Use of kdm6a in lung macrophages in the treatment, prevention or diagnosis of pulmonary fibrosis

Abstract

The application discloses application of a substance capable of up-regulating KDM6A protein level in lung macrophages in preparation of a drug for treating or preventing pulmonary fibrosis. Also disclosed is application of KDM6A protein in lung macrophages as a marker in preparation of a diagnostic or predictive kit for pulmonary fibrosis. The application finds that KDM6A is an important signal molecule of the macrophage-epithelial cell metabolic pathway, and by restoring the expression of KDM6A in lung macrophages, THBS1 secretion is inhibited, thereby repairing glycerophospholipid metabolism of ATII cells and improving pulmonary fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of substances that can upregulate the level of KDM6A protein in lung macrophages in the preparation of drugs for treating or preventing pulmonary fibrosis and kits for diagnosing pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a late-stage manifestation of interstitial lung disease characterized by fibroblast proliferation and extensive extracellular matrix deposition, 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, characterized by abnormal fibroblast activation, excessive extracellular matrix (ECM) deposition, and alveolar epithelial cell dysfunction. Currently, there is a lack of effective treatments. Existing drugs (such as pirfenidone and nintedanib) can only slow disease progression, not reverse fibrosis. Studies have shown that the incidence of IPF is significantly higher in men than in women, but the molecular mechanism remains unclear. As the most representative type of chronic progressive pulmonary fibrosis, understanding the pathogenesis of IPF is crucial for the treatment of all types of pulmonary fibrosis.

[0003] A key but poorly understood aspect of IPF pathogenesis involves the disruption of pulmonary surfactant homeostasis. Macrophages and alveolar type II epithelial (ATII) cells maintain pulmonary lipid metabolism homeostasis through dynamic interactions. 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 regulate macrophage polarization and lipolysis capacity by releasing lipid mediators. Disruption of this metabolic symbiosis triggers lipid accumulation, surfactant deficiency, and a pro-fibrotic signaling cascade. Therefore, macrophages are key regulators of fibrosis progression.

[0004] Histone demethylase KDM6A is an X-chromosome-encoded epigenetic regulator with a sex-specific expression pattern in macrophages. Existing research has demonstrated 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 remains unstudied. Summary of the Invention

[0005] In their research, the inventors discovered that KDM6A plays a crucial 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 (ATII) cells, thereby promoting pulmonary fibrosis. Conversely, increasing KDM6A levels can reverse the progression of pulmonary fibrosis. Therefore, this invention first provides the application of substances that can upregulate KDM6A protein levels in pulmonary macrophages in the preparation of drugs for the treatment or prevention of pulmonary fibrosis.

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

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

[0008] Preferably, the substance carries Kdm6a mRNA.

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

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

[0011] Furthermore, given that KDM6A deficiency causes macrophages to upregulate lipid biosynthesis while also causing comprehensive inhibition of glycerophospholipid metabolism in ATII cells, this invention also provides the application of substances that can upregulate KDM6A protein levels in lung macrophages in the preparation of drugs that restore glycerophospholipid metabolism in alveolar type II cells.

[0012] The present invention also provides a medicament for treating or preventing pulmonary fibrosis, the medicament comprising 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 includes a targeting carrier, and the targeting carrier is preferably a liposome.

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

[0016] Preferably, the drug is loaded with Kdm6a mRNA.

[0017] Preferably, the drug is administered via intratracheal injection or nebulized inhalation.

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

[0019] Furthermore, the present invention also provides the application 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 a biopsy sample from 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 males and females.

[0020] Based on the above applications, the present invention also provides a diagnostic or auxiliary diagnostic kit for pulmonary fibrosis, which includes reagents for detecting KDM6A levels in pulmonary macrophages.

[0021] On the other hand, the present invention provides a liposome that targets lung macrophages. The liposome has a bilayer self-assembled structure with a particle size of more than 1 μm and contains SPC (soybean lecithin), DOTAP ((2,3-dioleoyl-propyl)-trimethylammonium chloride), DSPE-PEG2000-mannose, and cholesterol.

[0022] Preferably, the liposomes have a particle size of 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 liposomes targeting lung macrophages are prepared as follows: SPC, DOTAP, DSPE-PEG2000-mannose, and cholesterol are mixed in a glycerol-water solution and incubated at 45-55°C for a period of time to allow self-assembly. After cooling, the mixture is shaken to form microbubbles. The bubbles are removed, the mixture is washed, and the precipitate is obtained by centrifugation.

[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 mixed 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, and more preferably 4:3:1:2.

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

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

[0029] Preferably, the oscillation frequency is 50–75 Hz; an amalgam mixer can be used.

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

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

[0032] Preferably, the liposomes have a targeting efficiency of over 70% for targeting lung macrophages.

[0033] The present invention also provides the use of the above-mentioned liposomes targeting lung macrophages in the preparation of drugs for the treatment or prevention of pulmonary fibrosis, wherein the liposomes are loaded with Kdm6a mRNA.

[0034] The present invention also provides the application of the above-mentioned liposomes targeting lung macrophages in the preparation of drugs for restoring glycerophospholipid metabolism in type II alveolar cells, as well as other drugs for fibrotic diseases related to abnormal lipid metabolism in macrophages, wherein the liposomes are loaded with Kdm6a mRNA.

[0035] Preferably, the liposomes that target lung macrophages target lung CD206+ macrophages.

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

[0037] Preferably, the drug is administered via intratracheal injection or nebulized inhalation.

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

[0039] This invention reveals that KDM6A acts as a gatekeeper for macrophage-epithelial metabolic crosstalk. Macrophage-specific Kdm6a knockout mice reproduce the pathology of human pulmonary fibrosis, exhibiting accelerated fibrosis and an expansion of the pro-fibrotic AM / IM population. Single-cell transcriptomics shows that KDM6A deficiency reprograms macrophages, a key pathway to addressing fibrosis. Based on this research, this invention uses KDM6A protein as a biomarker for diagnosing or predicting pulmonary fibrosis, upregulates KDM6A protein levels in lung macrophages as a therapeutic approach to combat pulmonary fibrosis, and prepares drugs for treating or preventing pulmonary fibrosis using nucleic acids encoded by the KDM6A protein targeting lung macrophages.

[0040] Furthermore, this invention provides micron-sized liposomes that can efficiently target lung macrophages, with a delivery efficiency 5.5 times higher than traditional nanoliposomes. These liposomes use mannose as a ligand for CD206, enhancing their ability to specifically target macrophages. In a bleomycin-induced mouse pulmonary fibrosis model, Kdm6a mRNA liposomes significantly reduced the fibrotic area and restored glycerophospholipid metabolism. In a human lung organoid model, its therapeutic effect was superior to pirfenidone, improving alveolar structural damage. Moreover, the components of this liposome exhibit high biocompatibility and no significant immunogenicity.

[0041] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0042] Figure 1 These are the results of scRNA-seq analysis of KDM6A mRNA expression levels in alveolar and interstitial macrophages;

[0043] Figure 2 The correlation analysis between KDM6A mRNA levels in alveolar and interstitial macrophages and lipid metabolism fraction;

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

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

[0046] Figure 5 These are Masson trichrome stained lung sections after BLM induction. The image below (scale bar: 50 μm) shows a magnified view of the fibrotic area in the image above (scale bar: 500 μm).

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

[0048] Figure 7 This is a representative immunofluorescence co-staining image of Kdm6a (red) and F4 / 80 (green) in mouse lung sections after BLM treatment. Scale bar: 10 μm.

[0049] Figure 8 Is it like this? Figure 7 Quantitative comparison of KDM6A fluorescence intensity in macrophages after immunofluorescence staining (n=4);

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

[0051] Figure 10 It is BLM-treated mice KDM6A + Statistical quantification plot of median fluorescence intensity of AM (left) and IM (right);

[0052] Figure 11 It is macrophage-specific Kdm6a knockout A schematic diagram of the process for constructing mouse and intratracheal BLM-induced pulmonary fibrosis models;

[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 Is it like this? Figure 12 Quantitative comparison of KDM6A fluorescence intensity in macrophages after immunofluorescence staining;

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

[0056] Figure 15 This is a schematic diagram of the construction process: Kdm6a is administered via intratracheal injection. F / Y and Kdm6a F / F Mice were administered two recombinant adeno-associated virus vectors and then subjected to BLM-induced fibrosis;

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

[0058] Figure 17 It is a macrophage-specific Kdm6a knock-in A schematic diagram of the process for constructing mouse and BLM-induced fibrosis models;

[0059] Figure 18 Males treated with BLM Representative micro-CT images of mouse lungs;

[0060] Figure 19Males treated with BLM Semi-quantitative Ashcroft score (left) and collagen area quantification (right) for the severity of pulmonary fibrosis in mice (n=4);

[0061] Figure 20 Males treated with BLM Representative immunofluorescence co-staining images of KDM6A (red) and F4 / 80 (green) in mouse lung sections, scale bar: 10 μM;

[0062] Figure 21 Is it like this? Figure 20 Quantitative comparison of KDM6A fluorescence intensity in macrophages after immunofluorescence staining (n=4);

[0063] Figure 22 It comes from The process and results of integrated proteomics analysis of mouse bone marrow-derived macrophages (BMDM) conditioned medium (CM) and single-cell transcriptomics revealed four significantly upregulated proteins (THBS1, B2M, PSAP, and CTSD), with THBS1 showing the most significant increase.

[0064] Figure 23 Kdm6a was analyzed using 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 is Kdm6a F / Y and qPCR quantification results of THBS1 mRNA in mouse BMDM (n=4);

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

[0067] Figure 26 This is a schematic diagram of the workflow for targeted lipid metabolomics analysis in ATII cells treated with Kdm6a knockout BMDM conditioned medium.

[0068] Figure 27 The stacked bar chart shows the relative abundance changes of 20 lipid species in ATII cells exposed to BMDM conditioned medium with WT, Kdm6a-KO, Kdm6a-KO+THBS1 neutralizing antibody or Kdm6a-KO+IgG control.

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

[0070] Figure 29 It is an in vitro fluorescence imaging of major organs after liposomes are administered via the airway;

[0071] Figure 30 The results are flow cytometry analysis comparing the targeting efficiency of 100nm and 1μm liposomes on macrophages.

[0072] Figure 31 The results are quantitative findings of 1μm liposomes targeting AM and IM.

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

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

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

[0076] Figure 35 The results are quantitative analysis of the KDM6A immunofluorescence intensity in the lungs after delivery of Kdm6a siRNA liposomes into the airways of male and female mice, n=4;

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

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

[0079] Figure 38 The results are quantitative results of Thbs1 fluorescence intensity in mouse lung slice macrophages treated with liposomes loaded with scramble or Kdm6a siRNA after BLM induction, n=4;

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

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

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

[0083] Figure 42 This is a representative Masson trichrome staining of BLM-induced fibrotic lung in mice after intratracheal injection of liposomes (Kdm6a mRNA) (the image below shows a magnified view of the fibrotic area in the image above). Scale bar: 500 μm (top), 50 μm (bottom).

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

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

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

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

[0088] Figure 47 The results are quantitative results of the collagen-positive area of ​​BLM-induced organoids treated with liposomes loaded with Kdm6a mRNA or pirfenidone after Masson staining, n=4;

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

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

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

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

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

[0094] This invention first analyzed a published single-cell RNA sequencing (scRNA-seq) dataset (GSE136831), which included 28 healthy lungs and 32 IPF lungs. Based on different transcriptional pathways, the single cells were grouped into 38 cell subpopulations. Two major macrophage subpopulations—alveolar macrophages (AM, 42.2%) and interstitial macrophages (IM, 21.4%)—together accounted for over 60% of all cells examined in the composite dataset.

[0095] Based on the expression of genes involved in lipid biosynthesis, degradation, and transport, we calculated a lipid metabolism score. The results showed that AM and IM were the two cell clusters with the highest metabolic activity in IPF. Conversely, type II alveolar epithelial (ATII) cells showed comprehensive inhibition of lipid metabolism pathways in IPF.

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

[0097] Existing research suggests that X-chromosome inactivation escape-mediated gene dosage effects may underlie the dominant role of males in the pathogenesis of pulmonary fibrosis. Five genes (XIST, DDX3X, KDM6A, EIF2S3, and KDM5C) have been identified as X-chromosome inactivation escape genes. Compared to female macrophages, all five genes are significantly downregulated in male macrophages, with KDM6A exhibiting the most significant inhibitory effect in both male and female IPF. Figure 1 Its expression level was significantly negatively correlated with lipid metabolism fraction in both IPF patients and controls. Figure 2Immunofluorescence quantification confirmed a gradual decrease in KDM6A protein: baseline expression was lower in male macrophages than in female macrophages, and further depletion was observed in IPF lungs. Figure 3 Timing analysis of macrophage differentiation revealed that KDM6A was gradually downregulated as lung monocytes matured at the interstitial and alveolar macrophage stages. Overall, these findings identify KDM6A deficiency as a potential driver of profibrotic macrophage reprogramming.

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

[0099] To verify the clinical relevance of KDM6A dysregulation, we established a bleomycin (BLM)-induced pulmonary fibrosis model in mice (C57BL / 6). In short, 8-10 week old mice were anesthetized intraperitoneally and then administered 1 mg / kg of BLM (HY-17565A, MedChemExpress, China, Shanghai) intratracheally via a high-pressure nebulizer (BioJane Trading Co., Ltd., Shanghai, China). Mice given the same volume of sterile saline served as controls.

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

[0101] Flow cytometry further confirmed that, compared to females, KDM6A... + The ratio of AM to IM is low, and fibrosis-induced depletion of KDM6A. + Percentage of macrophages in all living cells ( Figure 9 Furthermore, quantitative fluorescence analysis showed that the KDM6A protein level in male AM ​​and IM was lower than that in females. Figure 10These experimental results mechanistically reproduced the differences in human pulmonary fibrosis, indicating that both men and women can drive fibrosis progression through macrophage-specific downregulation of KDM6A. However, the baseline KDM6A level 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 mature Cre recombinase tool specifically designed for gene knockout in myeloid cells. We used Kdm6a F / F Mice were crossed with Lyz2-Cre mice to construct macrophage-specific Kdm6a knockout mice. ( Figure 11 ). Figure 12 Immunofluorescence co-staining images confirmed the effective elimination of KDM6A in lung macrophages, where Kdm6a... F / F Representing female Cre-Floxed mice, Kdm6a F / F Lyz2-Cre represents female Cre+Floxed mice, Kdm6a F / Y Representing male Cre-Floxed mice, Kdm6a F / Y Lyz2-Cre represents male Cre+Floxed mice. Statistical results are as follows: Figure 13 As shown. It is noteworthy that, Mice treated with BLM exhibited accelerated fibrosis progression, with micro-CT revealing larger lesion areas compared to the wild-type (WT) control group. Histopathological analysis indicated that... Increased collagen deposition and fibrotic remodeling in mice Figure 14 It is similar to the pathology of human pulmonary fibrosis.

[0103] Next, to minimize potential contamination from 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 then delivered to Kdm6a via intratracheal injection. F / Y and Kdm6a F / F In the lung tissue of mice ( Figure 15Compared to the F4 / 80Cre- control group, mice receiving F4 / 80Cre+ showed specific KDM6A elimination in lung macrophages. The F4 / 80Cre+ group exhibited accelerated fibrosis progression after BLM treatment, with micro-CT imaging showing enlarged lesion areas compared to the F4 / 80Cre- control group. Histopathological analysis further confirmed that, compared to the Cre- group, the F4 / 80Cre+ group showed significantly enhanced collagen deposition and fibrotic tissue remodeling after BLM treatment. Figure 16 The results indicate that specific KDM6A elimination in lung macrophages promotes fibrosis.

[0104] Next, in order to establish a reverse relationship, we will use Kdm6a KI / KI Mice were crossed with Lyz2-Cre mice to construct macrophage-specific Kdm6a knock-in mice. ( Figure 17 After BLM processing, it is compatible with Kdm6a. WT Animals (Kdm6a) KI / Y )compared to, Animals (Kdm6a) KI / Y Lyz2-Cre) showed a reduction in the area of ​​fibrotic lesions via micro-CT. Figure 18 Collagen deposition is reduced. Figure 19 Immunofluorescence confirmed the successful overexpression of KDM6A in macrophages. Figure 20 , Figure 21 These studies on gain-and-loss of function indicate that KDM6A is a major epigenetic regulator of macrophage-driven fibrosis. Furthermore, macrophage-specific Kdm6a knock-in has a certain preventive and mitigating effect on subsequent BLM-induced pulmonary fibrosis.

[0105] Example 3: Study on the mechanism by which KDM6A-deficient macrophages drive pulmonary fibrosis

[0106] We (Kdm6a F / Y Lyz2-Cre and control (Kdm6a) F / Y Single-cell RNA sequencing (scRNA-seq) was performed on the lung parenchyma of mice, isolating 32 distinct cell populations. Macrophage subsets showed significant expansion. In mice, AM increased by 1.93-fold and IM increased by 2.79-fold. UMAP visualization showed profound transcriptomic changes in AM and IM in the knockout animals, indicating that Kdm6a knockout significantly altered the function of AM and IM.

[0107] Next, lipid metabolism in 32 cell types was studied, and it was found that the top three cell types in lipid metabolism were AM, ATII, and IM cells. Although AM and IM cells exhibited enhanced lipid metabolism, but ATII cells showed overall inhibition of lipid metabolism. UMAP analysis of alveolar epithelial cells showed that Kdm6a... F / Y and Significant differences were found in the distribution of ATII among mice. GO enrichment analysis showed that... Mice exhibited more pronounced activation of biological processes in ATII cells, processes closely related to the development of pulmonary fibrosis. Meanwhile, 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... and Kdm6a F / Y (Control) Proteomics mass spectrometry analysis was performed on the conditioned medium of mouse bone marrow-derived macrophages (BMDM). Figure 22 Of the 31 upregulated proteins, four were significantly upregulated: THBS1, B2M, PSAP, and CTSD. The increase in THBS1 was the most pronounced, consistent with single-cell sequencing data. Figure 23 ). qPCR results from BMDM showed that THBS1 expression was increased in Kdm6a knockout mice. Figure 24 Immunofluorescence analysis showed that THBS1 levels were significantly elevated in lung macrophages of Kdm6a knockout mice. Figure 25 Conversely, compared to the wild-type control group, THBS1 expression was reduced in mice. Ligand-receptor interaction analysis showed that THBS1 secreted by KDM6A-deficient AM and IM cells significantly enhanced signal transduction in alveolar type II (ATII) cells in fibrotic lung.

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

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

[0111] I. Construction of Liposomes

[0112] To translate these findings into clinically feasible treatment methods, this invention developed liposomes for targeted delivery of Kdm6amRNA to lung macrophages. The raw materials used to prepare the liposomes were SPC, DOTAP, DSPE-PEG2000-mannose, and cholesterol, with DIO as the labeling agent. To select liposomes with stronger targeting ability to macrophages, this invention improved existing preparation methods, using two different methods to prepare liposomes of two different particle sizes.

[0113] ①100nm liposomes:

[0114] (1) Mix SPC, DOTAP, DSPE-PEG2000-mannose, cholesterol and DIO in a mass ratio of 4:3:1:2:0.5 and dissolve them in water. Then inject ethanol (volume not greater than 50% of the aqueous solution) into the aqueous solution and stir rapidly to make the lipid mixture self-assemble into liposomes.

[0115] (2) Then add PBS solution and centrifuge the mixture at 800 rpm for 1 minute to remove air bubbles;

[0116] (3) Centrifuge the solution at 30,000 rpm for 10 minutes using ultrafiltration.

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

[0118] (5) Redisperse the liposomes in PBS.

[0119] ②1μm liposomes:

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

[0121] (2) After cooling, use an amalgam mixer to shake the solution at 50-75 Hz for 2 minutes;

[0122] (3) Then add PBS solution and centrifuge the mixture at 800 rpm for 1 minute to remove air bubbles;

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

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

[0125] (6) Redisperse 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. The hydrodynamic diameter, polydispersity, zeta potential, and stability of the liposomes were measured using dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS, UK). The results are shown in Table 1 below (values ​​are expressed as Mean ± SD). The size distributions of the two liposomes are as follows: Figure 28 As shown.

[0128] Table 1

[0129]

[0130] II. 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 collected for in vitro fluorescence imaging. Furthermore, immunofluorescence was used to identify DIO-labeled macrophages, which may indicate that the liposomes can target macrophages. Flow cytometry was used to determine the proportion of DIO-positive macrophages among all DIO-positive cells, assessing the ability of the liposomes to target macrophages.

[0132] In vitro imaging ( Figure 29 The study showed that liposomes delivered intratracheally preferentially accumulated in lung tissue, but not in other major organs such as the heart, liver, spleen, and kidneys. Flow cytometry analysis indicated that 1 μm liposomes showed a 5.5-fold increase in macrophage targeting efficiency compared to 100 nm liposomes. Figure 30This is mainly due to the phagocytic function of macrophages; other cells are more capable of phagocytosing 100 nm liposomes than 1 μm liposomes, while macrophages can phagocytose 1 μm liposomes. Furthermore, the accumulation of 1 μm liposomes in AM (74.2%) is preferential than in 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 stronger profibrotic activity after KDM6A inhibition, we selected 1μm liposomes for subsequent therapeutic applications.

[0134] III. Characterization of drug-loaded liposomes

[0135] 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. Parameters for 1 μm liposomes are shown in Table 2 below.

[0136] Table 2

[0137]

[0138] IV. KDM6A Adjustment Function Verification

[0139] To verify the functional impact of KDM6A regulation, we first encapsulated Kdm6a siRNA in 1 μm liposomes, the structure of which is shown below. Figure 33 As shown, its electron microscope image is as follows: Figure 34 As shown, the medication is administered via high-pressure nebulizer injection into the airway. Figure 35 The results showed that this method effectively suppressed the level of KDM6A protein in macrophages. Mice in the BLM-treated group were administered the drug on day 14 after BLM-induced pulmonary fibrosis. Figure 36 and 37 The results showed that this method exacerbated the severity of pulmonary fibrosis. Mechanistically, liposomes loaded with Kdm6a siRNA significantly upregulated THBS1 expression in fibrotic mouse lung macrophages. Figure 38 This subsequently suppressed the protein levels of FASN and LPCAT1 in ATII cells. Conversely, liposomes encapsulating Kdm6a mRNA ( Figure 39 , Figure 40 The same method was used to administer the drug on day 18 after BLM-induced pulmonary fibrosis, which showed a therapeutic effect, with micro-CT imaging showing a significant reduction in the area of ​​fibrotic lesions. Figure 41 Masson's trichrome staining confirmed a reduction in the severity of fibrosis and a decrease in collagen deposition. 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 normalize glycerophospholipid metabolism in ATII cells, as evidenced by elevated FASN and LPCAT1 protein levels. Figure 46 These findings highlight the potential of Kdm6a mRNA delivery to improve macrophage fibrosis reprogramming and restore alveolar epithelial function.

[0140] Example 5: Therapeutic effect of Kdm6a mRNA liposomes in a human lung organoid fibrosis model

[0141] We further evaluated the therapeutic potential of liposomes loaded with Kdm6a mRNA using a human lung organoid model derived from pluripotent stem cells. The organoids were generated through a three-stage differentiation protocol (lung progenitor induction, branching morphogenesis, and maturation), producing structures containing 12 cell types (type 1 alveolar epithelial cells, type 2 alveolar epithelial cells, lung goblet cells, cilia, 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 a pre-defined protocol (PMID: 33631122). Fibrosis modeling was achieved by exposing the organoids at the gas-liquid interface to 100 μM BLM. Treatment intervention began on day 10 post-BLM induction, with continuous administration of either Kdm6a mRNA-loaded liposomes (1 μg / mL) or pirfenidone (50 μM; MedChemExpress, HY-B0673). On day 21 post-induction, the organoids were macroscopically recorded and then fixed in 4% paraformaldehyde (PFA) for 30 minutes. Subsequent histological analysis included Masson trichrome staining and multiplex immunofluorescence staining.

[0143] Results: BLM-induced fibrotic organoids exhibited significant volume reduction 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; liposomes were superior to pirfenidone in structural repair.

[0144] Masson trichrome staining confirmed reduced organoid fibrosis after liposome treatment, with significantly decreased collagen deposition compared to pirfenidone. Figure 47Immunofluorescence analysis showed that BLM-induced fibrosis elevated fibrosis markers (fibronectin, α-SMA, type I collagen), which were strongly inhibited by Kdm6a mRNA liposomes, showing superior efficacy compared to pirfenidone. Crucially, macrophages in fibrotic organoids exhibited decreased KDM6A levels, which were restored upon liposome treatment. Figure 48 Consistent with findings in mice, restoration of KDM6A in macrophages reduced THBS1 levels. Figure 49 This restored glycerophospholipid metabolism in ATII cells. Fibrotic organoids showed decreased levels of FASN and LPCAT1 proteins in ATII cells, which significantly increased after liposome treatment. Figure 50 This metabolic recovery is associated with functional alveolar regeneration, highlighting the dual mechanism of Kdm6a mRNA delivery in macrophage-driven fibrosis and epithelial repair. These human organoid data summarize key features of pulmonary fibrosis pathology and demonstrate the therapeutic advantage of Kdm6a mRNA liposomes over current clinical standards.

[0145] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of a substance capable of directly targeting lung macrophages and upregulating KDM6A protein levels in lung macrophages in the preparation of drugs for treating or preventing pulmonary fibrosis, characterized in that, The lung macrophages refer to alveolar macrophages and interstitial macrophages. The drug is delivered to the lung tissue via intratracheal injection or nebulized inhalation. The substance is loaded with... Kdm6a Micron-sized liposomes of mRNA, using mannose as a ligand for CD206.

2. A drug for treating or preventing pulmonary fibrosis, characterized in that, The drug comprises a substance that directly targets lung macrophages and can upregulate the level of KDM6A protein in lung macrophages. Lung macrophages refer to alveolar macrophages and interstitial macrophages. The drug is delivered to lung tissue via intratracheal injection or nebulized inhalation. The substance is loaded with… Kdm6a Micron-sized liposomes of mRNA, using mannose as a ligand for CD206.

Images