Application of KDM6A in lung macrophages in treatment, prevention or diagnosis of pulmonary fibrosis

Abstract

The invention discloses application of a substance capable of up-regulating the KDM6A protein level in lung macrophages in preparation of a medicine for treating or preventing pulmonary fibrosis. The invention also discloses application of the KDM6A protein in the lung macrophage as a marker in preparation of a pulmonary fibrosis diagnosis or prediction kit. KDM6A is found to be an important signal molecule of a macrophage-epithelial cell metabolic pathway, and the expression of KDM6A in lung macrophages is recovered, and THBS1 secretion is inhibited, so that glycerophospholipid metabolism of ATII cells is repaired, and pulmonary fibrosis is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a substance that can upregulate the level of KDM6A protein in lung macrophages in the preparation of a drug for treating or preventing pulmonary fibrosis and a kit for diagnosing pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a terminal manifestation of interstitial lung disease. This disease is characterized by fibroblast proliferation and excessive deposition of extracellular matrix, leading to the destruction of lung tissue structure. Its incidence is slightly higher in men than in women. Idiopathic pulmonary fibrosis (IPF) is the most representative type of chronic progressive pulmonary fibrosis. It is characterized by abnormal activation of fibroblasts, excessive deposition of extracellular matrix (ECM) and dysfunction of alveolar epithelial cells. There is currently a lack of effective treatment. Existing drugs (such as pirfenidone and nintedanib) can only delay disease progression but cannot reverse fibrosis. Studies have shown that the incidence of IPF in men is significantly higher than in women, but its molecular mechanism is still unclear. As the most representative type of chronic progressive pulmonary fibrosis, IPF has important implications for the treatment of all types of pulmonary fibrosis by clarifying its pathogenic mechanism.

[0003] A key yet understudied aspect of IPF pathogenesis involves disruption of pulmonary surfactant homeostasis. Macrophages and alveolar type II epithelial (ATII) cells maintain lipid metabolic homeostasis in the lungs through a dynamic interplay. On the one hand, macrophages coordinate the metabolic balance of pulmonary surfactant (synthesized by ATII cells) and clear oxidized lipid products; on the other hand, ATII cells feedback regulate the polarization state and lipolysis capacity of macrophages by releasing lipid mediators. Disruption of this metabolic symbiosis triggers lipid accumulation, surfactant deficiency, and a profibrotic signaling cascade. Therefore, macrophages are key regulators of fibrosis progression.

[0004] The histone demethylase KDM6A is an X-chromosome-encoded epigenetic regulator with sex-specific expression patterns in macrophages. Existing studies have shown that KDM6A is a key regulator of immune cell function, playing a role in resolving inflammation and maintaining genomic stability. KDM6A expression is affected by oxidative stress, but its relationship with macrophage dysfunction in pulmonary fibrosis has not been studied. Summary of the Invention

[0005] The present inventors discovered in their research that KDM6A plays a key role in the pathogenesis of pulmonary fibrosis. KDM6A deficiency leads to abnormal lipid metabolism in macrophages and, through the secretion of THBS1, disrupts glycerophospholipid metabolism in alveolar type II cells (ATII), thereby promoting pulmonary fibrosis. Conversely, increasing KDM6A levels can reverse the progression of pulmonary fibrosis. Therefore, the present invention first provides the use of a substance that can upregulate KDM6A protein levels in pulmonary macrophages in the preparation of a drug for treating or preventing pulmonary fibrosis.

[0006] Preferably, the substance has the ability to target macrophages; more preferably, it targets lung CD206+ macrophages.

[0007] Preferably, the substance capable of upregulating the KDM6A protein level in lung macrophages upregulates the KDM6A protein level by upregulating the expression of the KDM6A encoding gene.

[0008] Preferably, the substance carries Kdm6a mRNA.

[0009] Preferably, the medicament is used for the treatment or prevention of male or male pulmonary fibrosis.

[0010] Preferably, the drug is delivered to the lung tissue by intratracheal injection or aerosol inhalation.

[0011] Furthermore, given that KDM6A deficiency causes macrophages to upregulate lipid biosynthesis while also resulting in a comprehensive inhibition of glycerophospholipid metabolism in ATII cells, the present invention also provides the use of a substance that can upregulate KDM6A protein levels in lung macrophages in the preparation of a drug for restoring glycerophospholipid metabolism in alveolar type II cells.

[0012] The present invention also provides a drug for treating or preventing pulmonary fibrosis, which comprises a substance that can upregulate the level of KDM6A protein in pulmonary macrophages.

[0013] Preferably, the drug targets lung macrophages; more preferably, it targets lung CD206+ macrophages.

[0014] Preferably, the drug comprises a targeting carrier, and the targeting carrier is preferably a liposome.

[0015] Preferably, the medicament is used for the treatment or prevention of male or male pulmonary fibrosis.

[0016] Preferably, the drug carries Kdm6a mRNA.

[0017] Preferably, the drug is administered by intratracheal injection or aerosol inhalation.

[0018] Furthermore, the substance provided by the present invention that can upregulate the level of KDM6A protein in lung macrophages can also be used in the preparation of drugs for other fibrotic diseases related to abnormal macrophage lipid metabolism.

[0019] Furthermore, the present invention also provides the use of KDM6A protein in lung macrophages as a marker in the preparation of a diagnostic or predictive kit for pulmonary fibrosis; the diagnostic or predictive sample is preferably derived from a biopsy sample of lung tissue, and the diagnosis or prediction is preferably based on whether the KDM6A protein level is below a threshold, with different thresholds set for men (males) and women (females).

[0020] Based on the above application, the present invention also provides a diagnostic or auxiliary diagnostic kit for pulmonary fibrosis, which includes a reagent for detecting the level of KDM6A in pulmonary macrophages.

[0021] On the other hand, the present invention provides a liposome targeting lung macrophages, which is a double-layer self-assembled structure with a particle size of more than 1 μm, and contains SPC (soy lecithin), DOTAP ((2,3-dioleoyl-propyl)-trimethylammonium-chloride), DSPE-PEG2000-mannose, and cholesterol.

[0022] Preferably, the particle size of the liposome is 1 to 2 μm, more preferably 1.2 to 1.8 μm.

[0023] Preferably, the liposomes are loaded with Kdm6a mRNA or Kdm6a siRNA.

[0024] Furthermore, the preparation method of the liposome targeting lung macrophages is as follows: SPC, DOTAP, DSPE-PEG2000-mannose, and cholesterol are mixed in a glycerol-water solution, incubated at 45-55° C. for a period of time to allow self-assembly, and then shaken after cooling to form microbubbles, the bubbles are removed, washed, and centrifuged to obtain a precipitate.

[0025] Furthermore, the step of loading Kdm6a mRNA or Kdm6a siRNA is as follows: after redispersing the aforementioned precipitate, Kdm6a mRNA or Kdm6a siRNA is added and incubated to obtain liposomes loaded with Kdm6a mRNA or Kdm6a siRNA.

[0026] Preferably, the mixing mass ratio of SPC, DOTAP, DSPE-PEG2000-mannose and cholesterol is 3.5-4.5:2.6-3.4:0.8-1.2:1.8-2.2, more preferably 4:3:1:2.

[0027] Preferably, the volume ratio of glycerol to aqueous solution is 1:15-25.

[0028] Preferably, the incubation time is more than 20 minutes.

[0029] Preferably, the oscillation frequency is 50-75 Hz, and can be performed using a silver-mercury blender.

[0030] Preferably, the method for removing bubbles is centrifugation, and the centrifugal speed is preferably 600-1000 rpm, more preferably 700-900 rpm.

[0031] Preferably, the centrifugal speed used in the step of obtaining the precipitate by centrifugation is greater than 2000 rpm, more preferably greater than 4000 rpm.

[0032] Preferably, the targeting efficiency of the liposomes to lung macrophages is higher than 70%.

[0033] The present invention also provides the use of the above-mentioned liposomes targeting lung macrophages in the preparation of a drug for treating or preventing pulmonary fibrosis, wherein the drug is loaded with Kdm6a mRNA.

[0034] The present invention also provides the use of the above-mentioned lung macrophage-targeted liposomes in the preparation of drugs for restoring alveolar type II cell glycerophospholipid metabolism and other drugs for fibrotic diseases associated with abnormal macrophage lipid metabolism, wherein the loaded drug is Kdm6a mRNA.

[0035] Preferably, the lung macrophage-targeted liposomes target lung CD206+ macrophages.

[0036] Finally, the present invention also provides a drug for treating or preventing pulmonary fibrosis, wherein the drug comprises the above-mentioned liposomes targeting lung macrophages, wherein the drug is loaded with Kdm6a mRNA.

[0037] Preferably, the drug is administered by intratracheal injection or aerosol inhalation.

[0038] Preferably, the drug further comprises other pharmaceutically acceptable carriers.

[0039] The present invention discovered that KDM6A is a gatekeeper for macrophage-epithelial metabolic crosstalk. Macrophage-specific Kdm6a knockout mice reproduce human pulmonary fibrosis pathology, with accelerated fibrosis and an expansion of the pro-fibrotic AM / IM population. Single-cell transcriptomics revealed that KDM6A deficiency reprograms macrophages, a key pathway for addressing fibrosis. Based on this research, the present invention uses KDM6A protein as a diagnostic or prognostic marker for pulmonary fibrosis, and upregulating KDM6A protein levels in pulmonary macrophages as a therapeutic approach to combat pulmonary fibrosis. This therapeutic objective is achieved by preparing a drug for treating or preventing pulmonary fibrosis using nucleic acids encoding KDM6A protein targeted to pulmonary macrophages.

[0040] Furthermore, the present invention provides micron-sized liposomes that can effectively target lung macrophages, achieving a delivery efficiency 5.5 times higher than traditional nanoliposomes. These liposomes utilize mannose as a ligand for CD206, enhancing their ability to specifically target macrophages. In a bleomycin-induced mouse model of pulmonary fibrosis, Kdm6a mRNA liposomes significantly reduced the area of fibrosis and restored glycerophospholipid metabolism. In a human lung organoid model, their efficacy was superior to that of pirfenidone, ameliorating alveolar structural damage. Furthermore, these liposome components exhibit high biocompatibility and are non-immunogenic.

[0041] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the result of scRNA-seq analysis of KDM6A mRNA expression levels in alveolar and interstitial macrophages;

[0043] Figure 2 is the correlation analysis between KDM6A mRNA levels in alveolar and interstitial macrophages and lipid metabolism scores;

[0044] Figure 3 is a quantitative comparison of the KDM6A fluorescence staining intensity of macrophages in human lung sections (n=5);

[0045] Figure 4 are representative micro-CT images of mouse lungs after bleomycin (BLM)-induced pulmonary fibrosis;

[0046] Figure 5 Masson trichrome-stained lung sections after BLM induction; the lower panel (scale bar: 50 μm) shows an enlarged view of the fibrotic area in the upper panel (scale bar: 500 μm);

[0047] Figure 6 Semi-quantitative Ashcroft score (left) and collagen area quantification (right) in the lungs of mice after BLM treatment (n = 4 per group);

[0048] Figure 7 Representative immunofluorescence co-staining images of Kdm6a (red) and F4 / 80 (green) in lung sections of mice after BLM treatment, scale bar: 10 μm;

[0049] Figure 8 Yes Figure 7 Quantitative comparison of KDM6A fluorescence intensity in macrophages after immunofluorescence staining (n=4);

[0050] Figure 9Flow cytometry analysis of mouse KDM6A after BLM treatment + Statistical quantification of the percentages of alveolar macrophages (AM, left) and interstitial macrophages (IM, right);

[0051] Figure 10 KDM6A in mice after BLM treatment + Statistical quantification of the median fluorescence intensity of AM (left) and IM (right);

[0052] Figure 11 Macrophage-specific Kdm6a knockout Schematic diagram of the process of establishing the pulmonary fibrosis model in mice and induced by intratracheal BLM;

[0053] Figure 12 After BLM processing Representative immunofluorescence co-staining images of KDM6A (red) and F4 / 80 (green) in mouse lung sections, scale bar: 5 μM;

[0054] Figure 13 Yes Figure 12 Quantitative comparison of KDM6A fluorescence intensity in macrophages after immunofluorescence staining;

[0055] Figure 14 It was knocked out by Lyz2-Cre after BLM treatment Semi-quantitative Ashcroft score (left) and collagen area quantification (right) of the severity of lung fibrosis in mice (n=4);

[0056] Figure 15 Schematic diagram of the construction process: Kdm6a is injected into the trachea F / Y and Kdm6a F / F Mice were administered two recombinant adeno-associated viral vectors and then subjected to BLM-induced fibrosis;

[0057] Figure 16 AAV vector knockout after BLM treatment Semi-quantitative Ashcroft score (left) and collagen area quantification (right) of the severity of lung fibrosis in mice (n=4);

[0058] Figure 17 Macrophage-specific Kdm6a knock-in Schematic diagram of the process of establishing mice and BLM-induced fibrosis model;

[0059] Figure 18 It is a male after BLM treatment Representative microCT images of mouse lungs;

[0060] Figure 19It is a male after BLM treatment Semi-quantitative Ashcroft score (left) and collagen area quantification (right) of the severity of lung fibrosis in mice (n=4);

[0061] Figure 20 It is a male after BLM treatment Representative immunofluorescence co-staining images of KDM6A (red) and F4 / 80 (green) in mouse lung sections, scale bar: 10 μM;

[0062] Figure 21 Yes Figure 20 Quantitative comparison of KDM6A fluorescence intensity in macrophages after immunofluorescence staining (n=4);

[0063] Figure 22 Is from The comprehensive proteomic analysis and identification process and results of mouse bone marrow-derived macrophage (BMDM) conditioned medium (CM) and single-cell transcriptomics revealed four significantly upregulated proteins (THBS1, B2M, PSAP, and CTSD), among which THBS1 was the most significantly increased.

[0064] Figure 23 Kdm6a was characterized by proteomics (left) and scRNA-seq (right). F / Y and Results of comparative analysis of THBS1 expression levels in mice (n=4);

[0065] Figure 24 It's Kdm6a F / Y and qPCR quantification results of THBS1 mRNA in mouse BMDM (n=4);

[0066] Figure 25 It's Kdm6a F / Y and Quantification of THBS1 fluorescence intensity in mouse lung macrophages (n=4);

[0067] Figure 26 Schematic diagram of the workflow for targeted lipid metabolomics analysis in ATII cells treated with conditioned medium from Kdm6a knockout BMDMs;

[0068] Figure 27 are stacked bar graphs showing the relative abundance changes of 20 lipid species in ATII cells exposed to BMDM conditioned medium from WT, Kdm6a-KO, Kdm6a-KO+THBS1 neutralizing antibody, or Kdm6a-KO+IgG control;

[0069] Figure 28 is the size distribution of liposomes (100 nm and 1 μm);

[0070] Figure 29 It is ex vivo fluorescence imaging of major organs after trans-airway administration of liposomes;

[0071] Figure 30 The results of flow cytometric analysis comparing the targeting efficiency of 100 nm and 1 μm liposomes to macrophages;

[0072] Figure 31 These are the quantitative results of 1 μm liposomes targeting AM and IM;

[0073] Figure 32 Representative immunofluorescence co-staining images of DIO-labeled liposomes (green), CD206+ (yellow), and F4 / 80+ (purple) macrophages in lung sections of mice after BLM treatment. Scale bars: 10 μm (left); 5 μm (right).

[0074] Figure 33 This is a schematic diagram of the construction of Kdm6a siRNA-loaded liposomes;

[0075] Figure 34 are transmission electron microscopy (TEM) images of blank liposomes, scramble siRNA liposomes, and Kdm6a siRNA liposomes;

[0076] Figure 35 Quantitative analysis of KDM6A immunofluorescence intensity in the lungs of male and female mice after airway delivery of Kdm6a siRNA liposomes, n = 4;

[0077] Figure 36 Semi-quantitative Ashcroft score (left) and quantification of collagen-positive area (right) of lung fibrosis severity in male mice treated with scramble or Kdm6a siRNA-loaded liposomes after BLM induction, n = 4;

[0078] Figure 37 Semi-quantitative Ashcroft score (left) and quantification of collagen-positive area (right) of lung tissue fibrosis severity in female mice treated with scramble or Kdm6a siRNA-loaded liposomes after BLM induction, n = 4;

[0079] Figure 38 Quantification of Thbs1 fluorescence intensity in macrophages from mouse lung sections treated with scramble or Kdm6a siRNA-loaded liposomes after BLM induction, n = 4;

[0080] Figure 39This is a schematic diagram of the work of constructing Kdm6a mRNA encapsulated liposomes;

[0081] Figure 40 is a TEM image of Kdm6a mRNA liposomes;

[0082] Figure 41 are representative microCT images of the lungs of BLM-induced mice after intratracheal injection of liposomes (Kdm6a mRNA);

[0083] Figure 42 Representative Masson trichrome staining of BLM-induced fibrotic lungs in mice after intratracheal injection of liposomes (Kdm6a mRNA) (the lower panel shows an enlarged view of the fibrotic area in the upper panel). Scale bars: 500 μm (upper panel), 50 μm (lower panel);

[0084] Figure 43 Yes Figure 42 Semi-quantitative Ashcroft score (left) and quantitative collagen-positive area (right) of stained mouse lung sections, n = 4;

[0085] Figure 44 The quantitative results of KDM6A fluorescence intensity in macrophages of lung sections of BLM-induced mice treated with liposomes (Kdm6a mRNA), n = 4;

[0086] Figure 45 The quantitative results of Thbs1 fluorescence intensity in macrophages of lung sections of BLM-induced mice treated with liposomes (Kdm6a mRNA), n = 4;

[0087] Figure 46 The quantitative results of FASN and LPCAT1 protein fluorescence intensity in ATII cells of lung sections of BLM-induced mice treated with liposomes (Kdm6a mRNA), n = 4;

[0088] Figure 47 Quantification of collagen-positive areas after Masson staining in BLM-induced organoids treated with Kdm6a mRNA-loaded liposomes or pirfenidone, n = 4;

[0089] Figure 48 is the quantitative result of KDM6A fluorescence intensity in BLM-induced organoid CD68+ macrophages treated with liposomes loaded with Kdm6a mRNA, n = 4;

[0090] Figure 49 is the quantitative result of THBS1 fluorescence intensity in BLM-induced organoid CD68+ macrophages treated with liposomes loaded with Kdm6a mRNA, n = 4;

[0091] Figure 50 Quantification of FASN (right) and LPCAT1 (left) fluorescence intensities in BLM-induced organoid ATII cells treated with liposomes loaded with Kdm6a mRNA, n = 4;

[0092] The statistical data in the above figures are expressed as mean ± SEM, *: p < 0.05, **: p < 0.01, ***: p < 0.001. DETAILED DESCRIPTION

[0093] Example 1 Male-associated downregulation of KDM6A in lung macrophages drives the pathogenesis of IPF

[0094] The researchers first analyzed a published single-cell RNA sequencing (scRNA-seq) dataset (GSE136831) comprising 28 healthy lungs and 32 lungs with IPF. Based on their transcriptome, the single cells were grouped into 38 subpopulations. Two major macrophage subpopulations, alveolar macrophages (AM, 42.2%) and interstitial macrophages (IM, 21.4%), collectively accounted for over 60% of all cells examined in the combined dataset.

[0095] We calculated a lipid metabolism score based on the expression of genes involved in lipid biosynthesis, degradation, and trafficking, and our results showed that AM and IM were the top two cell clusters with increased metabolic activity in IPF. In contrast, alveolar type II epithelial (ATII) cells showed a global suppression of lipid metabolism pathways in IPF.

[0096] Comparative analysis revealed sex-specific lipid metabolism reprogramming in IPF. Compared with controls, macrophages from IPF patients exhibited enhanced lipid metabolism, with increases of 1.13-fold in AM compared to 1.19-fold in men and 1.31-fold in IM compared to 1.52-fold in women. These findings suggest that sex-specific dysregulation of lipid metabolism in macrophages is a potential mechanism contributing to the progression of fibrosis.

[0097] Existing studies have shown that gene dosage effects mediated by X chromosome inactivation escape may be the basis for the male dominance in the pathogenesis of pulmonary fibrosis. Five genes (XIST, DDX3X, KDM6A, EIF2S3, KDM5C) have been identified as X chromosome inactivation escape genes. Compared with female macrophages, all five genes are significantly downregulated in male macrophages, among which KDM6A shows the most obvious inhibitory effect in both male and female IPF ( Figure 1 ), whose expression levels were significantly negatively correlated with lipid metabolism scores in both IPF patients and controls ( Figure 2Immunofluorescence quantification confirmed a progressive reduction in KDM6A protein: baseline expression was lower in male macrophages than in female macrophages, with further depletion observed in IPF lungs ( Figure 3 Temporal analysis during macrophage differentiation revealed that KDM6A is progressively downregulated as lung monocytes mature through the interstitial and alveolar macrophage stages. Collectively, these findings position KDM6A deficiency as a potential driver of pro-fibrotic macrophage reprogramming.

[0098] Example 2 Macrophage-specific KDM6A regulation directly controls fibrosis outcomes

[0099] To examine the clinical relevance of KDM6A dysregulation, we established a bleomycin (BLM)-induced pulmonary fibrosis model in C57BL / 6 mice. Briefly, 8-10-week-old mice were anesthetized intraperitoneally and intratracheally administered 1 mg / kg of BLM (HY-17565A, MedChemExpress, Shanghai, China) dissolved in 30 μL of PBS using a high-pressure atomizer (BioJane Trading Co., Ltd., Shanghai, China). Mice receiving the same volume of sterile saline served as controls.

[0100] Results: Micro-CT imaging showed that the fibrotic lesions in male mice were significantly larger than those in female mice ( Figure 4 ). Histopathological analysis was performed by Masson trichrome staining, and the staining results were as follows. Figure 5 The statistical results are shown in Figure 6 As shown in Figure 2, males showed increased fibrosis severity and collagen deposition compared to females. Immunofluorescence staining of mouse lungs was performed, where we used F4 / 80 (a macrophage marker) to label macrophages. The staining images are shown in Figure 2. Figure 7 The results also showed that there was gender-related KDM6A inhibition in lung macrophages, and the statistical results were as follows Figure 8 As shown, baseline KDM6A protein levels in male macrophages were lower than those in female macrophages, and fibrosis induction further reduced KDM6A levels in both sexes.

[0101] Flow cytometry further confirmed that KDM6A + The proportion of AM and IM was lower, and fibrosis was induced by depletion of KDM6A + The percentage of macrophages in all living cells ( Figure 9 Furthermore, fluorescence quantitative analysis showed that KDM6A protein levels in male AM and IM were lower than those in females ( Figure 10These experimental results mechanistically reproduce the differences in human pulmonary fibrosis, indicating that both men and women can drive fibrosis progression through macrophage-specific downregulation of KDM6A, but the baseline KDM6A in male macrophages is lower than that in females, suggesting that men are more likely to control fibrosis progression through upregulation of KDM6A.

[0102] Lyz2-Cre is a well-established Cre recombinase tool specifically used for gene knockout in myeloid cells. F / F Mice were crossed with Lyz2-Cre mice to construct macrophage-specific Kdm6a knockout mice ( Figure 11 ). Figure 12 The immunofluorescence co-staining images in the figure confirmed the effective elimination of KDM6A in lung macrophages, where Kdm6a F / F Representative female Cre-floxed mice, Kdm6a F / F ; Lyz2-Cre represents female Cre+floxed mice, Kdm6a F / Y Representative male Cre-floxed mice, Kdm6a F / Y ; Lyz2-Cre represents male Cre+Floxed mice. The statistical results are as follows Figure 13 As shown. It is noteworthy that Mice treated with BLM showed accelerated fibrosis progression, with micro-CT demonstrating larger lesion areas compared to wild-type (WT) controls. Histopathological analysis showed that Collagen deposition and fibrotic remodeling were exacerbated in mice ( Figure 14 ), which is similar to the pathology of human pulmonary fibrosis.

[0103] Next, to minimize the potential confounding effects of other myeloid cell populations, we again used F4 / 80 to construct two recombinant adeno-associated virus (AAV) vectors: HBAAV2 / 6-F4 / 80-cre-ZsGreen (F4 / 80Cre+) and HBAAV2 / 6-F4 / 80-ZsGreen (F4 / 80Cre-), which were delivered to Kdm6a cells via airway injection. F / Y and Kdm6a F / F In the lung tissue of mice ( Figure 15). Compared with the F4 / 80Cre- control group, mice receiving F4 / 80Cre+ showed specific KDM6A elimination in lung macrophages. The F4 / 80Cre+ group showed accelerated fibrosis progression after BLM treatment, and micro-CT imaging showed that the lesion area was enlarged relative to the F4 / 80Cre- control group. Histopathological analysis further confirmed that collagen deposition and fibrotic tissue remodeling were significantly enhanced in the F4 / 80Cre+ group animals after BLM treatment compared with the Cre- group ( Figure 16 ), indicating that specific KDM6A ablation in lung macrophages promotes fibrosis.

[0104] Next, to establish the reverse relationship, we pass Kdm6a KI / KI Mice were crossed with Lyz2-Cre mice to construct macrophage-specific Kdm6a knock-in mice. ( Figure 17 ). After BLM treatment, Kdm6a WT Animal (Kdm6a KI / Y )compared to, Animal (Kdm6a KI / Y ; Lyz2-Cre) showed a reduction in the area of fibrotic lesions by micro-CT ( Figure 18 ), collagen deposition decreased ( Figure 19 ). Immunofluorescence confirmed the successful overexpression of KDM6A in macrophages ( Figure 20 、 Figure 21 These gain- and loss-of-function studies indicate that KDM6A is a major epigenetic regulator of macrophage-driven fibrosis. Furthermore, macrophage-specific Kdm6a knockdown has a protective and mitigating effect on subsequent BLM-induced pulmonary fibrosis.

[0105] Example 3 Study on the mechanism of KDM6A-deficient macrophages driving pulmonary fibrosis

[0106] We are (Kdm6a F / Y ; Lyz2-Cre) and control (Kdm6a F / Y Single-cell RNA sequencing (scRNA-seq) was performed on the lung parenchyma of ) mice, and 32 different cell populations were isolated. The macrophage subset showed a significant expansion, The AM of the mice increased by 1.93-fold and the IM increased by 2.79-fold. UMAP visualization revealed profound transcriptomic changes in the AM and IM of the knockout animals, indicating that Kdm6a knockout significantly altered the function of the AM and IM.

[0107] Next, the lipid metabolism of 32 cell types was studied and it was found that the top three cells in lipid metabolism were AM, ATII cells and IM cells. AM and IM cells showed enhanced lipid metabolism, but ATII cells showed a comprehensive inhibition of lipid metabolism. UMAP of alveolar epithelial cells showed that Kdm6a F / Y and There are significant differences in the distribution of ATII between mice. GO enrichment analysis showed that The mice showed more pronounced activation of biological processes in ATII cells, which are closely related to the development of pulmonary fibrosis. The lipid metabolism pathway in mouse ATII cells showed a significant inhibitory effect.

[0108] To elucidate the mechanism by which KDM6A-deficient macrophages affect alveolar epithelial cells, we conducted and Kdm6a F / Y The conditioned medium of (control) mouse bone marrow-derived macrophages (BMDM) was subjected to proteomic mass spectrometry analysis ( Figure 22 Among the 31 upregulated proteins, four were significantly upregulated, namely THBS1, B2M, PSAP, and CTSD, among which THBS1 was the most significantly increased, which was consistent with the single-cell sequencing data ( Figure 23 ). BMDM qPCR results showed that THBS1 expression increased in Kdm6a knockout mice ( Figure 24 Immunofluorescence analysis showed that THBS1 levels in lung macrophages of Kdm6a knockout mice were significantly increased ( Figure 25 ). In contrast, compared with the wild-type control group, THBS1 expression was reduced in mice. Ligand-receptor interaction analysis revealed that THBS1 secreted by KDM6A-deficient AMs and IMs significantly enhanced signaling in alveolar type II (ATII) cells in fibrotic lungs.

[0109] THBS1 (thrombospondin-1) is a multifunctional glycoprotein belonging to the extracellular matrix protein family. It interacts with cell surface receptors such as CD36, CD47, and integrins, affecting cell signaling, migration, and apoptosis. To investigate the functional consequences of THBS1 secretion, ATII cells ( Figure 26 Targeted lipid metabolomics identified 20 lipids, 18 of which were reduced in ATII cells exposed to Kdm6a knockout BMDM culture medium, while the addition of a THBS1 neutralizing antibody ameliorated these effects, restoring the levels of 17 lipids ( Figure 27Integrated analysis of single-cell sequencing and metabolomics data revealed that glycerophospholipid metabolism was significantly inhibited in ATII cells treated with culture medium from Kdm6a-knockout macrophages, while the expression levels of many genes involved in the glycerophospholipid metabolism pathway were altered. These findings suggest that KDM6A-deficient macrophages disrupt glycerophospholipid metabolism in ATII cells by secreting THBS1.

[0110] Example 4: Microliposomes encapsulating Kdm6a mRNA as a targeted therapeutic strategy for pulmonary fibrosis

[0111] 1. Liposome Construction

[0112] To translate these findings into a clinically viable treatment, the present inventors developed liposomes for targeted delivery of Kdm6a mRNA to lung macrophages. The raw materials used to prepare the liposomes are SPC, DOTAP, DSPE-PEG2000-mannose, and cholesterol, and the marker is DIO. To select liposomes with stronger macrophage targeting, the present inventors improved existing preparation methods and produced liposomes of two different particle sizes using two different methods.

[0113] ①100nm liposomes:

[0114] (1) SPC, DOTAP, DSPE-PEG2000-mannose, cholesterol, and DIO in a mass ratio of 4:3:1:2:0.5 were dissolved in water, and then ethanol (no more than 50% of the volume of the aqueous solution) was injected into the aqueous solution while rapidly stirring to allow the lipid mixture to self-assemble into liposomes;

[0115] (2) PBS solution was then added, and the mixture was centrifuged at 800 rpm for 1 minute to remove bubbles;

[0116] (3) The solution was ultrafiltered and centrifuged at 30,000 rpm for 10 minutes;

[0117] (4) After washing and centrifuging the precipitate several times, liposomes are obtained;

[0118] (5) Re-disperse the liposomes in PBS.

[0119] ②1μm liposomes:

[0120] (1) SPC, DOTAP, DSPE-PEG2000-mannose, cholesterol, and DIO were mixed in a glycerol-water solution (v / v = 1:20) at a mass ratio of 4:3:1:2:0.5 and incubated at 50°C for 30 min;

[0121] (2) After cooling, the solution was shaken at 50-75 Hz for 2 minutes using a mercury amalgamator.

[0122] (3) PBS solution was then added, and the mixture was centrifuged at 800 rpm for 1 min to remove bubbles;

[0123] (4) Centrifuge the solution at 5000 rpm for 3 minutes;

[0124] (5) After washing and centrifuging the precipitate several times, liposomes are obtained.

[0125] (6) Re-disperse the liposomes in PBS.

[0126] When siRNA or mRNA needs to be loaded, siRNA or mRNA powder is added to the solution and incubated to obtain mannosylated and DIO-labeled liposomes loaded with siRNA or mRNA; uncaptured siRNA or mRNA is removed by ultrafiltration centrifugation.

[0127] To optimize delivery efficiency, we systematically compared 100 nm and 1 μm liposome formulations and measured the hydrodynamic diameter, polydispersity, zeta potential, and stability of the liposomes by dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS, UK). The results are shown in Table 1 below (each value is expressed as Mean ± SD). Figure 28 shown.

[0128] Table 1

[0129]

[0130] 2. Biodistribution of liposomes in vivo

[0131] DIO-labeled liposomes were constructed and then injected intratracheally into anesthetized mice 14 days after BLM induction. Six days later (day 21 after BLM induction), the mice were euthanized, and organs were harvested for ex vivo fluorescence imaging. Furthermore, immunofluorescence was used to identify DIO-labeled macrophages, suggesting that the liposomes can target macrophages. Flow cytometry was used to measure the proportion of DIO-positive macrophages among all DIO-positive cells to assess the macrophage-targeting ability of the liposomes.

[0132] In vitro imaging ( Figure 29 ) showed that intratracheally delivered liposomes preferentially accumulated in lung tissue but not in other major organs such as the heart, liver, spleen, and kidney. Flow cytometric analysis demonstrated that the macrophage targeting efficiency of 1 μm liposomes was 5.5-fold higher than that of 100 nm liposomes ( Figure 30This is mainly due to the phagocytic function of macrophages. Other cells have a stronger ability to internalize 100nm liposomes than to phagocytize 1μm liposomes, while macrophages can phagocytize 1μm liposomes. In addition, the accumulation of 1μm liposomes in AM (74.2%) is preferentially compared to IM (25.8%) ( Figure 31 ).

[0133] Immunofluorescence analysis confirmed the specific targeting effect of 1 μm liposomes on profibrotic macrophages (CD206+ population) ( Figure 32 Given that AM exhibits enhanced profibrotic activity upon KDM6A inhibition, we selected 1 μm liposomes for subsequent therapeutic applications.

[0134] 3. Characterization of drug-loaded liposomes

[0135] The mRNA encapsulation efficiency was calculated using the RiboGreen assay. Liposomes were characterized by transmission electron microscopy (TEM, Jeol, Japan) after staining with 2% phosphotungstic acid. The parameters of 1 μm liposomes are shown in Table 2 below.

[0136] Table 2

[0137]

[0138] 4. Verification of KDM6A regulatory function

[0139] To verify the functional impact of KDM6A regulation, we first encapsulated Kdm6a siRNA in 1 μm liposomes with the structure as Figure 33 As shown in the electron microscope photo Figure 34 As shown, and administered by airway high-pressure atomization needle injection, Figure 35 The results showed that this method effectively inhibited the level of KDM6A protein in macrophages. The BLM-treated mice were injected with the drug on the 14th day after BLM-induced pulmonary fibrosis. Figure 36 and 37 The results showed that this approach exacerbated the severity of pulmonary fibrosis. Mechanistically, liposomes loaded with Kdm6a siRNA significantly upregulated the expression of THBS1 in lung macrophages of fibrotic mice ( Figure 38 ), subsequently inhibited the protein levels of FASN and LPCAT1 in ATII cells. In contrast, liposomes encapsulating Kdm6a mRNA ( Figure 39 、 Figure 40 ) was injected on the 18th day after BLM-induced pulmonary fibrosis using the same method, which showed a therapeutic effect. Micro-CT imaging showed that the area of fibrotic lesions was significantly reduced ( Figure 41 ), while Masson trichrome staining confirmed that the severity of fibrosis was reduced and collagen deposition was reduced ( Figure 42 、 Figure 43 ). Liposomes loaded with Kdm6a mRNA restored KDM6A protein levels in macrophages ( Figure 44 ), inhibited the expression of THBS1 ( Figure 45 ), and normalized glycerophospholipid metabolism in ATII cells, as evidenced by increased FASN and LPCAT1 protein levels ( Figure 46 These findings highlight the potential of Kdm6a mRNA delivery to improve fibrotic reprogramming of macrophages and restore alveolar epithelial function.

[0140] Example 5 Therapeutic Effect of Kdm6a mRNA Liposomes in Human Lung Organoid Fibrosis Model

[0141] We further evaluated the therapeutic potential of Kdm6a mRNA-loaded liposomes using a human lung organoid model derived from pluripotent stem cells. Organoids were generated through a three-stage differentiation protocol (lung progenitor bud induction, branching morphogenesis, and maturation) that yielded structures containing 12 cell types (type 1 alveolar epithelial cells, type 2 alveolar epithelial cells, goblet cells, ciliated cells, basal cells, endocrine cells, Clara cells, lung progenitor cells, macrophages, mesenchymal stromal cells, smooth muscle cells, and fibroblasts).

[0142] Specifically, human iPSC-derived lung organoids were purchased from Seven Plus Biotech Co., Ltd (Tianjin, China) according to an established protocol (PMID: 33631122). Fibrosis modeling was achieved by exposing the organoids at the air-liquid interface to 100 μM BLM. Therapeutic intervention began on the 10th day after BLM induction, with continuous administration of Kdm6a mRNA-loaded liposomes (1 μg / mL) or pirfenidone (50 μM; MedChemExpress, HY-B0673). On the 21st day after induction, the organoids were macroscopically recorded and then fixed in 4% paraformaldehyde (PFA) for 30 minutes. Subsequent histological analysis included Masson trichrome staining and multiple immunofluorescence staining.

[0143] Results: BLM-induced fibrotic organoids exhibited significant volume loss and structural collapse, with loss of alveolar structure. Treatment with liposomes loaded with Kdm6a mRNA or the clinical control, pirfenidone, restored organoid volume and reconstructed alveolar morphology, with liposomes being superior to pirfenidone in structural repair.

[0144] Masson trichrome staining confirmed that liposome-treated organoids showed attenuated fibrosis, and collagen deposition was significantly reduced compared with pirfenidone ( Figure 47Immunofluorescence analysis revealed that BLM-induced fibrosis elevated fibrotic markers (fibronectin, α-SMA, type I collagen), which were strongly suppressed by Kdm6a mRNA liposomes, which were more effective than pirfenidone. Crucially, macrophages in fibrotic organoids exhibited reduced levels of KDM6A, which were restored after liposome treatment ( Figure 48 Consistent with the findings in mice, KDM6A restoration in macrophages reduced THBS1 levels ( Figure 49 ), thereby restoring glycerophospholipid metabolism in ATII cells. Fibrotic organoids showed that FASN and LPCAT1 protein levels were reduced in ATII cells and significantly increased after liposome treatment ( Figure 50 This metabolic restoration was associated with functional alveolar regeneration, highlighting the dual mechanistic potential of Kdm6a mRNA delivery in targeting macrophage-driven fibrosis and epithelial repair. These human organoid data recapitulate key features of pulmonary fibrosis pathology and demonstrate the therapeutic advantages of Kdm6a mRNA liposomes over the current clinical standard.

[0145] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. Use of a substance that can upregulate KDM6A protein levels in pulmonary macrophages in the preparation of a drug for treating or preventing pulmonary fibrosis.

2. Use of the substance capable of upregulating the level of KDM6A protein in lung macrophages according to claim 1 in the preparation of a drug for treating or preventing pulmonary fibrosis, characterized in that: The substance has the ability to target lung macrophages.

3. Use of the substance capable of upregulating the level of KDM6A protein in lung macrophages according to claim 1 in the preparation of a drug for treating or preventing pulmonary fibrosis, characterized in that: The material is loaded with Kdm6a mRNA.

4. The use of substances that can upregulate KDM6A protein levels in lung macrophages in the preparation of drugs for restoring glycerophospholipid metabolism in alveolar type II cells.

5. A drug for treating or preventing pulmonary fibrosis, comprising a substance that can upregulate the level of KDM6A protein in pulmonary macrophages.

6. The drug for treating or preventing pulmonary fibrosis according to claim 5, wherein The drug targets lung macrophages.

7. The drug for treating or preventing pulmonary fibrosis according to claim 6, wherein The drug is loaded with Kdm6a mRNA.

8. The drug for treating or preventing pulmonary fibrosis according to claim 5 or 6, characterized in that The drug is delivered to the lung tissue by intratracheal injection or aerosol inhalation.

9. Use of substances that can upregulate KDM6A protein levels in lung macrophages in the preparation of drugs for fibrotic diseases associated with abnormal macrophage lipid metabolism.

10. Application of KDM6A protein in pulmonary macrophages as a marker in the preparation of a diagnostic or predictive kit for pulmonary fibrosis.

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