating oxidative stress and inhibiting fibroblast activation. These results not only validate the therapeutic efficacy of Ebselen as a GPX3 mimic but also provide strong evidence for its preclinical development as a candidate drug for treating pulmonary fibrosis.

[0103] In summary, AAV-mediated GPX3 overexpression effectively alleviates bleomycin-induced pulmonary fibrosis in mice by reducing oxidative stress levels and inhibiting TGF-β1 activation and its downstream signaling pathways. These results further confirm the crucial protective role of GPX3 in the development and progression of pulmonary fibrosis, providing experimental evidence for its potential therapeutic target.

[0104] The therapeutic effects of Ebselen: In two mouse models of pulmonary fibrosis, nebulized Ebselen significantly improved the pathological manifestations of fibrosis, reduced collagen deposition and oxidative stress levels, demonstrating its effectiveness as a GPX3 agonist.

[0105] This invention clarifies the importance of GPX3 as a novel therapeutic target for pulmonary fibrosis and verifies the outstanding efficacy of Ebselen as an agonist of this target in treating pulmonary fibrosis. This discovery provides important theoretical basis and experimental support for the development of targeted therapies for pulmonary fibrosis.

[0106] Matters not covered in this invention are common knowledge.

[0107] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of glutathione peroxidase 3 as a therapeutic target in the preparation of pulmonary fibrosis drugs, including the application of glutathione peroxidase 3 agonists in the preparation of pulmonary fibrosis drugs for prevention and / or treatment, wherein glutathione peroxidase 3 is GPX3.

2. The application according to claim 1, characterized in that, The glutathione peroxidase 3 agonist is ebselen.

3. A pharmaceutical composition for the prevention and / or treatment of pulmonary fibrosis, characterized in that, It contains a therapeutically effective amount of GPX3 agonist and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition for the prevention and / or treatment of pulmonary fibrosis as described in claim 3, characterized in that, The pharmaceutical composition is prepared into a dosage form suitable for pulmonary administration.

5. The pharmaceutical composition for the prevention and / or treatment of pulmonary fibrosis as described in claim 4, characterized in that, The dosage form suitable for pulmonary administration is an inhalation preparation, including nebulized inhalation liquid, dry powder inhaler, or aerosol.

6. The pharmaceutical composition for the prevention and / or treatment of pulmonary fibrosis as described in claim 5, characterized in that, The pharmaceutical composition comprises liposomes loaded with the GPX3 agonist.

7. Use of a pharmaceutical composition according to any one of claims 3 to 6 in the preparation of a medicament for the prevention and / or treatment of pulmonary fibrosis.

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