Use of daxx and / or setdb2 in anti-pulmonary fibrosis

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of DAXX and / or SETDB2 in preparation of an anti-pulmonary fibrosis drug. It is found through a large number of experiments that DAXX and SETDB2 have a synergistic effect in co-overexpression. It is proved through in-vivo and in-vitro experiments that the co-overexpression of DAXX and SETDB2 is significantly superior to single overexpression of DAXX and SETDB2 in inhibiting iron death of lung epithelial cells, reducing collagen deposition and reducing expression of fibrosis markers (alpha-SMA, COL1A1 and FAP alpha), and presents a synergistic effect. The application uses an AAV-6 vector driven by an SPC promoter to realize specific overexpression of DAXX and SETDB2 in alveolar epithelial cells, avoids side effects of systemic administration, and has high clinical conversion potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of DAXX and / or SETDB2 in anti-pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a progressive interstitial lung disease characterized by fibroblast proliferation, excessive extracellular matrix deposition, and alveolar structural destruction. Currently, first-line clinical drugs (such as pirfenidone and nintedanib) can slow the decline in lung function, but they cannot reverse the fibrotic process and are often accompanied by side effects such as gastrointestinal reactions or hepatotoxicity.

[0003] Recent studies have revealed that ferroptosis, an iron-dependent form of programmed cell death driven by lipid peroxidation, plays a crucial role in the pathological process of pulmonary fibrosis. Activated lung fibroblasts exhibit unique iron metabolism characteristics, with abnormal mitochondrial morphology and resistance to ferroptosis; while persistent lipid peroxidation stress not only directly damages alveolar epithelial cells but also creates a pro-fibrotic microenvironment. Inhibition of ferroptosis (such as using the ferroptosis inhibitor Fer-1) has been confirmed by multiple studies to effectively reduce collagen deposition in bleomycin-induced pulmonary fibrosis models.

[0004] DAXX (Death domain-associated protein, also known as DAP6, BING2, or EAP1) was initially identified in 1997 as a protein that interacts with the Fas death domain and can activate the JNK and apoptosis pathways. As a transcriptional co-repressor, DAXX specifically recognizes and binds to SUMOylated transcription factors (such as ETS1, GR, and SMAD4) through its SIMs, thereby inhibiting the activity of these transcription factors. This process is dynamically regulated by the PML nucleosome: within the PML nucleosome, DAXX is isolated and inactivated; when DAXX is released from the PML nucleosome and translocates to the nucleoplasm, its transcriptional repression is exerted. DAXX gene mutations are common in pancreatic neuroendocrine tumors and, together with ATRX mutations, lead to the telomere-selective elongation (ALT) phenotype.

[0005] SETDB2 (SET domain bifurcated 2, also known as CLLD8, C13orf4, KMT1F) is a member of the SETDB family. SETDB2 possesses a unique atypical methyl-CpG binding domain (MBD). Unlike the classic MBD, SETDB2's MBD has lost its ability to bind methylated DNA, evolving into a protein-protein interaction surface. This MBD domain contains a conserved MBD core and a unique N-terminal extension region, capable of recognizing and binding cysteine-rich domains of proteins such as C11orf46. SETDB2 represses gene transcription by adding three methyl groups (H3K9me3) to the 9th lysine residue of histone H3, forming an epigenetic marker of heterochromatin. This function is closely related to gene silencing, transposon repression, and the maintenance of genome stability. SETDB2 may play an important role in various biological processes, but research on it is still in its early stages.

[0006] Currently, there is no existing technology to study the role of DAXX and SETDB2 in the pathological process of pulmonary fibrosis. Summary of the Invention

[0007] The purpose of this invention is to provide the application of DAXX and / or SETDB2 in the treatment of pulmonary fibrosis.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The use of DAXX and / or SETDB2 in the preparation of drugs for pulmonary fibrosis.

[0009] DAXX binds to and activates SETDB2 in a SUMOylation-dependent manner. SETDB2 catalyzes H3K9me3 modification of the TP53 promoter region, inhibiting TP53 transcription, thereby suppressing ferroptosis in lung epithelial cells and alleviating pulmonary fibrosis.

[0010] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows: In one preferred embodiment, DAXX and / or SETDB2 are overexpressed in the drug.

[0011] In one preferred embodiment, DAXX and SETDB2 are co-overexpressed in the drug.

[0012] Based on the same inventive concept, this invention also claims protection for the use of DAXX overexpression reagent and / or SETDB2 overexpression reagent in the preparation of drugs for pulmonary fibrosis.

[0013] In one preferred embodiment, the DAXX overexpression reagent includes a DAXX overexpression vector; the SETDB2 overexpression reagent includes a SETDB2 overexpression vector.

[0014] In one preferred embodiment, the DAXX overexpression vector is a viral vector or a non-viral vector.

[0015] In one preferred embodiment, the SETDB2 overexpression vector is a viral vector or a non-viral vector.

[0016] In one preferred embodiment, the viral vector is selected from adeno-associated virus, lentivirus, adenovirus, or retrovirus; adeno-associated virus is preferred.

[0017] In one preferred embodiment, the adeno-associated virus is AAV6, AAV9, or AAV-DJ serotype.

[0018] In one preferred embodiment, the DAXX overexpression reagent is an adeno-associated virus carrying the DAXX encoding gene.

[0019] In one preferred embodiment, the SETDB2 overexpression reagent is an adeno-associated virus carrying the SETDB2 encoding gene.

[0020] Based on the same inventive concept, this invention also claims a pharmaceutical composition comprising an overexpression reagent of DAXX and / or an overexpression reagent of SETDB2, as well as pharmaceutically acceptable excipients.

[0021] Based on the same inventive concept, the present invention also claims protection for a gene delivery tool comprising an adeno-associated virus carrying a DAXX coding gene and / or a SETDB2 coding gene.

[0022] In one preferred embodiment, the adeno-associated virus is driven by the SPC promoter.

[0023] Based on the same inventive concept, this invention also claims the use of DAXX overexpression reagent and / or SETDB2 overexpression reagent in the preparation of drugs that inhibit ferroptosis.

[0024] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention reveals a novel mechanism by which DAXX / SETDB2 acts as a negative regulator of ferroptosis: This invention is the first to discover that SETDB2 is expressed at low levels in pulmonary fibrosis, and that DAXX activates SETDB2 in a SUMOylation-dependent manner, filling a research gap in the field of SETDB2 regulation of pulmonary fibrosis and ferroptosis.

[0025] 2. Through extensive experiments, this invention has found that co-overexpression of DAXX and SETDB2 has a synergistic effect: In vitro and in vivo experiments have confirmed that co-overexpression of DAXX and SETDB2 is significantly better than overexpression of either alone in inhibiting ferroptosis in lung epithelial cells, reducing collagen deposition, and reducing the expression of fibrosis markers (α-SMA, COL1A1, FAPα), showing a synergistic effect.

[0026] 3. This invention provides a gene delivery tool that specifically targets alveolar epithelial cells: This invention uses an AAV-6 vector driven by the SPC promoter to achieve specific overexpression of DAXX and SETDB2 in alveolar epithelial cells, avoiding the side effects of systemic administration and having high potential for clinical translation. Attached Figure Description

[0027] Figure 1 These are H&E staining images of alveolar structure, interstitial thickening, and abnormal lung markings in the lung tissues of mice in the control group and on days 14, 21, and 28 after BLM induction, as well as the control group in Example 1.

[0028] Figure 2 This is a Masson trichrome staining image of collagen deposition in the lung tissue of mice in the control group and on days 14, 21, and 28 after BLM induction, as shown in Example 1. The scale bar in the image is 25 μm.

[0029] Figure 3 This is a Western blot diagram showing the protein expression levels of α-SMA, FAPα, N-cadherin, TGF-β, and E-cadherin in the lung tissues of mice in the control group and on days 14, 21, and 28 after BLM induction, as described in Example 1.

[0030] Figure 4 The graph shows the flow cytometry results of lipid ROS levels in the lung tissues of mice in the control group and on days 14, 21, and 28 after BLM induction, in Example 1.

[0031] Figure 5 The Fe content of the lung tissues of mice in the control group and on days 14, 21, and 28 after BLM induction in Example 1 is shown. 2+ Levels and 4-HNE levels; among which, Figure 5 a represents Fe in the lung tissue of mice from the control group and on days 14, 21, and 28 after BLM induction. 2+ level, Figure 5 b represents the 4-HNE level in the lung tissue of mice in the control group and on days 14, 21, and 28 after BLM induction.

[0032] Figure 6The values ​​represent the relevant protein levels in the lung tissues of mice in the control group and on days 14, 21, and 28 after BLM induction, as in Example 1; among them, Figure 6 a represents the protein expression levels of GPX4 and SLC7A11 in the lung tissues of mice in the control group and on days 14, 21, and 28 after BLM induction. Figure 6 b represents the protein expression levels of SETDB2 and H3K9me3 in the lung tissues of mice in the control group and on days 14, 21, and 28 after BLM induction.

[0033] Figure 7 This is an immunofluorescence image showing the positive rate of SETDB2 in the lung tissue of mice in the control group and on days 14, 21, and 28 after BLM induction, as well as in Example 1.

[0034] Figure 8 This is a Western blot image of oe-SETDB2 transfection in MLE-12 and Beas-2B cells.

[0035] Figure 9 This is a Western blot diagram of H3K9me3 levels in each group of Beas-2B cells.

[0036] Figure 10 This is a Western blot diagram of H3K9me3 levels in each group of MLE-12 cells.

[0037] Figure 11 The lipid ROS levels in each group of Beas-2B and MLE-12 cells; among them, Figure 11 a shows flow cytometry images of lipid ROS levels in different groups of Beas-2B cells; Figure 11 b is a flow cytometry plot of lipid ROS levels in each group of MLE-12 cells.

[0038] Figure 12 Fe in each group of MLE-12 cells 2+ Levels of 4-HNE, GPX4, and SLC7A11 protein expression; among which, Figure 12 a represents Fe in each group of MLE-12 cells. 2+ Horizontal map; Figure 12 b shows the 4-HNE levels in each group of MLE-12 cells; Figure 12 c is a Western blot plot showing the expression levels of GPX4 and SLC7A11 proteins in each group of MLE-12 cells.

[0039] Figure 13 These are TEM images of mitochondrial morphology in different groups of MLE-12 cells; the arrows indicate mitochondrial shrinkage and increased membrane density.

[0040] Figure 14 These are TEM images of mitochondrial morphology in different groups of Beas-2B cells; the arrows indicate mitochondrial shrinkage and increased membrane density.

[0041] Figure 15 The expression levels of α-SMA, COL1A1, and FAPα in L929 fibroblasts within the co-culture system were measured; among them, Figure 15 a is a Western blot plot showing the protein expression level of α-SMA in L929 fibroblasts; Figure 15 b is a Western blot plot showing the protein expression levels of COL1A1 and FAPα in L929 fibroblasts; Figure 15 c is an immunofluorescence image of α-SMA expression in L929 fibroblasts. The scale bar in the image is 25 μm.

[0042] Figure 16 This is a flow cytometry image of lipid ROS levels in each group of Beas-2B cells in Example 3.

[0043] Figure 17 The Fe groups in each group of Beas-2B cells in Example 3 2+ And the levels of 4-HNE, GPX4 and SLC7A11; among them, Figure 17 a represents Fe in each group of Beas-2B cells. 2+ A bar chart with 4-HNE levels; Figure 17 b is a bar chart showing the levels of GPX4 and SLC7A11 in each group of Beas-2B cells.

[0044] Figure 18 This is a TEM image of mitochondrial morphology in Beas-2B cells.

[0045] Figure 19 This is a Western blot diagram showing the knockdown efficiency of the three si-DAXX groups in MLE-12 and Beas-2B cells.

[0046] Figure 20 The co-IP assay was used to verify the interaction between SETDB2 and DAXX in MLE-12 and Beas-2B cells; among which, Figure 20 Figure a shows the interaction results between SETDB2 and DAXX in Beas-2B cells; Figure 20 b shows the interaction results between SETDB2 and DAXX in MLE-12 cells.

[0047] Figure 21 This is a Western blot plot showing the effects of WT DAXX and ΔSIM mutants on the expression of H3K9me3, TP53, and SETDB2.

[0048] Figure 22 This is a Western blot diagram showing the expression levels of SETDB2, TP53, and DAXX proteins in the BLM+oe-SETDB2+oe-NC-2 group, the BLM+oe-SETDB2+WT DAXX group, and the BLM+oe-SETDB2+ΔSIM group in MLE-12 cells.

[0049] Figure 23 Flow cytometry images of lipid ROS levels in the BLM+oe-SETDB2+oe-NC-2 group, the BLM+oe-SETDB2+WT DAXX group, and the BLM+oe-SETDB2+ΔSIM group.

[0050] Figure 24 Fe of BLM+oe-SETDB2+oe-NC-2 group, BLM+oe-SETDB2+WT DAXX group, and BLM+oe-SETDB2+ΔSIM group 2+ The expression levels of 4-HNE, GPX4, and SLC7A11; among them, Figure 24 a represents Fe from the BLM+oe-SETDB2+oe-NC-2 group, the BLM+oe-SETDB2+WT DAXX group, and the BLM+oe-SETDB2+ΔSIM group. 2+ 4-HNE horizontal diagram; Figure 24 b is a plot showing the expression levels of GPX4 and SLC7A11 in the BLM+oe-SETDB2+oe-NC-2 group, the BLM+oe-SETDB2+WT DAXX group, and the BLM+oe-SETDB2+ΔSIM group.

[0051] Figure 25 These are transmission electron microscope images of mitochondrial morphology in the BLM+oe-SETDB2+oe-NC-2 group, the BLM+oe-SETDB2+WT DAXX group, and the BLM+oe-SETDB2+ΔSIM group.

[0052] Figure 26 This is an immunofluorescence image of AAV-6-mediated overexpression of SETDB2 and DAXX in alveolar epithelial cells.

[0053] Figure 27 This is a Western blot image showing the protein expression levels of SETDB2, DAXX, and TP53 in the lung tissues of mice in each group after BLM / SiO2 treatment; among them, Figure 27 a is a Western blot plot showing the protein expression levels of SETDB2, DAXX, and TP53 in the lung tissue of mice in each group after BLM treatment; Figure 27b is a Western blot diagram showing the protein expression levels of SETDB2, DAXX, and TP53 in the lung tissue of mice in each group after SiO2 treatment.

[0054] Figure 28 These are H&E staining images of lung damage in the lung tissues of mice in each group after BLM treatment.

[0055] Figure 29 This is a Masson trichrome staining image showing collagen deposition in the lung tissues of mice in each group after BLM treatment.

[0056] Figure 30 These are H&E staining images of lung damage in the lung tissues of mice in each group after SiO2 treatment.

[0057] Figure 31 This is a Masson trichrome staining image showing the collagen deposition in the lung tissues of mice in each group after SiO2 treatment.

[0058] Figure 32 This is a Western blot image showing the expression of α-SMA, COL1A1, and FAPα in the lung tissues of mice in each group after BLM treatment.

[0059] Figure 33 This is a Western blot diagram showing the expression of α-SMA, COL1A1, and FAPα in the lung tissues of mice in each group after SiO2 treatment.

[0060] Figure 34 This is a flow cytometry image of lipid ROS levels in the lung tissues of mice in each group after SiO2 treatment.

[0061] Figure 35 The Fe in the lung tissue of mice in each group after BLM treatment 2+ Expression levels of 4-HNE, GPX4, and SLC7A11.

[0062] Figure 36 The Fe in the lung tissue of mice in each group after SiO2 treatment 2+ Expression levels of 4-HNE, GPX4, and SLC7A11. Detailed Implementation

[0063] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0064] In this invention, the handling of surgical specimens complied with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of Zhengzhou University People's Hospital (2019(28)). All patients signed written informed consent forms. In this study, 20 normal lung tissue samples were mainly from non-cancerous areas of the lungs of lung cancer patients. 20 fibrotic lung tissue samples were from lung transplant patients. Inclusion criteria were: age 18–80 years; clear pathological diagnosis; qualified tissue specimens; and signed informed consent. Exclusion criteria were: concurrent acute infection, active phase of autoimmune disease; preoperative radiotherapy, chemotherapy, targeted or immunotherapy; unqualified tissue specimens; severe dysfunction of vital organs; pregnancy or lactation.

[0065] The clinical characteristics of the patients are summarized in Table 1. All lung tissue samples used in this study were obtained from Henan Provincial People's Hospital.

[0066] Table 1 Summary of patients' clinical characteristics

[0067] C57BL / 6 mice (7-8 weeks old, 18-22 g) were purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd. All animals were housed under specific pathogen-free (SPF) conditions with controlled environmental parameters (temperature 20-25°C, humidity 45-60%, 12:12 hour light-dark cycle). Food and water were readily available before the experiment. Six mice were used per group. To establish the animal model, mice were intratracheally infused with 3 mg / kg BLM (B802467, Shanghai Maclean's), 2.5 mg / kg SiO2 (S5631, Sigma-Aldrich), or sterile saline (vector control). Mice were humanely sacrificed on days 14, 21, and 28 post-exposure, and bilateral lung tissue was immediately collected for subsequent analysis. The adeno-associated virus 6 (AAV-6) vector effectively mediated the overexpression of transgenes DAXX or SETDB2 under the control of the surfactant protein C (SPC) promoter, showing specificity towards alveolar epithelial cells. To determine the effects of DAXX and SETDB2 on pulmonary fibrosis, AAV-6 (1.1 × 10^12 vg / mL) was administered intratracheally two weeks prior to BLM or SiO2 injection. All experimental procedures and animal handling were approved by the Biomedical Research Ethics Committee of Nanhua University (2024-491) and conducted in accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals. All experimental evaluations were performed by uninformed investigators to ensure objectivity.

[0068] The detection involved in this invention is as follows: Hematoxylin-eosin (H&E) staining Lung tissue was fixed in 4% paraformaldehyde (P0099, Beyotime) and embedded in paraffin. Sections 4 μm thick were prepared using a paraffin microtome (YD-315, Yidi Medical Equipment Co., Ltd., Jinhua, China). The tissues were then dewaxed and rehydrated. Subsequently, the tissues were stained sequentially with hematoxylin (G1080, Solarbio, Beijing, China) and eosin (G1100, Solarbio). After mounting, the results were observed under an optical microscope (BA210T, MOTIC, Xiamen, China). In the figures, the scale bar for 20× field of view is 500 μm, for 100× field of view it is 100 μm, and for 400× field of view it is 25 μm.

[0069] Masson Tricolor Staining Masson's trichrome staining was performed using a commercial kit (C0189S, Beyotime). Tissue sections were dewaxed, hydrated, and stained with hematoxylin, acid fuchsin, and aniline blue. After destaining with ethanol and rinsing with xylene, the sections were mounted with neutral resin. In the figure, the scale bar for the field of view is 25 μm.

[0070] Immunofluorescence Tissue or cell sections were fixed with 4% paraformaldehyde for 30 minutes. After permeation with 0.3% Triton X-100 (P0096, Beyotime) at 37°C for 30 minutes and blocking with 5% BSA (SW3015, Solarbio) at 37°C for 60 minutes, samples were incubated overnight at 4°C with the primary antibody α-SMA (BM0002, 1:50, BOSTER, Wuhan, China). Subsequently, samples were incubated with diluted secondary antibody, goat anti-mouse IgG (H+L) (AWS0004a, 1:100, Abiowell, China), at 37°C for 90 minutes. Cell nuclei were stained with DAPI working solution at 37°C for 10 minutes. Samples were washed with PBS for 5 minutes, repeated three times. Finally, slides were mounted with buffered glycerol and observed under a fluorescence microscope (BA410T, MOTIC).

[0071] Flow cytometry Cells were extracted from lung tissue by digestion at 37°C for 2 hours. The collected cells were resuspended in RPMI-1640 medium (R8758, Sigma, USA). Cells from the tissue, as well as MLE-12 and Beas-2B cells, were digested using trypsin digestion solution. Subsequently, the cells were incubated with 5 μM C11-BODIPY (MX5211-1MG, Shanghai Mokang Biotechnology, China) at 37°C for 60 minutes. After washing twice with PBS and resuspending, the samples were analyzed using a flow cytometer (A00-1-1102, Beckman, USA).

[0072] Fe2+ and 4-HNE level assessment According to the kit instructions, use Fe 2+ The assay kit (JL-T1255, Jonlnbio, Shanghai, China) assesses Fe in tissues and cells. 2+ The levels of 4-HNE were measured in tissue and cell samples using a human 4-HNE assay kit (CSB-E16214h, Cusabio, Wuhan, China) and a mouse 4-HNE assay kit (CSB-E13412m, Cusabio).

[0073] Western blot Proteins were extracted using RIPA buffer (Cell Signaling Technology, USA) containing protease / phosphatase inhibitors, quantified by BCA assay, and separated by SDS-PAGE (P1200, Solarbio). After transfer, membranes were blocked with 5% skim milk / TBST and incubated with primary antibody (4°C, overnight), followed by incubation with HRP-conjugated secondary antibody (room temperature, 90 min). Protein bands were visualized using ECL (K-12049-D50, Advansta, USA) and quantified using ImageJ (National Institutes of Health, USA). Information on the first and second antibodies used in this study is shown in Table 2.

[0074] Table 2 Information on the first and second antibodies used in this study

[0075] Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from tissues and cells according to the protocols provided in the Trizol reagent manual (15596026, Thermo Fisher Scientific, USA). Total RNA was reverse transcribed into cDNA using a high-efficiency mRNA reverse transcription kit (CW2569, CWBlO, Beijing, China). The resulting cDNA was then analyzed by RT-qPCR using the SuperScript™ III Platinum™ One-Step qRT-PCR Kit (Thermo Fisher Scientific (USA)) and QuantStudio1 (ABI, USA). The mRNA levels of target genes were calculated using the 2^(-ΔΔCt) method. The specific primer sequences used are shown in Table 3.

[0076] Table 3 Primer sequence information

[0077] Transmission electron microscopy (TEM) Cells were fixed with 2.5% glutaraldehyde (12 h, DF0156, Beijing Regen, China) and 1% osmium tetroxide (2 h, 18456, Ted Pella Inc, USA), then dehydrated by a series of ethanol solutions, embedded in epoxy resin, and polymerized (60 °C, 48 h). Ultrathin sections were prepared and stained with uranyl acetate, and examined by TEM (JEM1400, JEOL, Japan).

[0078] Immunoprecipitation-mass spectrometry (IP-MS) Cells were lysed and clarified. Protein A / G magnetic beads were incubated with anti-SETDB2 antibody (2 μg / mg lysis buffer) at 4°C for 1 hour. IgG was used as a control. The lysis buffer was incubated with antibody-bound magnetic beads at 4°C overnight. Bound proteins were eluted with Laemmli buffer (95°C, 5 min). Proteins were reduced with 5 mM DTT, alkylated with 15 mMIAA, and digested with trypsin (1:50, 37°C, overnight). Peptides were desalted using C18 pipette tips. Peptides were separated on a C18 column (75 μm × 25 cm) at a 90-minute gradient. MS data were acquired in DDA mode on a Q-Exactive HF-X (IQLAAEGAAPFALGMBDK, Thermo Fisher Scientific, USA). Raw files were processed using MaxQuant (MaxQuant v2.6.2.0, Matthias Mann Lab, Germany). Significant interaction factors were identified compared with the IgG control (fold change ≥ 2, p < 0.05).

[0079] Bioinformatics Analysis The idiopathic pulmonary fibrosis (IPF) dataset was obtained from the GSE24206 database. Data analysis was then performed using R ×64 3.5.3 software. Differentially expressed genes between control and IPF tissues were analyzed using the edgeR package in R, with selection parameters set to |logFC|>0.4 and adjusted p-value <0.05. Ferroplasm-related genes were obtained from the FerrDB database (FerrDB 10.1093 / nar / gkac935). Proteins potentially interacting with SETDB2 were predicted using the Biogrid website. IP-MS analysis was performed on Beas-2B cells to identify proteins potentially interacting with SETDB2. Proteins overlapping between the Biogrid and IP-MS analyses were then determined.

[0080] Chromatin immunoprecipitation (ChIP) Cells were cross-linked with formaldehyde and lysed by sonication. Chromatin was fragmented and immunoprecipitated with anti-SETDB2 or anti-H3K9me3 antibody (4°C overnight). Antibody-protein-DNA complexes were captured using agarose beads, washed, and reverse cross-linked (98°C). DNA was purified and analyzed by RT-qPCR.

[0081] Co-immunoprecipitation (Co-IP) Total protein (1 mg) was immunoprecipitated using anti-SETDB2 or anti-DAXX antibody (5 μg, overnight at 4°C), with IgG as a control. Immune complexes were analyzed by Western blot. To assess SUMOylation dependence, HA-labeled DAXX was analyzed with and without the SUMOylation inhibitor ML-792. Antibodies: anti-SETDB2 (ab13712, 1:1000, Abcam, UK), anti-DAXX (67879-1-Ig, 1:1000, Abcam), anti-HA (51064-2-AP, 1:10000, Proteintech, USA), anti-H3K9me3 (ab176916, 1:1000, Abcam).

[0082] The statistical analysis method for the data in this invention is as follows: Data were processed using GraphPad Prism8 software (8.0.2.263, San Diego, USA) and analyzed using t-tests or one-way ANOVA.

[0083] Measurement data are expressed as mean ± standard deviation. Experiments were repeated three times. In all experiments, a p-value < 0.05 was considered statistically significant. Correlation analysis was performed using the Pearson correlation coefficient.

[0084] Example 1 Low expression of SETDB2 and occurrence of ferroptosis in animal models of pulmonary fibrosis First, an animal model of pulmonary fibrosis was established using bleomycin BLM. C57BL / 6 mice (7-8 weeks old, 18-22 g) were purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd. All animals were housed under specific pathogen-free (SPF) conditions with controlled environmental parameters (temperature 20-25°C, humidity 45-60%, 12:12 hour light-dark cycle), and had free access to food and water before the experiment. Six mice were used per group. To establish the animal model, mice were intratracheally infused with 3 mg / kg bleomycin BLM (B802467, Shanghai Maclean's), 2.5 mg / kg SiO2 (S5631, Sigma-Aldrich), or sterile saline (carrier control). Mice were humanely sacrificed on days 14, 21, and 28 post-exposure, and bilateral lung tissue was immediately collected for subsequent H&E staining, Masson's trichrome staining, Western blot, flow cytometry, and Fe2+ staining. 2+ Assessment of 4-HNE levels and immunofluorescence analysis.

[0085] After H&E staining and analysis of Ashcroft scores, the results showed that, compared with the control group, the BLM group exhibited alveolar structure destruction, interstitial thickening, abnormal lung markings, and increased Ashcroft scores, with the severity of damage increasing over time. Figure 1 Masson trichrome staining results also showed that BLM increased collagen deposition, and pulmonary fibrosis increased in a gradient with the duration of BLM induction. Figure 2 Furthermore, Western blot analysis showed increased levels of α-SMA, FAPα, N-cadherin, and TGF-β proteins in the BLM group, but decreased levels of E-cadherin. These changes were partially reversed between days 21 and 28 compared to day 14. Figure 3 These results indicate that BLM has successfully established an animal model of pulmonary fibrosis.

[0086] Then, ferroptosis was explored in an animal model of pulmonary fibrosis. Flow cytometry results showed that iron was significantly reduced. 2+ Evaluation results of the detection kit and the 4-HNE detection kit showed that, compared with the control group, BLM increased lipid ROS and Fe. 2+The levels of 4-HNE were reduced, while the levels of GPX4 and SLC7A11 were also decreased. These results indicate that ferroptosis occurs in pulmonary fibrosis tissue. However, with prolonged BLM induction time, ferroptosis attenuated on days 21 and 28. Figure 4 , Figure 5 , Figure 6 ).in, Figure 5 middle, Figure 5 a represents the Fe2+ level in lung tissue of mice in the control group and on days 14, 21, and 28 after BLM induction. Figure 5 b represents the 4-HNE level in the lung tissue of mice in the control group and on days 14, 21, and 28 after BLM induction. Figure 6 middle, Figure 6 a represents the protein expression levels of GPX4 and SLC7A11 in the lung tissues of mice in the control group and on days 14, 21, and 28 after BLM induction. Figure 6 b represents the protein expression levels of SETDB2 and H3K9me3 in the lung tissues of mice in the control group and on days 14, 21, and 28 after BLM induction. Western blot results showed that, compared with the control group, SETDB2 expression was downregulated in the BLM group, and its ability to form H3K9me3 was reduced. Figure 6 b). Immunofluorescence assay results showed that the positivity rate of SETDB2 was also reduced in the BLM group. Figure 7 Therefore, SETDB2 is expressed at low levels in fibrotic lung. SETDB2 expression showed the most significant downregulation on day 14 after BLM induction, and this time point was chosen for subsequent studies.

[0087] Example 2 SETDB2 overexpression inhibits fibroblast activation by suppressing ferroptosis in lung epithelial cells. Cell Culture and Treatment: MLE-12 (mouse alveolar epithelial cells, AW-MNM486, Abiowell, Changsha, China) and Beas-2B (human lung epithelial cells, AW-CNH004, Abiowell, China) were cultured in DMEM / F12 medium. An in vitro model of pulmonary fibrosis was established by inducing both cell lines with 10 μg / mL BLM for 24 hours. For cell transfection, plasmids were transfected into MLE-12 and Beas-2B cells using Lipofectamine 2000 (11668030, Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The plasmids were oe-SETDB2 (HG-HO031915, HonorGene) and oe-TP53 (HG-HO000546, HonorGene). To further investigate the effects of SETDB2 and DAXX on ferroptosis, cells were treated with either the ferroptosis inducer erastin (SC0224, Beyotime Biotechnology, Shanghai, China) or the ferroptosis inhibitor Ferrostatin-1 (Fer-1). The treatment methods were as follows: 1 hour before BLM treatment, Fer-1 was added to the culture medium to a final concentration of 1 μM, followed by co-treatment with BLM for 24 hours. Alternatively, 1 hour before BLM treatment, erastin was added to the culture medium to a final concentration of 10 μM, followed by co-treatment with BLM for 24 hours.

[0088] Co-culture system: L929 (mouse fibroblasts, AW-CNM125, Abiowell, China) and MRC-5 (human lung fibroblasts, AW-CNH069, Abiowell, China) were cultured in MEM medium. MLE-12 and Beas-2B cells transfected and treated as described above were co-cultured with mouse fibroblasts (L929) and human lung fibroblasts (MRC-5) for 48 hours to investigate the effects of these treatments on fibroblast activation. Analytical methods included: transmission electron microscopy, Western blot, and flow cytometry for lipid ROS and Fe. 2+ Assessment of 4-HNE levels and immunofluorescence detection of α-SMA.

[0089] Western blot results showed that the oe-NC group had elevated SETDB2 levels compared to the oe-SETDB2 group, indicating that MLE-12 and Beas-2B cells were successfully transfected with oe-SETDB2. Figure 8 BLM reduced H3K9me3 levels in MLE-12 and Beas-2B cells. Overexpression of SETDB2 reversed the effects of BLM and increased H3K9me3 levels. As a ferroptosis activator, erastin did not affect H3K9me3 levels. Figure 10 and Figure 9 BLM increased cellular lipid ROS levels. Overexpression of SETDB2 reversed this effect and decreased lipid ROS levels. Erastin counteracted the effect of oe-SETDB2 and increased lipid ROS levels. Figure 11 ,in, Figure 11 a shows flow cytometry images of lipid ROS levels in different groups of Beas-2B cells; Figure 11 b is a flow cytometry plot of lipid ROS levels in each group of MLE-12 cells. Furthermore, oe-SETDB2 reversed BLM-induced Fe... 2+ Increased 4-HNE levels and decreased GPX4 and SLC7A11 expression. Erastin blocked these effects of oe-SETDB2 ( Figure 12 ,in, Figure 12 a represents Fe in each group of MLE-12 cells. 2+ Horizontal map; Figure 12 b shows the 4-HNE levels in each group of MLE-12 cells; Figure 12 c shows the Western blot plot of GPX4 and SLC7A11 protein expression levels in each group of MLE-12 cells. TEM revealed that MLE-12 and Beas-2B cells in the BLM group exhibited mitochondrial shrinkage and increased membrane density, which are morphological features of ferroptosis. Overexpression of SETDB2 attenuated these phenomena. However, Erastin exacerbated the ferroptosis phenotype. Figure 13 and Figure 14 These results indicate that overexpression of SETDB2 inhibits BLM-induced ferroptosis.

[0090] In a cell co-culture system, immunofluorescence and Western blot analysis demonstrated that BLM significantly upregulated the expression of α-SMA in fibroblasts, as well as increased levels of fibrosis markers (COL1A1, FAPα). SETDB2 overexpression effectively reversed these BLM-induced pro-fibrotic effects, while the ferroptosis inducer erastin counteracted the protective effect of SETDB2 and increased the expression of fibrosis markers. Figure 15 ,in, Figure 15 a is a Western blot plot showing the protein expression level of α-SMA in L929 fibroblasts; Figure 15 b is a Western blot plot showing the protein expression levels of COL1A1 and FAPα in L929 fibroblasts; Figure 15 c is an immunofluorescence image showing α-SMA expression in L929 fibroblasts. Similar results were obtained in human lung fibroblasts. These results suggest that SETDB2 overexpression may inhibit fibroblast activation by suppressing BLM-induced ferroptosis.

[0091] Example 3 SETDB2 inhibits fibroblast activation by suppressing BLM-induced ferroptosis in lung epithelial cells. Cell transfection: The groups are as follows: BLM+oe-NC+oe-NC-1, BLM+oe-SETDB2+oe-NC-1, BLM+oe-NC+oe-TP53, and BLM+oe-SETDB2+oe-TP53.

[0092] 1. Experimental materials Cell lines: MLE-12 (mouse alveolar epithelial cells, AW-MNM486, Abiowell), Beas-2B (human lung epithelial cells, AW-CNH004, Abiowell). Transfection reagent: Lipofectamine 2000 (11668030, Thermo Fisher Scientific); Reduced serum medium: Opti-MEM I Reduced Serum Medium (used for diluting DNA and transfection reagents) Expression plasmids: oe-SETDB2 and oe-TP53 (SETDB2 and oe-TP53 overexpression plasmids, HG-HO031915, HonorGene, HG-HO000546, HonorGene), oe-NC (negative control plasmid, HonorGene). Culture containers: 24-well plates (Western blot, flow cytometry, Fe) 2+ / 4-HNE detection) or co-culture system with the corresponding specifications.

[0093] 2. Cell preparation before transfection (Day 1) One day before transfection, MLE-12 or Beas-2B cells were digested with trypsin and then counted. Cells were counted at 0.5–2 × 10⁶ cells per well. 5 Cells were seeded at a density in 24-well plates, with 500 μL of antibiotic-free growth medium (DMEM / F12) added to each well. Cells were incubated overnight at 37°C in a 5% CO2 incubator. Cell confluence reached 70-90% on the day of transfection.

[0094] 3. Preparation of transfection complex (Day 2) Taking a single hole of a 24-hole plate as an example: Step 1: Dilute plasmid DNA. Take a 1.5 mL sterile centrifuge tube and add 50 μL of Opti-MEM I serum-depleted medium (without serum and antibiotics). Add 0.8–1.0 μg of oe-SETDB2 plasmid (or an equal amount of oe-NC control plasmid). Gently pipette to mix and incubate at room temperature.

[0095] Step 2: Dilute Lipofectamine 2000. Take another 1.5 mL sterile centrifuge tube and add 50 μL of Opti-MEM I serum-depleted medium. Gently mix the Lipofectamine 2000 stock solution (do not vortex). Add 2.0 μL of Lipofectamine 2000 to Opti-MEM. Gently pipette to mix and let stand at room temperature for 5 minutes.

[0096] Step 3: Formation of the DNA-Lipofectamine 2000 complex. Add the DNA dilution from Step 1 to the Lipofectamine 2000 dilution from Step 2. Gently pipette to mix 3-5 times. Incubate at room temperature for 20 minutes to allow the DNA to fully bind with the liposomes and form a complex.

[0097] 4. The transfection complex is added to the cells. Add 100 μL of the DNA-Lipofectamine 2000 complex dropwise to each well of the cell culture. Gently shake the culture plate back and forth and side to side to distribute the complex evenly in the culture medium. Return the culture plate to a 37°C, 5% CO2 incubator for further incubation.

[0098] 5. Post-transfection treatment Four to six hours after transfection, the culture medium can be switched to complete medium (containing serum, but without antibiotics) to reduce the cytotoxicity of Lipofectamine 2000. Alternatively, cells can be collected 48-72 hours after transfection to directly assess transfection efficiency. Another option is to treat cells with BLM (10 μg / mL) for 24 hours after transfection, and then collect the cells for subsequent experiments.

[0099] Detection indicators: lipid ROS (flow cytometry), Fe 2+ The levels of 4-HNE, GPX4 and SLC7A11 (Western blot), mitochondrial morphology were observed by TEM, and fibroblast α-SMA, FAPα and COL1A1 were detected by co-culture system (immunofluorescence and Western blot). The operation method was the same as in Examples 1 and 2.

[0100] Compared with the BLM+oe-NC+oe-NC-1 group, SETDB2 overexpression decreased the level of lipid ROS in cells, while TP53 overexpression increased the level of lipid ROS. Compared with the BLM+oe-SETDB2+oe-NC-1 group, TP53 overexpression reversed the effect of oe-SETDB2 and increased the level of lipid ROS. Figure 16 Overexpression of SETDB2 reduced Fe. 2+ The levels of 4-HNE were increased, along with the levels of GPX4 and SLC7A11. Conversely, overexpression of TP53 increased Fe. 2+ The levels of 4-HNE were reduced, and the levels of GPX4 and SLC7A11 were decreased. Overexpression of TP53 also counteracted these effects of oe-SETDB2. Figure 17 ,in, Figure 17 a represents Fe in each group of Beas-2B cells. 2+ A bar chart with 4-HNE levels; Figure 17 b is a bar graph showing the levels of GPX4 and SLC7A11 in each group of Beas-2B cells. Mitochondrial damage in cells was alleviated after oe-SETDB2, but was further aggravated after oe-TP53. oe-TP53 reversed the inhibitory effect of oe-SETDB2 on mitochondrial damage. Figure 18 These results indicate that the SETDB2 / TP53 axis inhibits BLM-induced ferroptosis in lung epithelial cells.

[0101] In the cell co-culture system, overexpression of SETDB2 reduced the levels of α-SMA, FAPα, and COL1A1 in fibroblasts. Overexpression of TP53 increased the expression of α-SMA, FAPα, and COL1A1 and reversed the effect of oe-SETDB2. These results suggest that SETDB2 can inhibit fibroblast activation by suppressing BLM-induced ferroptosis in lung epithelial cells.

[0102] Example 4 DAXX stimulates SETDB2 activity in a SUMOylation-dependent manner. Experimental process Immunoprecipitation-mass spectrometry (IP-MS): Cells were lysed and clarified. Protein A / G magnetic beads were incubated with anti-SETDB2 antibody (2 μg / mg lysis buffer) at 4°C for 1 hour. IgG was used as a control. The lysis buffer was incubated with antibody-bound magnetic beads at 4°C overnight. Bound proteins were eluted with Laemmli buffer (95°C, 5 min). Proteins were reduced with 5 mM DTT, alkylated with 15 mM IAA, and digested with trypsin (1:50, 37°C, overnight). Peptides were desalted using C18 pipette tips. Peptides were separated on a C18 column (75 μm × 25 cm) at a gradient of 90 min. MS data were acquired in DDA mode on a Q-Exactive HF-X (IQLAAEGAAPFALGMBDK, Thermo Fisher Scientific, USA). Raw files were processed using MaxQuant (MaxQuant v2.6.2.0, Matthias Mann Lab, Germany). Significant interaction factors were identified compared with the IgG control (fold change ≥2, p<0.05).

[0103] Bioinformatics analysis: The Biogrid website was used to predict proteins that may interact with SETDB2. IP-MS analysis was performed on Beas-2B cells to identify proteins that may interact with SETDB2. Proteins overlapping between the Biogrid and IP-MS analyses were then determined.

[0104] Co-immunoprecipitation (Co-IP): Total protein (1 mg) was immunoprecipitated overnight at 4°C using 5 μg of anti-SETDB2 (ab13712, 1:1000, Abcam, UK) or anti-DAXX antibody (67879-1-Ig, 1:1000, Abcam), with IgG as a control. Immune complexes were analyzed by Western blot. To assess SUMOylation dependence, HA-labeled DAXX was analyzed with and without the SUMOylation inhibitor ML-792. Antibodies: anti-SETDB2 (ab13712, 1:1000, Abcam, UK), anti-DAXX (67879-1-Ig, 1:1000, Abcam), anti-HA (51064-2-AP, 1:10000, Proteintech, USA), and anti-H3K9me3 (ab176916, 1:1000, Abcam).

[0105] Cell transfection: The siRNA sequence targeting DAXX is as follows: si-DAXX#2,5'-AGTGCTACAAGTTGGATAATG-3' (SEQ ID NO.33).

[0106] Experimental results showed that, compared with si-NC, the si-DAXX#2 construct (constructed by HonorGene based on the siRNA sequence targeting DAXX, a commercially available plasmid; the corresponding models for si-DAXX#2 are pHG-HomoDAXX-sh1, HG-shMO007829, HonorGene; wild-type DAXX: oe-DAXX, HG-MO007829, HonorGene) effectively reduced DAXX levels in MLE-12 and Beas-2B cells. si-DAXX#2 exhibited the highest knockdown efficiency and was selected for subsequent experiments. Figure 19 Subsequently, WT DAXX and its mutant variant (ΔSIM) were transfected.

[0107] The Biogrid website predicted a total of 17 proteins that may interact with SETDB2. A Venn diagram showed that the overlapping protein was DAXX. BLM had no significant effect on DAXX expression in MLE-12 and Beas-2B cells. Co-IP showed that SETDB2 interacts with DAXX. Figure 20 ,in, Figure 20 Figure a shows the interaction results between SETDB2 and DAXX in Beas-2B cells; Figure 20 Figure b shows the interaction between SETDB2 and DAXX in MLE-12 cells. Compared with the Control-IP-SETDB2 group, BLM weakened the interaction between SETDB2 and DAXX.

[0108] Knockdown of DAXX resulted in decreased H3K9me3 expression. DAXX immunoprecipitation (anti-DAXX-si-NC) showed significant TP53 enrichment, indicating an association between DAXX and TP53 enrichment. Furthermore, DAXX knockdown reduced TP53 enrichment regardless of whether DAXX or H3K9me3 antibody was used. HA-DAXX increased H3K9me3 expression compared to the oe-NC group. ML-792 blocked the effect of HA-DAXX and downregulated H3K9me3 expression. DAXX expression was increased in both the WT and ΔSIM groups compared to the oe-NC group, indicating successful transfection of the WTDAXX and SUMO-binding defective mutant into cells. DAXX knockdown decreased H3K9me3 while increasing TP53, while WTDAXX showed the opposite effect. The ΔSIM mutant had no effect, indicating that DAXX regulates H3K9me3 and TP53 levels in a SUMOylation-dependent manner. Notably, SETDB2 expression was unaffected by DAXX manipulation. Figure 21Co-IP results showed that WT DAXX enhanced the interaction between DAXX and SETDB2. However, ΔSIM reduced the binding of DAXX and SETDB2, indicating that DAXX mediates its interaction with SETDB2 through SUMOylation. These results suggest that DAXX depends on SUMOylation to stimulate SETDB2 activity.

[0109] Example 5 DAXX inhibits fibroblast activation by SUMOylation-dependent binding to SETDB2 to suppress ferroptosis. Cell grouping: Under BLM induction, MLE-12 and Beas-2B cells were divided into the following groups: BLM+oe-SETDB2+oe-NC-2, BLM+oe-SETDB2+WT DAXX, and BLM+oe-SETDB2+ΔSIM.

[0110] The experimental procedure was the same as above. First, oe-NC and oe-SETDB2, oe-SETDB2 and WT DAXX, oe-SETDB2 and ΔSIM were transfected for 48 h, and then BLM was added for 24 h.

[0111] Detection indicators: Western blot analysis of SETDB2, TP53, DAXX, H3K9me3, GPX4, and SLC7A11 levels; flow cytometry analysis of lipid ROS; Fe... 2+ The levels of 4-HNE were assessed; mitochondrial morphology was observed using TEM; and α-SMA, COL1A1, and FAPα in fibroblasts were detected using a co-culture system (immunofluorescence and Western blot). An additional Fer-1 intervention group and a si-DAXX knockdown group were also included. The procedures were the same as those described in the previous examples.

[0112] Experimental results showed that BLM-induced overexpression of SETDB2 increased SETDB2 levels, decreased TP53 levels, and did not affect DAXX levels. Compared with the BLM+oe-SETDB2+oe-NC-2 group, both WT DAXX and ΔSIM increased DAXX levels but did not affect SETDB2 levels. WT DAXX decreased TP53 levels, while ΔSIM did not. Figure 22 Under BLM induction, overexpression of SETDB2 and WT DAXX enhanced H3K9me3 production with a synergistic effect. However, ΔSIM did not have this effect. Flow cytometry results indicated that WT DAXX, but not ΔSIM, further enhanced the lipid ROS-reducing effect of oe-SETDB2. Figure 23 In addition, WT DAXX reduced Fe. 2+The levels of 4-HNE increased the expression of GPX4 and SLC7A11 and showed a synergistic effect with oe-SETDB2, while ΔSIM did not. Figure 24 ,in, Figure 24 a represents Fe from the BLM+oe-SETDB2+oe-NC-2 group, the BLM+oe-SETDB2+WT DAXX group, and the BLM+oe-SETDB2+ΔSIM group. 2+ 4-HNE horizontal diagram; Figure 24 b shows the expression levels of GPX4 and SLC7A11 in the BLM+oe-SETDB2+oe-NC-2 group, the BLM+oe-SETDB2+WT DAXX group, and the BLM+oe-SETDB2+ΔSIM group. WT DAXX alleviated mitochondrial damage and had a synergistic effect with oe-SETDB2, manifested as inhibition of mitochondrial shrinkage and increased membrane density. However, the SUMOylation-deficient ΔSIM lost this function of DAXX. Figure 25 ).

[0113] In the cell co-culture system, WT DAXX, rather than ΔSIM, further enhanced oe-SETDB2-mediated reduction in α-SMA fluorescence and inhibition of α-SMA / COL1A1 / FAPα expression. Fer-1 synergistically inhibited the fibrosis markers (α-SMA / COL1A1 / FAPα) with oe-SETDB2, while DAXX knockdown reversed this protective effect. Fer-1 blocked this effect of si-DAXX and reduced the expression of α-SMA, COL1A1, and FAPα. These results suggest that DAXX can inhibit fibroblast activation by SUMOylation-dependent binding to SETDB2, thereby suppressing TP53-mediated transcriptional regulation of ferroptosis in lung epithelial cells.

[0114] Example 7 DAXX and SETDB2 synergistically reduce ferroptosis and pulmonary fibrosis in vivo. Experimental process AAV-6 Vector Construction and Administration: The adeno-associated virus 6 (AAV-6) vector efficiently mediated the overexpression of transgenic DAXX or SETDB2 under the control of the surfactant protein C (SPC) promoter, demonstrating specificity for alveolar epithelial cells. To determine the effects of DAXX and SETDB2 on pulmonary fibrosis, AAV-6 (1.1 × 10¹¹ mg / L) was administered intratracheally 2 weeks prior to BLM or SiO2 injection. 2The groups were as follows: control group (physiological saline), BLM / SiO2+AAV-6-NC, BLM / SiO2+AAV-6-SETDB2, BLM / SiO2+AAV-6-DAXX, and BLM / SiO2+AAV-6-SETDB2+AAV-6-DAXX. Plasmids AAV-6-NC, AAV-6-SETDB2, AAV-6-DAXX, and AAV-6-SETDB2+AAV-6-DAXX were all purchased from Gilead Sciences.

[0115] Detection indicators: Immunofluorescence was used to verify the expression of SETDB2 and DAXX in alveolar epithelial cells; Western blot was used to detect SETDB2, DAXX, TP53, H3K9me3, α-SMA, COL1A1, FAPα, GPX4, and SLC7A11; H&E staining and Masson staining were used to assess lung injury and collagen deposition; Flow cytometry was used to detect lipid ROS; Fe 2+ And 4-HNE level assessment. The operation method is the same as the previous embodiment.

[0116] Experimental results: Immunofluorescence showed increased expression of SETDB2 and DAXX in alveolar epithelial cells in vivo. Figure 26 BLM / SiO2 treatment in mice specifically decreased SETDB2 and increased TP53 without altering DAXX. While overexpression of either SETDB2 or DAXX selectively increased their respective protein levels, the two interventions synergistically suppressed TP53 expression. Figure 27 ,in, Figure 27 a is a Western blot plot showing the protein expression levels of SETDB2, DAXX, and TP53 in the lung tissue of mice in each group after BLM treatment; Figure 27 b is a Western blot plot showing the protein expression levels of SETDB2, DAXX, and TP53 in the lung tissue of mice in each group after SiO2 treatment. BLM and SiO2 reduced H3K9me3 formation. Overexpression of SETDB2 and DAXX reversed this effect of BLM and SiO2 and increased H3K9me3 levels. Overexpression of DAXX further enhanced the ability of SETDB2 AAV-6 to form H3K9me3. Overexpression of SETDB2 and DAXX alleviated BLM- or SiO2-induced lung injury and pulmonary fibrosis. Based on SETDB2 overexpression, DAXX overexpression further inhibited lung injury and pulmonary fibrosis. Figure 28 , Figure 29 , Figure 30 , Figure 31BLM and SiO2 enhanced the expression of α-SMA, COL1A1, and FAPα in mice. Overexpression of SETDB2 and DAXX blocked this effect of BLM and SiO2, showing an additive effect. Figure 32 and Figure 33 BLM and SiO2 increased lipid ROS levels in mice, while overexpression of SETDB2 or DAXX reversed this effect. Combined overexpression of SETDB2 and DAXX showed an additive inhibitory effect on lipid ROS accumulation. Figure 34 BLM and SiO2 increased Fe 2+ The levels of 4-HNE were reduced, and GPX4 and SLC7A11 proteins were decreased. Overexpression of SETDB2 and DAXX reversed these changes and had a synergistic effect. Figure 35 , Figure 36 These results indicate that DAXX and SETDB2 can synergistically reduce ferroptosis and pulmonary fibrosis in BLM mice.

[0117] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. Application of DAXX and / or SETDB2 in the preparation of drugs for treating pulmonary fibrosis.

2. Application of DAXX overexpression reagent and / or SETDB2 overexpression reagent in the preparation of anti-pulmonary fibrosis drugs.

3. The application according to claim 2, characterized in that, The DAXX overexpression reagent includes the DAXX overexpression vector; the SETDB2 overexpression reagent includes the SETDB2 overexpression vector.

4. The application according to claim 3, characterized in that, DAXX overexpression vectors can be viral or non-viral vectors; SETDB2 overexpression vectors can be viral or non-viral vectors.

5. The application according to claim 4, characterized in that, The viral vector is selected from adeno-associated virus, lentivirus, adenovirus, or retrovirus; adeno-associated virus is preferred.

6. The application according to claim 5, characterized in that, The adeno-associated virus is AAV6, AAV9, or AAV-DJ serotype.

7. The application according to claim 2, characterized in that, The DAXX overexpression reagent is an adeno-associated virus carrying the DAXX coding gene; the SETDB2 overexpression reagent is an adeno-associated virus carrying the SETDB2 coding gene.

8. A pharmaceutical composition, characterized in that, It contains reagents for overexpressing DAXX and / or SETDB2, as well as pharmaceutically acceptable excipients.

9. A gene delivery tool, characterized in that, It includes adeno-associated viruses carrying the DAXX coding gene and / or the SETDB2 coding gene.

10. Application of DAXX overexpression reagent and / or SETDB2 overexpression reagent in the preparation of drugs that inhibit ferroptosis.

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