Application of KAT7 as pulmonary fibrosis diagnosis and prognosis biomarker and therapeutic target

Abstract

The invention discloses application of KAT7 as a pulmonary fibrosis diagnosis and prognosis biomarker and a treatment target. The research finds that the lysine acetyltransferase KAT7 is remarkably up-regulated in lung tissues of human pulmonary fibrosis patients and mouse fibrosis models, and is mainly expressed in macrophages; kat7 is specifically knocked out from myeloid cells, so that lung inflammation and fibrosis processes of mice can be remarkably relieved; further research finds that the fibrosis promoting function of macrophages can be down-regulated by inhibiting the activity of KAT7, and the generation and deposition of collagen can be reduced. Research results of the invention reveal the key driving effect of KAT7 in pulmonary fibrosis, a new marker is provided for diagnosis and prognosis of pulmonary fibrosis, and a new target is provided for prevention and treatment of pulmonary fibrosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of KAT7 as a pulmonary fibrosis diagnosis, prognosis biomarker and treatment target. BACKGROUND

[0002] Pulmonary fibrosis (PF) is a chronic, progressive, fatal interstitial lung disease characterized by abnormal proliferation of fibroblasts and excessive deposition of extracellular matrix (ECM), which eventually leads to destruction of lung tissue structure, gas exchange dysfunction and respiratory failure. Although research on its pathogenesis is continuously deepening, there is still a lack of effective treatment at present, and lung transplantation is the only choice for patients in the end stage. Therefore, it is of great clinical significance to clarify the key regulatory mechanism of pulmonary fibrosis and find new therapeutic targets.

[0003] In recent years, a large number of studies have shown that epigenetic modification plays a key role in the pathological process of pulmonary fibrosis, and post-translational modification of proteins, such as acetylation, is involved in the regulation of gene transcription and cell function. The lysine acetyltransferase (KAT) family is a key enzyme that catalyzes protein acetylation, which regulates the activity and function of target proteins by transferring acetyl groups to lysine residues. There are many members of the KAT family, and their specific roles and molecular mechanisms in pulmonary fibrosis are not fully understood.

[0004] KAT7 is a member of the MYST (MOZ, Ybf2 / Sas3, Sas2, Tip60) acetyltransferase family, which has been proven to play a role in various biological processes such as cell cycle regulation, DNA damage repair and transcriptional activation. Previous studies have focused on its role in tumor development, but its function in fibrotic diseases, especially pulmonary fibrosis, remains to be elucidated. The pathological core of pulmonary fibrosis is persistent tissue damage and abnormal repair response, and abnormal activation of immune cells such as macrophages is considered to be a key factor driving inflammation and fibrosis progression. However, whether KAT7 is expressed in these key cells and regulates their function to affect the progression of pulmonary fibrosis is a scientific problem that needs to be solved. SUMMARY

[0005] The purpose of the present application is to provide KAT7 as a pulmonary fibrosis diagnosis, prognosis biomarker and treatment target in view of the above problems.

[0006] In order to achieve the purpose of the present application, the technical scheme adopted by the present application is:

[0007] The first aspect of the present application provides use of a reagent for detecting expression of biomarker KAT7 in preparation of a product for assisting diagnosis or prognosis of a subject with pulmonary fibrosis.

[0008] The method for diagnosis comprises obtaining a test sample from a patient, determining the expression level of KAT7 in the test sample, and if the expression level of KAT7 is higher than that of a normal control, it indicates that the subject is a patient with pulmonary fibrosis.

[0009] The method for prognosis comprises obtaining a test sample from a patient with pulmonary fibrosis, determining the expression level of KAT7 in the test sample, and if the expression level of KAT7 is high, it indicates that the patient has a poor prognosis.

[0010] Preferably, the test sample is lung tissue.

[0011] The second aspect of the present application provides use of KAT7 as a target in screening drugs for treating pulmonary fibrosis.

[0012] The drug inhibits the expression or activity of KAT7, and a decrease in the expression or activity level of KAT7 inhibits or alleviates the development of pulmonary fibrosis.

[0013] The third aspect of the present application provides use of a reagent for inhibiting the expression or activity of KAT7 in preparation of a drug for treating pulmonary fibrosis.

[0014] The reagent for inhibiting the expression or activity of KAT7 comprises a nucleic acid molecule, a KAT7 protein specific binding agent, or a compound.

[0015] The compound is a small molecule or a low molecular weight compound, preferably WM-3835.

[0016] The KAT7 protein specific binding agent is a selective inhibitor, an antibody, or an antagonist against KAT7 protein.

[0017] The nucleic acid molecule targets KAT7, interferes with or inhibits the expression of KAT7.

[0018] The drug inhibits the expression or activity of KAT7 in lung tissue macrophages, inhibits the pro-fibrotic function, reduces the release of pro-inflammatory cytokines and extracellular matrix deposition, reduces collagen deposition, reduces inflammation, and alleviates or inhibits the development of pulmonary fibrosis.

[0019] The beneficial effects of the present application are: the present application researches and finds that lysine acetyltransferase KAT7 is significantly up-regulated in human lung fibrosis patients and mouse fibrosis model lung tissues, and mainly expressed in macrophages; myeloid cell-specific knockout Kat7 can significantly alleviate lung inflammation and fibrosis progression in mice; further research finds that inhibiting the activity of KAT7 can down-regulate the pro-fibrosis function of macrophages, and reduce the production and deposition of collagen. The research results of the present application reveal the key driving role of KAT7 in lung fibrosis, and provide a new marker for lung fibrosis diagnosis and prognosis, and provide a new target for preventing and treating lung fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The expression of KAT7 is up-regulated in human lung fibrosis patients and mouse lung fibrosis models: A. Wayne diagram analysis of lysine acetyltransferase (KATs) family members related to lung fibrosis in public databases (GSE76808, GSE83717, GSE163740); B. Immunohistochemical (IHC) staining and scoring of KAT7 protein expression in lung tissues of non-fibrosis control group and lung fibrosis (PF) patients; C. Correlation analysis of KAT7 mRNA expression level and fibrosis gene (COL1A1, COL3A1, ACTA2) expression level in human idiopathic pulmonary fibrosis (IPF) patient tissues (data source: GSE231893); D. Correlation analysis of KAT7 mRNA expression level and lung function index carbon monoxide diffusion capacity (DLCO) in IPF patients (data source: GSE213001); E-G. Western blot detection of KAT7 in lung tissues of male mice after intratracheal injection of bleomycin (BLM) or normal saline (Sal) for 21 days (E), protein quantification analysis (F) and RT-qPCR detection of mRNA level (G); H-I. Western blot and protein quantification analysis of KAT7 in lung tissues of mice after intratracheal injection of silica dioxide (SiO2) or normal saline for 50 days (H) and RT-qPCR detection of mRNA level (I); J. Immunofluorescence (IF) staining and quantification analysis of KAT7 in lung tissues of Sal and BLM treated mice; KAT7 is green, and the nucleus is counterstained by DAPI (blue); K. RT-qPCR detection of Kat7 mRNA expression level in lung tissues of BLM treated female mice.

[0021] Figure 2KAT7 is mainly distributed in macrophages in fibrotic lung tissues: A-B. Analysis of human lung tissue single-cell sequencing data showed the expression distribution of KAT7 in different cell lineages (immune cells, epithelial cells, stromal cells) (A) and high expression in each type of lung cell UMAP map (B); C. Immunofluorescence co-localization staining of KAT7 (green) with macrophage marker F4 / 80, endothelial cell marker VE-cadherin, epithelial cell marker E-cadherin and fibroblast marker a-SMA (all red) in mouse fibrotic lung tissues; D. RT-qPCR detection of Kat7 mRNA expression levels in different primary lung cells of mice (alveolar epithelial cells AECs, macrophages, endothelial cells, fibroblasts); E. RT-qPCR comparison of the expression difference of Kat7 mRNA in mouse epithelial cells after Sal or BLM treatment; F. Correlation analysis of KAT7 and macrophage marker MRC1 expression levels in human lung tissue samples (data source: GSE231893); G. Western blot detection of KAT7 protein levels in mouse primary alveolar macrophages (AMs) after BLM or SiO2 treatment.

[0022] Figure 3 Myeloid cell-specific knockout of KAT7 can alleviate BLM-induced pulmonary fibrosis: A-B. Lung histopathology of WT and Kat7 ΔLyz2 Survival curve (A) and lung weight (B) of mice; C-D. RT-qPCR detection of Tgf-β1 mRNA expression levels in lung tissues of WT and Kat7 ΔLyz2 F4 / 80 + Immunofluorescence staining (C) and quantitative analysis (D) of macrophages; E-F. RT-qPCR detection of Tgf-β1 mRNA expression levels in lung tissues of WT and Kat7 ΔLyz2 Representative histopathology images of mouse lung tissues, including HE staining (G), Masson's trichrome staining (H) and a-SMA immunohistochemical staining (I); J-K. Quantitative analysis of the degree of pulmonary fibrosis, including Ashcroft score (J) and hydroxyproline content detection (K); L. ELISA detection of TGF-β levels in bronchoalveolar lavage fluid (BALF) of WT and Kat7 ΔLyz2 Micro-CT image comparison of mouse lungs.

[0023] Figure 4This study demonstrated that KAT7 deficiency in myeloid cells inhibits the pro-fibrotic function of macrophages: WT and Kat7 levels after AD. BLM treatment. ΔLyz2 Transcriptome sequencing (RNA-seq) analysis of mouse lung tissue, including experimental design diagram (A), principal component analysis (PCA) diagram (B), differentially expressed gene heatmap (C), and expression heatmap of genes related to immune response and macrophage activation (D); E. Gene set enrichment analysis (GSEA) results of RNA-seq data; FG. Comparison with Kat7 ΔLyz2 Immunofluorescence staining and quantification of α-SMA expression in primary fibroblasts after co-culture with mouse BALF (F), and Western blot detection and quantification (G).

[0024] Figure 5 The study demonstrated that reinfusion of KAT7-deficient macrophages could alleviate pulmonary fibrosis: AB. Quantitative analysis of the degree of pulmonary fibrosis in each group of mice during the macrophage reinfusion experiment, including Ashcroft score (A) and hydroxyproline content (B); CD. Histopathological analysis of lung tissue in each group of mice during the macrophage reinfusion experiment, including HE staining and α-SMA immunohistochemical staining (C), and Western blot detection of α-SMA protein expression (D).

[0025] Figure 6 The following images illustrate the efficacy of the pharmacological inhibitor WM-3835 in alleviating bleomycin (BLM)-induced pulmonary fibrosis: A. Schematic diagram of the therapeutic dosing regimen of the KAT7 inhibitor WM-3835 in a BLM-induced pulmonary fibrosis model; BC. Curves showing the effect of WM-3835 treatment on body weight change (B) and lung index (C) in BLM-treated mice; DF. Effects of WM-3835 treatment on inflammation and fibrosis-related molecules in mice: RT-qPCR detection of mRNA expression levels of inflammatory factors (D) and fibrosis-related genes (F) in lung tissue, and detection of serum protein levels of inflammatory cytokines (IL-6, TNF-α, TGF-β) (E); G. Immunohistochemical staining of α-SMA in lung tissue of BLM-induced mice treated with WM-3835. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0027] Unless otherwise specified, the experimental methods in the following examples are conventional methods; the chemical and biological materials and reagents used are conventional materials and reagents in the art and are commercially available unless otherwise specified.

[0028] Example 1

[0029] I. Materials and Methods

[0030] 1. KAT7 expression in human patients with pulmonary fibrosis

[0031] Publicly available datasets GSE76808, GSE83717, and GSE163740 were obtained from the GEO database to analyze the expression differences of KAT7 in patients with pulmonary fibrosis and healthy controls. These datasets contain gene and protein expression data from lung tissue or serum samples of patients with pulmonary fibrosis and healthy controls. The data were acquired and preprocessed using R language, and statistical analysis was performed on the expression differences between the pulmonary fibrosis group and the healthy control group.

[0032] 2. Human lung tissue analysis

[0033] Thirty normal lung tissue samples from lung cancer patients and 34 lung tissue samples from IPF patients were collected from the Department of Pathology, China-Japan Friendship Hospital, Beijing. Immunohistochemical staining analysis was performed on the lung tissues.

[0034] 3. Animal models

[0035] Pulmonary fibrosis models were induced by intratracheal infusion of bleomycin (BLM, MedChemExpress) or silica (SiO2, Sigma-Aldrich). Mice were anesthetized by intraperitoneal injection of afotenol (400 mg / kg), followed by intratracheal infusion of 2.5 mg / kg BLM or 125 mg / kg silica suspension. Control group mice received an equal volume of sterile saline. Unless otherwise specified, mice were sacrificed on day 21 after BLM treatment and on day 50 after SiO2 treatment. In the pharmacodynamic studies of the KAT7 inhibitor WM-3835 (Selleck), mice were intranasally infused with 5 mg / kg WM-3835 daily starting from day 7 after BLM administration, while the control group received an equal volume of DMSO.

[0036] Using Kat7 flox / flox Mice were crossed with Lyz2-Cre mice to create conditionally macrophage-specific KAT7-deficient mice (Kat7...). ΔLyz2 ).

[0037] 4. Isolation of primary fibroblasts

[0038] After euthanasia of mice, lung tissue was aseptically removed and rinsed several times with PBS. Lung lobes were cut into approximately 1 mm³ tissue blocks and evenly spread in 10 cm culture dishes. The dishes were incubated at 37°C with 5% CO2 for 2 hours until the tissue blocks adhered to the dish. DMEM medium containing 15% FBS was then slowly added. After 4-5 days of culture, once fibroblasts had emerged from and merged around the tissue blocks, the dishes were passaged or used for downstream experiments.

[0039] 5. Isolation of primary alveolar epithelial cells

[0040] After deep anesthesia, mice were perfused with saline via the right ventricle to purge pulmonary blood. One mL of an enzyme solution containing 1 mg / mL dispase (Sigma-Aldrich, D4693) and 50 U / mL DNase I (Roche, 10104159001) was slowly perfused into the lungs via the trachea, followed by perfusion with 1% low-melting-point agarose and rapid freezing with ice. The lung tissue was transferred to PBS containing dispase and incubated at 37°C for 45 minutes. Subsequently, the lung tissue was thoroughly minced in DMEM containing 10% FBS and DNase I and filtered sequentially through 70 μm and 40 μm cell sieves. After centrifugation, erythrocytes were removed using ACK lysis buffer (Biosharp, BL503A). Finally, highly purified alveolar epithelial cells were sorted using fluorescence-activated cell sorting (FACS).

[0041] 6. Isolation of mouse alveolar macrophages (AM)

[0042] Alveolar macrophages were isolated via bronchoalveolar lavage (BALF). The recovered BALF was centrifuged at 500 g for 10 min, and the cell pellet was resuspended in RPMI 1640 medium containing 10% FBS. The cell suspension was seeded into culture dishes and incubated for 45 min to allow macrophages to adhere. Afterward, the supernatant was carefully aspirated to remove non-adherent cells, and the adherent macrophages were collected after washing with PBS for subsequent analysis.

[0043] 7. Macrophage reinfusion experiment

[0044] Two days prior to BLM treatment, clodronate liposomes were administered intratracheally to deplete macrophages in the lungs of WT mice. On day 7 after BLM treatment, macrophages were depleted from the lungs of WT or Kat7 mice. ΔLyz2 Primary alveolar macrophages isolated from mice (1×10⁻⁶) 6 (Number of mice) were injected intratracheally into macrophage-depleted WT mice. Mice were sacrificed on day 14 after infusion (day 21 after BLM treatment) for analysis.

[0045] 8. Histology and Immunohistochemistry / Immunofluorescence Staining

[0046] Lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin-eosin (HE) staining and Masson's trichrome staining were performed to assess histopathological changes and collagen deposition. The Ashcroft scoring system was used for semi-quantitative assessment of fibrosis. For immunohistochemical staining, after dewaxing and hydration of paraffin sections, antigen retrieval and endogenous peroxidase blocking were performed. Sections were incubated overnight at 4°C with primary antibody (KAT7, α-SMA), followed by detection with HRP-labeled secondary antibody and the DAB chromogenic system. For immunofluorescence staining, sections or cell slides were fixed, permeabilized, and blocked, then incubated overnight at 4°C with primary antibody, followed by incubation at room temperature for 1 hour with the corresponding Alexa Fluor-labeled secondary antibody. After counterstaining the cell nuclei with DAPI, images were observed and acquired under a confocal microscope.

[0047] 9. Western Blot

[0048] Total protein was extracted from tissues or cells using RIPA lysis buffer (containing protease and phosphatase inhibitors). After quantification using the BCA method, proteins were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and incubated overnight at 4°C with the corresponding primary antibodies (KAT7, α-SMAβ-actin, Tubulin). After washing, the membranes were incubated with HRP-conjugated secondary antibody at room temperature for 1 hour. Finally, protein bands were detected using an ECL chemiluminescent imaging system.

[0049] 10. Real-time quantitative PCR (RT-qPCR)

[0050] Total RNA was extracted from tissues or cells using TRIzol reagent (TaKaRa). 1 μg of total RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (TaKaRa). qPCR was performed using the SYBR Green PCR Mix kit (TaKaRa) on a Bio-Rad CFX real-time PCR system. 18S rRNA was used as an internal reference gene, and a 2... -ΔΔCt The method calculates the relative expression level of genes.

[0051] 11. Detection of cytokines in bronchoalveolar lavage fluid (BALF)

[0052] Mice lungs were lavaged twice with 1 mL PBS via endotracheal intubation, and the lavage fluid was collected. After centrifugation at 4°C and 1000 rpm for 10 minutes, the supernatant was collected. The concentrations of IL-6, TNF-α, and TGF-β in the supernatant were detected using an ELISA kit (eBioscience).

[0053] 12. Flow cytometry analysis

[0054] Lung tissue was minced and digested into single-cell suspensions using collagenase IV and DNase I. The cell suspensions were filtered through a 70 μm filter and incubated at 4°C for 30 minutes in the dark with corresponding fluorescently labeled antibodies (CD45, Ly6G, Siglec-F, CD11b, F4 / 80). Flow cytometry was used for analysis, and data were analyzed using FlowJo software.

[0055] 13. RNA sequencing (RNA-seq)

[0056] Total RNA was extracted from fibrotic lung tissue. After quality control, the RNA was used by Novogene for library construction and sequencing. Differentially expressed genes were analyzed using the DESeq2 R package. Functional pathway enrichment analysis was performed on the differentially expressed genes using GSEA (Gene Set Enrichment Analysis).

[0057] 14. Statistical Analysis

[0058] All data are expressed as mean ± standard deviation (SD). Two-tailed Student's t-tests were used for comparisons between two groups. One-way ANOVA was performed followed by Tukey's or Dunnett's multiple comparison tests for comparisons among multiple groups. All statistical analyses were performed using GraphPad Prism software. P < 0.05 was considered statistically significant.

[0059] II. Results

[0060] 1. KAT7 expression is upregulated in human pulmonary fibrosis patients and mouse pulmonary fibrosis (PF) models.

[0061] Pulmonary fibrosis (PF) is a disease that severely disrupts gas exchange and immune homeostasis in the lungs. Recent epigenetic studies have shown that the lysine acetyltransferase (KAT) family, as key transcriptional activators, may play a crucial role in the pathological progression of PF. To systematically investigate the expression changes of this family members in pulmonary fibrosis, we analyzed lung tissue samples from human patients and mouse models. First, we found that in patients with idiopathic pulmonary fibrosis (IPF), systemic sclerosis-associated interstitial lung disease (SSc-ILD), and BLM-induced mouse pulmonary fibrosis models, only KAT7 expression levels were significantly higher than in healthy controls. Figure 1 A). Furthermore, we collected normal lung tissue from lung cancer patients and lung tissue from IPF patients. Immunohistochemical (IHC) analysis showed that KAT7 was generally upregulated in the lung tissue of fibrotic patients. Figure 1B). Simultaneously, clinical correlation analysis revealed that in IPF patients, the expression level of KAT7 was significantly positively correlated with the expression of key fibrosis genes (Col1a1, Col3a1, Acta2), and significantly negatively correlated with carbon monoxide diffusing capacity (DLco), a pulmonary function indicator reflecting gas exchange efficiency. Figure 1 (C-1D). This indicates that high KAT7 expression is closely related to the degree of fibrosis and the deterioration of lung function.

[0062] To validate these findings in animal models, we constructed two mouse models of pulmonary fibrosis induced by bleomycin (BLM) or silica, respectively, with different etiologies. The results were highly consistent with observations in human samples: in both models, the levels of Kat7 mRNA and protein in mouse lung tissue were significantly elevated. Figure 1 EI). Immunofluorescence (IF) analysis also confirmed that KAT7 protein was significantly increased in the lung tissue of fibrotic mice. Figure 1 J). Furthermore, to investigate whether this upregulation of expression differs by sex, we supplemented the study with a female mouse model. The results showed that Kat7 mRNA expression also increased with the progression of fibrosis, suggesting that KAT7 upregulation may not be sex-specific. Figure 1 K).

[0063] In summary, evidence from human patients and two mouse models collectively suggests that upregulation of KAT7 expression is a stable feature in the pathological process of pulmonary fibrosis and is closely related to disease severity, suggesting that it may play a key role in the initiation and progression of the disease.

[0064] 2. KAT7 is mainly distributed in macrophages in fibrotic lung tissue.

[0065] To clarify the cellular origin of upregulated KAT7 in fibrotic lung tissue, we first investigated the distribution of KAT7 in human lung tissue. The results showed that KAT7 was highly expressed in macrophages and epithelial cells, but expressed very little in fibroblasts and endothelial cells. Figure 2 A-2B). This distribution pattern was also similarly validated in mouse fibrotic lung tissue: immunofluorescence colocalization analysis showed that KAT7 was mainly colocalized with macrophage marker F4 / 80 and epithelial cell marker E-cadherin. Figure 2 C). Furthermore, analysis of primary mouse lung cells also confirmed that Kat7 mRNA expression levels were highest in macrophages and epithelial cells. Figure 2D). Although the above results consistently indicate that KAT7 is primarily localized in macrophages and epithelial cells, it is crucial to identify which cell type "upregulates" KAT7 expression during fibrosis. To this end, we specifically compared macrophages and epithelial cells from control and bleomycin (BLM)-induced fibrosis model mice. Notably, BLM treatment did not significantly induce Kat7 expression in epithelial cells compared to the control group. Figure 2 E). We also found that the expression level of KAT7 was significantly positively correlated with the macrophage marker MRC1 (E). Figure 2 Consistent with this finding, we observed significantly increased KAT7 expression levels in both BLM and silica (SiO2)-treated mouse primary alveolar macrophages. Figure 2 G). In summary, macrophages are the key cellular source of abnormally upregulated KAT7 expression in the process of pulmonary fibrosis, suggesting that abnormal KAT7 function in macrophages may promote the occurrence and progression of the disease.

[0066] 3. Myeloid-specific loss of KAT7 alleviates inflammation and fibrosis.

[0067] Given the high expression of KAT7 in macrophages, we hypothesized that it might play a crucial role in lung inflammation and fibrosis. Therefore, we utilized KAT7... flox / flox Mice were crossed with Lyz2-Cre mice to create conditionally macrophage-specific KAT7-deficient mice (Kat7...). ΔLyz2 Subsequently, wild-type (WT) and Kat7 were tested. ΔLyz2 Mice were administered BLM intratracheally, and lung tissue was collected for analysis on day 7 (inflammatory phase) and day 21 (fibrotic phase) after treatment. Results showed that, compared with WT mice, Kat7 mice... ΔLyz2 Mice treated with high doses of BLM (5 mg / kg) showed a significant reduction in mortality, and those treated with low doses (2.5 mg / kg) experienced less weight loss. Figure 3 (A-3B). This overall protective effect is closely associated with a significant reduction in early inflammatory responses. Under saline treatment conditions, WT and Kat7... ΔLyz2 F4 / 80 in mouse lungs + There was no significant difference in macrophage numbers. However, 7 days after BLM-induced injury, Kat7... ΔLyz2 F4 / 80 of mice + The number of macrophages was significantly reduced. Figure 3 C-3D). Meanwhile, the expression levels of the inflammatory factor TGF-β in lung tissue and bronchoalveolar lavage fluid (BALF) were significantly decreased. Figure 3E-3F). Regarding fibrosis, Kat7 ΔLyz2 Mice showed reduced collagen deposition and significantly decreased expression of α-SMA, a marker of fibroblast activation. Figure 3 G-3I). This is consistent with the decrease in Ashcroft score and hydroxyproline content in lung tissue (G-3I). Figure 3 J-3K). Furthermore, Micro-CT three-dimensional imaging visually confirmed the reduction in pulmonary fibrosis. Figure 3 L).

[0068] 4. KAT7 deficiency in myeloid cells alleviates pulmonary fibrosis by inhibiting the pro-fibrotic function of macrophages.

[0069] To further elucidate the molecular mechanism behind the protective effect of KAT7 deficiency, we investigated the effects of BLM treatment on wild-type (WT) and Kat7... ΔLyz2 Transcriptome sequencing (RNA-seq) was performed on mouse lung tissue. Figure 4 A). Principal component analysis (PCA) showed a clear separation between the two groups of samples in terms of transcriptional profiles. Figure 4 B), the differentially expressed gene heatmap visually demonstrates the global gene expression changes caused by Kat7 knockout. Figure 4 C). We found that the absence of Kat7 remodeled the transcriptional profile of the lung immune microenvironment: in Kat7 ΔLyz2 In mouse lung tissue, the expression of genes related to pro-fibrotic macrophage function was significantly suppressed, while genes related to type I immune response were activated. Figure 4 D). Gene set enrichment analysis (GSEA) further confirmed that key pro-fibrotic signaling pathways, such as the TGF-β signaling pathway, are present in Kat7. ΔLyz2 Significant inhibition was observed in mice ( Figure 4 E).

[0070] To verify whether this alteration in transcriptional profile could functionally inhibit fibroblast activation, we conducted an in vitro co-culture experiment. We co-cultured primary lung fibroblasts from WT mice with BLM-treated WT or Kat7 mice. ΔLyz2 Co-culture with bronchoalveolar lavage fluid (BALF) from mice. Results showed that co-culturing with BALF from Kat7 mice... ΔLyz2 Co-culture with BALF in mice significantly inhibited the expression of α-SMA in fibroblasts. Figure 4 FG).

[0071] In summary, these results indicate that KAT7 in myeloid cells is a key molecule driving lung inflammation and fibrosis. Specific knockout of Kat7 in macrophages can effectively block early inflammation and curb subsequent fibrotic lesions by inhibiting its pro-fibrotic transcriptional program and weakening its ability to activate fibroblasts.

[0072] 5. Reinfusion of KAT7-deficient macrophages has a protective effect against fibrosis.

[0073] To further evaluate the role of endogenous KAT7 in macrophages in fibrosis, we conducted a macrophage reinfusion experiment. Two days after intratracheal infusion of clodronate liposomes to deplete macrophages in the lungs of wild-type (WT) mice, mice were administered BLM to induce pulmonary fibrosis. On day 7 of fibrosis progression, we reinfused macrophages from WT mice with either clodronate liposomes or KAT7. ΔLyz2 Primary alveolar macrophages isolated from mice (1×10⁻⁶) 6 (1) Kat7 cells were reinfused into WT mice with depleted macrophages. Results showed that, compared to mice infused with WT macrophages, mice infused with Kat7 cells... ΔLyz2 Macrophages significantly reduced the degree of fibrosis in mice, and this protective effect was confirmed by several key fibrosis indicators, including reduced collagen deposition, decreased α-SMA expression, reduced Ashcroft score, and significantly decreased hydroxyproline levels in lung tissue. Figure 5 A-5D).

[0074] In summary, the combination of in vivo cell reinfusion experiments and in vitro functional validation has demonstrated that endogenous KAT7 in macrophages is a key driver of fibrosis. Its deficiency can remodel macrophage function, weaken its ability to activate fibroblasts, and thus exert an anti-fibrotic effect.

[0075] 6. Therapeutic potential of pharmacological inhibition of KAT7

[0076] To translate the potential of KAT7 as a therapeutic target into practical applications, we further evaluated the therapeutic efficacy of its specific small molecule inhibitor, WM-3835, in a BLM-induced mouse model of pulmonary fibrosis. We employed a dosing regimen that mimicked clinical treatment: mice were given once-daily nasal administration of WM-3835 for 14 days, starting 7 days after BLM-induced pulmonary fibrosis, a critical window for the transition from inflammation to fibrosis. Figure 6 A). The results showed that WM-3835 treatment did not cause significant changes in body weight or lung weight, suggesting that it has good safety. Figure 6B-6C). At the molecular level, it not only reduces the expression of pro-inflammatory factors such as IL-1β and TGF-β in lung tissue, but also effectively reduces the levels of IL-6, TNF-α, and TGF-β in serum. Consistent with this anti-inflammatory effect, WM-3835 also inhibits the expression of key fibrosis-related genes such as α-SMA and Col1a1 in lung tissue. Figure 6 D-6F). This suggests that the anti-fibrotic effect of WM-3835 is closely related to KAT7 targeted inhibition. Further histological analysis showed that α-SMA expression was reduced in the lung tissue of the WM-3835 treatment group, and the degree of fibrosis was significantly lower than that of the control group. Figure 6 G).

[0077] In summary, these results demonstrate that KAT7 is not only a key pathogenic driver of pulmonary fibrosis but also a potential therapeutic target that can be effectively targeted by small molecule drugs. WM-3835 exhibits significant potential in inhibiting fibrosis progression in animal models, providing strong experimental evidence for the development of anti-fibrotic drugs targeting KAT7.

Claims

1. Application of reagents for detecting the expression of the biomarker KAT7 in the preparation of products for the auxiliary diagnosis, diagnosis or prognosis of pulmonary fibrosis.

2. The application according to claim 1, characterized in that, The diagnostic method includes: obtaining a test sample from the patient, determining the expression level of KAT7 in the test sample, and if the expression level is higher than that of normal controls, it suggests that the patient has pulmonary fibrosis. The prognostic method includes obtaining test samples from patients with pulmonary fibrosis, determining the expression level of KAT7 in the test samples, and high KAT7 expression indicating a poor prognosis.

3. The application according to claim 2, characterized in that: The test sample was lung tissue.

4. Application of KAT7 as a target in screening drugs for the treatment of pulmonary fibrosis.

5. The application according to claim 4, characterized in that: The drug inhibits the expression or activity of KAT7, and the decrease in KAT7 expression or activity levels inhibits or alleviates the development of pulmonary fibrosis.

6. Application of reagents that inhibit KAT7 expression or activity in the preparation of drugs for treating pulmonary fibrosis.

7. The application according to claim 6, characterized in that: The reagents that inhibit KAT7 expression or activity include nucleic acid molecules, KAT7 protein-specific binders, or compounds.

8. The application according to claim 7, characterized in that: The compound is a small molecule or low molecular weight compound, preferably WM-3835; The KAT7 protein-specific binder is a selective inhibitor, antibody, or antagonist targeting the KAT7 protein; The nucleic acid molecules target KAT7, interfering with or inhibiting KAT7 expression.

9. The application according to claim 4 or 6, characterized in that: The drug inhibits the expression or activity of KAT7 in lung tissue macrophages, suppresses its pro-fibrotic function, reduces the release of pro-inflammatory cytokines and extracellular matrix deposition, reduces collagen deposition, lowers the inflammatory response, and alleviates or inhibits the development of pulmonary fibrosis.

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