Use of tarm1 in preparation of drugs for treating acute respiratory distress syndrome

Abstract

The application discloses application of TARM1 in preparation of a drug for treating acute respiratory distress syndrome, and relates to the technical field of biological medicine; the application of TARM1 in preparation of the drug for treating acute respiratory distress syndrome, wherein the TARM1 inhibitor is a short hairpin RNA targeting a TARM1 gene, the short hairpin RNA is carried by a recombinant adeno-associated virus serotype 6 vector, and the recombinant adeno-associated virus vector contains a macrophage-specific CD68 promoter, which is used to drive expression of the short hairpin RNA in alveolar macrophages. The application integrates three independent mouse ARDS transcriptome data sets and single-cell RNA sequencing data, and for the first time, screens out TARM1 as a key gene common to ARDS and M1-type macrophage polarization, thereby providing a new intervention direction for targeted treatment of ARDS.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of TARM1 in the preparation of drugs for treating acute respiratory distress syndrome. Background Technology

[0002] Acute respiratory distress syndrome (ARDS) is a severe inflammatory lung disease caused by a variety of direct or indirect damaging factors. Clinically, patients present with persistent hypoxemia and respiratory failure, and have an extremely high mortality rate of 30% to 40%. Although progress has been made in supportive treatment strategies such as lung-protective ventilation, targeted therapies against the underlying pathogenesis of ARDS remain extremely limited.

[0003] The development of ARDS involves complex interactions between numerous target cells and effector cells. As the primary sentinel cells of the lung's innate immune system, alveolar macrophages play a crucial role in the occurrence and progression of ARDS. Increasing research indicates that macrophage polarization is essential to the pathogenesis of ARDS. Upon activation, macrophages can polarize into either the classic activating M1 phenotype or the alternative activating M2 phenotype. M1 macrophages mediate tissue damage by secreting large amounts of pro-inflammatory cytokines and expressing inducible nitric oxide synthase; while M2 macrophages play a vital role in mitigating inflammation and promoting tissue repair. In the early stages of ARDS, the directed differentiation of alveolar macrophages into the M1 lineage is considered a key event initiating a "cytokine storm" and disrupting the alveolar-capillary barrier. Therefore, elucidating the core molecular regulatory pathways behind M1 polarization is crucial for developing novel ARDS therapies.

[0004] The rapid development of high-throughput sequencing technology has revolutionized the systematic discovery of key genes related to disease pathogenesis. However, traditional transcriptome analysis can only reflect changes in overall gene expression within tissues and cannot distinguish the roles of specific cell subpopulations. Single-cell RNA sequencing can reveal cellular heterogeneity, but innovative strategies are still needed to effectively link cell type-specific expression characteristics with overall pathological phenotypes. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of TARM1 in the preparation of drugs for treating acute respiratory distress syndrome.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The application of TARM1 in the preparation of drugs for treating acute respiratory distress syndrome, wherein the TARM1 inhibitor is a short hairpin RNA that targets the TARM1 gene, the short hairpin RNA is carried by a recombinant adeno-associated virus serotype 6 vector, the recombinant adeno-associated virus vector contains a macrophage-specific CD68 promoter to drive the expression of the short hairpin RNA in alveolar macrophages.

[0007] Preferably, the recombinant adeno-associated virus vector further comprises a reporter gene ZsGreen or an enhanced green fluorescent protein gene, wherein the reporter gene is linked to the short hairpin RNA via a self-cleaving peptide for monitoring macrophage transduction efficiency.

[0008] Preferably, the TARM1 inhibitor is a small interfering RNA that targets the TARM1 gene and is used to knock down TARM1 expression in an in vitro macrophage model; the small interfering RNA is transfected with liposome Lipofectamine 3000, the macrophage confluence is 70% at the time of transfection, and the knockdown effect is detected 48 hours after transfection.

[0009] Preferably, the in vitro macrophage model is the mouse RAW264.7 macrophage line, which is stimulated with 1 μg / mL lipopolysaccharide for 24 hours to induce M1 polarization in order to simulate the activation state of macrophages in acute respiratory distress syndrome.

[0010] Preferably, the anti-inflammatory effect of the drug is achieved by inhibiting macrophage M1 polarization, specifically manifested as follows: in lipopolysaccharide-stimulated RAW264.7 cells, knockdown of TARM1 leads to a decrease in the expression level of inducible nitric oxide synthase protein, and a decrease in the mRNA expression levels of interleukin-1β and tumor necrosis factor-α.

[0011] Preferably, the acute respiratory distress syndrome is a mouse model of acute respiratory distress syndrome induced by intratracheal instillation of lipopolysaccharide (LPS). The LPS dose is 5 mg / kg body weight, administered once intratracheally, and lung injury is assessed 48 hours after administration.

[0012] A pharmaceutical composition comprising a therapeutically effective amount of the recombinant adeno-associated virus vector and a pharmaceutically acceptable nebulized carrier, said nebulized carrier being composed of one or more of mannitol, sucrose, and polysorbate 80 in sterile saline or phosphate buffer, for local airway delivery.

[0013] Preferably, it further comprises a second therapeutic agent selected from one or more of glucocorticoids, IL-6 receptor antagonists, NF-κB pathway inhibitors, pulmonary surfactant, or antibacterial drugs.

[0014] An in vitro method for screening candidate drugs for the treatment of acute respiratory distress syndrome, comprising the following steps: Step (1) Culturing mouse RAW264.7 macrophages and stimulating them with 1 μg / mL lipopolysaccharide for 24 hours to construct an M1 polarization model; Step (2) Add the test compound to the model and incubate; Step (3) The mRNA expression levels of TARM1, IL-1β, and TNF-α were detected by qRT-PCR, and the iNOS protein expression level was detected by Western blot, with β-actin as an internal reference. Step (4) If the test compound can downregulate the expression level of TARM1 mRNA compared with the model group, and at least one of iNOS, IL-1β, and TNF-α is simultaneously downregulated, then the compound is determined to be a candidate drug for the treatment of acute respiratory distress syndrome.

[0015] Preferably, in step (3), qRT-PCR is normalized with the internal reference gene Gapdh; Western blot uses β-actin as the loading control, and the primary antibody dilution ratios are TARM1 1:500, iNOS 1:1000, and β-actin 1:20000.

[0016] The beneficial effects of this invention are as follows: 1. This invention integrates three independent mouse ARDS transcriptome datasets and single-cell RNA sequencing data to identify TARM1 as a key gene shared by ARDS and M1 macrophage polarization for the first time, providing a new direction for targeted therapy of ARDS.

[0017] 2. This invention confirms through immunofluorescence double staining experiments that TARM1 and the macrophage marker CD68 exhibit significant co-localization in ARDS lung tissue; combined with single-cell sequencing data, it clarifies that TARM1 is specifically highly expressed in the M1 macrophage subset; it reveals that TARM1 has good cell specificity as an intervention target, providing solid histopathological evidence for the subsequent design of macrophage-targeted therapeutic strategies.

[0018] 3. In an LPS-stimulated RAW264.7 cell model, this invention used Lipofectamine 3000 to transfect siRNA targeting TARM1, demonstrating that knocking down TARM1 significantly inhibited the protein expression of iNOS, a marker of M1 macrophages, and simultaneously downregulated the mRNA levels of pro-inflammatory cytokines IL-1β and TNF-α. The results indicate that TARM1 is a key regulator driving macrophage differentiation into the pro-inflammatory M1 phenotype, and inhibiting TARM1 can effectively block the initiation of the "cytokine storm".

[0019] 4. This invention constructs an AAV6-shTARM1 recombinant viral vector driven by a macrophage-specific CD68 promoter; in vivo experiments have demonstrated that after intratracheal infusion of this vector for 4 weeks, TARM1 expression can be efficiently and specifically knocked down in alveolar macrophages, and the reporter gene ZsGreen clearly shows transduction efficiency; the natural tropism of AAV6 for lung tissue is utilized to avoid the off-target risk of systemic gene editing.

[0020] 5. Based on the LPS-stimulated RAW264.7 cell model, this invention establishes a screening system with TARM1 expression level, iNOS protein level, IL-1β and TNF-α expression level as the core evaluation indicators; it provides a standardized experimental platform for subsequent high-throughput screening of small molecule compounds or specific antibodies targeting TARM1, and has good prospects for industrial application. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating how TARM1 was identified as a candidate gene upregulated in ARDS lung tissue in this invention. Figure 2 This is a schematic diagram illustrating the upregulation of TARM1 expression and co-localization with macrophages in the lung tissue of ARDS mice in this invention; Figure 3 This is a schematic diagram illustrating how TARM1 gene knockdown can inhibit M1 macrophage polarization and pro-inflammatory cytokine expression in this invention. Figure 4 This is a schematic diagram illustrating macrophage-specific TARM1 gene knockdown achieved via AAVshRNA in this invention. Figure 5 This is a schematic diagram illustrating how macrophage-specific TARM1 gene knockout can alleviate lung injury in ARDS mice in this invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0023] Example 1: 1.1 Acquisition and Processing of Transcriptome Datasets

[0024] The transcriptome datasets were obtained from the NCBI Gene Expression Database (GEO). This invention downloaded three independent LPS-induced ARDS mouse model lung tissue transcriptome datasets: GSE193958, GSE216943, and GSE263867, with sample sizes of 3, 6, and 5 pairs (ARDS group and control group), respectively.

[0025] 1.2 Screening of differentially expressed genes (DEGs)

[0026] The limma package in SangerBox 3.0 software (https: / / www.sangerbox.com / ) was used for analysis, with a threshold set at |log2(fold change)| ≥ 2 and a corrected p-value < 0.05. All gene expression values ​​were normalized to quantiles before DEG analysis. Subsequently, Venn diagram analysis was used to identify overlapping genes among the three datasets, revealing a set of common genes associated with ARDS.

[0027] 1.3 scRNA-seq data analysis

[0028] The scRNA-seq dataset (GSE217324) derived from ARDS mouse lung tissue was obtained from the same GEO database. All subsequent analyses were performed using R software (version 4.5.1) and the SeuratR package (version 5.3.0). To ensure data quality, cells were excluded if they met any of the following criteria: number of detected genes <300 or >5000, mitochondrial gene proportion >10%, hemoglobin gene proportion >3%, or UMI count in the top 3% or <1000. The Harmony algorithm was used to eliminate batch effects between samples; unsupervised clustering was performed using the first 30 principal components (resolution = 0.05), and dimensionality reduction visualization was performed using t-SNE and UMAP; the FindAllMarkers function (Wilcoxon rank-sum test; |log2FC|>0.25 and min.pct>0.25) was used to identify the characteristic genes corresponding to each cluster; finally, cell types were determined based on the identified marker genes.

[0029] 1.4 Macrophage subset analysis and target gene screening

[0030] Based on classic functional marker genes, macrophages were divided into M1 and M2 phenotypes. Subsequently, differential expression profiling was performed on M1 and M2 macrophages using |log2FC|≥2 and a corrected P-value <0.05 (Benjamini-Hochberg correction) as threshold criteria to determine a complete list of differentially expressed genes (DEGs). This set of differentially expressed genes was then subjected to intersection analysis with DEGs previously identified in three ARDS-related transcriptome datasets (GSE193958, GSE216943, and GSE263867) to construct a final candidate target gene library. The expression of these candidate genes in ARDS mouse lung tissue was validated by qRT-PCR, thus identifying TARM1 as the core target gene of this study. Furthermore, visualization tools such as feature maps, violin plots, and dot plots were used to evaluate the specific expression patterns of TARM1 in different macrophage subsets based on single-cell datasets, and the data was visualized using ggplot2 and patchwork software packages.

[0031] 1.5 Preliminary experimental verification and screening of candidate genes

[0032] Through comprehensive bioinformatics analysis, 15 differentially expressed genes (DEGs) were identified across four datasets. Preliminary experimental validation was conducted to determine the key molecules with the greatest research value. Seven genes (MEFV, TARM1, OASL1, IFI205, OAS3, LACC1, and SLFN4) with insufficient data or controversial mechanisms of action in acute respiratory distress syndrome (ARDS) were prioritized for quantitative real-time PCR (qRT-PCR) validation. The selection criteria were based on two main aspects: first, gene novelty—the functions of these genes in the pathogenesis of ARDS were not yet clearly defined or were controversial in existing literature; second, avoiding duplicate studies—other genes (such as IRF7, CCL2, and CXCL3) have been well-established to show significant and consistent expression changes in ARDS; therefore, this study aimed to explore new potential regulatory targets. Comparative analysis of gene expression in lung tissues of control mice and lipopolysaccharide-induced ARDS mice revealed that TARM1 exhibited the most significant and obvious expression changes. Therefore, TARM1 was identified as the core target gene for all subsequent functional and mechanistic studies.

[0033] 1.6 Laboratory Animals and Ethical Standards

[0034] Male C57BL / 6 mice (6-8 weeks old, weighing 20-22 grams) were purchased from the Experimental Animal Resource Center of Chongqing Medical University. The animals were housed in a 12-hour light / dark cycle environment, with free access to standard feed and sterile water. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Chongqing Medical University.

[0035] 1.7 Key Reagents and Antibodies

[0036] LPS (E. coli O111:B4 strain, catalog number L2630) and Lipofectamine 3000 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and Invitrogen (California, USA), respectively. Protein extraction kits were provided by Bestbio (Shanghai, China); ELISA kits for IL1β and TNFα were purchased from Credit Suisse Biotechnology (Fujian, China). One-step TUNEL apoptosis detection kit was purchased from Servicebio (Wuhan, China). Wright-Giemsa (WG) staining kit (catalog number G1021) was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Biosharp (Hefei, China) provided 5×SDS-PAGE loading buffer (BL502B), ECL substrate (BL520B), and penicillin-streptomycin solution (BL505A). RNA extraction and qRT-PCR kits were from GenesandBiotech (Beijing, China). HyCytoBiotech (Suzhou, China) supplied high-glucose DMEM medium and fetal bovine serum (FBS). BCA kits, DAPI, and anti-fading mounting media were purchased from Beyotime Biotechnology (Shanghai, China). PVDF membranes were purchased from Merck Millipore (Cork, Ireland). TARM1 antibody (sc514218) and iNOS antibody (340668) were purchased from Santa Cruz Biotechnology and ZenBio, respectively; βActin antibody (AC038) was purchased from ABclonal (Wuhan, China). HRP-labeled secondary antibodies were purchased from Biosharp (rabbit, BL003A) and Proteintech (mouse, SA000011); AntiCD68 antibody (GB11067100), Cy3-labeled anti-mouse antibody (GB21301), and Alexa Fluor594-labeled anti-rabbit antibody (GB28301) were all purchased from Servicebio (Wuhan, China). AAV6 shRNA virus (AAV6hCD68TARM1shRNAzsgreen) and control virus were purchased from Newheal Biotechnology (Shanghai, China). TARM1 siRNA and control siRNA were purchased from Cysophar Biosciences (Jiangsu Province, China). Primers were synthesized by General Biol (Shanghai, China).

[0037] 1.8 LPS-stimulated ARDS mouse model

[0038] Mice were anesthetized using a small animal anesthesia machine by inhalation of 5% isoflurane, followed by intratracheal administration of LPS (1 mg / mL, 5 mg / kg) or sterile phosphate-buffered saline (PBS) as a control. Body weight was measured at 0 hours (baseline), 24 hours, and 48 hours after LPS injection, and weight changes were recorded. Clinical signs were assessed daily upon weighing. No deaths occurred during the 48-hour observation period prior to euthanasia. Forty-eight hours after LPS stimulation, mice were euthanized by cervical dislocation after inducing deep anesthesia via intraperitoneal injection of sodium pentobarbital (50 mg / kg). Depth of anesthesia was confirmed by the disappearance of the plantar withdrawal reflex. Lung tissue, bronchoalveolar lavage fluid (BALF), and serum samples were collected according to established protocols and stored at -80°C until use.

[0039] 1.9 Recombinant AAVshRNA Vector

[0040] To achieve TARM1 gene knockdown, researchers constructed a recombinant adeno-associated virus serotype 6 (AAV6) vector. 50 μL of TARM1-targeting AAV-shRNA or control AAV-shRNA (5.0 × 10¹¹ viral particles per mouse) was administered via intratracheal infusion. Experimental procedures were performed on mice 4 weeks after AAV administration to ensure viral transduction efficiency.

[0041] 1.10BALF

[0042] After anesthetizing mice, a 20-gauge catheter was inserted into the trachea. A syringe was connected to the catheter, and the lungs were lavaged three times with 1 mL of pre-cooled sterile phosphate-buffered saline (PBS), collecting bronchoalveolar lavage fluid (BALF). BALF samples were centrifuged at 800 rpm for 15 minutes at 4°C. The total cell count in BALF was determined using a cell counter; the number of neutrophils and macrophages was determined by Wright-Giemsa staining; and the protein concentration in BALF was determined using the BCA assay. The supernatant was preserved for subsequent cytokine assays.

[0043] 1.11 Lung wet / dry weight ratio: Weigh the wet weight of the right upper lobe of the lung, dry it at 65°C for 48 hours to a constant dry weight, and calculate its wet / dry weight ratio.

[0044] 1.12HE

[0045] Lung tissue samples were stained with hematoxylin and eosin (H&E) according to the kit manufacturer's instructions. The severity of lung injury was assessed using a semi-quantitative scoring system based on four parameters (edema, hemorrhage, neutrophil infiltration, and alveolar wall thickening), ranging from 0 (no injury) to 4 (diffuse injury), as described previously. The total score (0-16) was the sum of the individual scores. Scoring was performed independently by two blinded researchers, and the average score was used as the final result.

[0046] 1.13 Immunohistochemistry (IHC)

[0047] For immunohistochemical analysis, paraffin sections underwent antigen retrieval after dewaxing and rehydration. Endogenous peroxidase was blocked with 3% hydrogen peroxide solution; subsequently, sections were blocked with goat serum and incubated overnight at 4°C with anti-TARM1 primary antibody (dilution 1:200). The following day, sections were incubated with secondary antibody at room temperature for 2 hours, developed using 3,3'-diaminobenzidine (DAB), and mounted with neutral resin. Quantitative analysis was performed using ImageJ software: a uniform threshold was set for all images to identify TARM1-positive (brown) regions, and the positive area was calculated as a percentage of the total tissue area in each field of view. Three high-power fields were randomly selected from each section for analysis, and the final average value for each mouse was calculated.

[0048] 1.14 Immunofluorescence staining

[0049] Lung tissue sections were double-stained overnight at 4°C with anti-TARM1 (1:200) and anti-CD68 (1:200) secondary antibodies, followed by secondary staining at room temperature. Cell nuclei were counterstained with DAPI and observed using a confocal microscope.

[0050] 1.15 TUNEL test

[0051] TUNEL assay was performed on paraffin-embedded lung tissue sections. Apoptotic cells were detected using an in situ cell death assay kit, following the manufacturer's instructions.

[0052] 1.16-week protein was extracted from lung tissue or cultured cells using a commercial kit, and its concentration was determined by the BCA method. Equal volumes of sample were separated by 10% SDS-PAGE electrophoresis and transferred to PVDF membranes, which were then blocked with 5% skim milk. The membranes were co-incubated overnight at 4°C with primary antibodies against TARM1 (1:500), iNOS (1:1000), and βactin (1:20,000), followed by incubation at room temperature with horseradish peroxidase-labeled secondary antibody for 1 hour. Bands were visualized by ECL and quantified using ImageJ software; βactin was used as a loading control.

[0053] 1.17ELISA

[0054] The IL1β and TNFα values ​​of elisabalf were quantified using a commercial ELISA kit and in accordance with the manufacturer's instructions.

[0055] 1.18 Cell Culture and Treatment

[0056] RAW264.7 mouse macrophages were purchased from Haixing Biotechnology Co., Ltd. (Suzhou, China). Cells were cultured in a 37°C, 5% CO2 incubator using high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. To induce M1 polarization, cells were stimulated with LPS (1 μg / mL) for 24 hours.

[0057] 1.19 siRNA transfection

[0058] When RAW264.7 macrophages reached 70% confluence, they were transfected using Lipofectamine 3000 reagent according to the manufacturer's specifications. All subsequent in vitro experiments were performed 48 hours after transfection to ensure optimal gene silencing.

[0059] 1.20 qRT-PCR analysis

[0060] Total RNA was extracted using a commercial kit and detected by qRT-PCR according to the manufacturer's instructions. GAPDH was used as an internal control.

[0061] 1.21 Data Analysis

[0062] Data were analyzed using GraphPadPrism (10.4.0). Data are expressed as mean ± standard deviation. Student's t-test or Mann-Whitney U-test was used for comparisons between two groups; one-way ANOVA followed by Tukey's post-hoc test was used for comparisons among multiple groups; Pearson correlation coefficient analysis was used for linear relationships. Statistical significance was set at P < 0.05.

[0063] result: 2.1 TARM1 expression levels were elevated in ARDS lung tissue and enriched in M1 macrophages.

[0064] like Figure 1In the figure: (A) Overlap Venn diagram of differentially expressed genes (DEGs) between the three control groups and the whole RNA sequencing dataset of lung tissue from ARDS mice. (B) UMAP distribution of cell types in lung tissue from single-cell RNA sequencing (scRNAseq). (C) Stacked bar chart showing the average cell type composition of the control and ARDS groups. (D) Venn diagram showing the overlap between DEGs from the three whole RNA sequencing datasets of lung tissue and DEGs from M1 / M2 macrophages in one scRNAseq dataset. (E) qRT-PCR detection results of candidate genes in lung tissue of control and LPS-induced ARDS mice (n=3 mice per group), data are expressed as mean ± standard deviation; *p<0.05 indicates significant difference compared with the control group, and each sample was tested in triplicate. (F) Plot of TARM1 expression distribution in scRNAseq for cell type identification. (G) UMAP plot showing the expression level of TARM1 in different cell populations, indicating that it is mainly expressed in M1 macrophages and neutrophils.

[0065] To screen for potential genes involved in ARDS development, three independent transcriptome datasets from LPS-induced mouse ARDS model lung tissue were first compared, identifying a total of 81 common genes that were significantly altered in ARDS. Figure 1 A). Meanwhile, single-cell RNA sequencing analysis of ARDS lung tissue showed that macrophages are one of the main immune cell populations during ARDS, with a significantly increased proportion of macrophages differentiating towards the M1 phenotype. Figure 1 B, C). To further narrow down the candidate gene list, these 81 ARDS-related common genes were cross-referenced with differentially expressed genes between M1 and M2 macrophages in the single-cell dataset, ultimately identifying 15 candidate genes (B, C). Figure 1 D). Seven genes with unknown or poorly studied functions in ARDS (MEFV, TARM1, OASL1, IFI205, OAS3, LACC1, and SLFN4) were preferentially selected for qRT-PCR validation. Consistent with these results, in the ARDS mouse model, TARM1 was confirmed to be the most significantly upregulated gene in lung tissue after LPS stimulation. Figure 1 E). Further analysis of single-cell RNA sequencing data showed that TARM1 expression was significantly enriched in M1 macrophages compared to M2 macrophages and other cell subsets; in addition, TARM1 was also highly expressed in the neutrophil population. Figure 1 F, G).

[0066] 2.2 TARM1 is highly and specifically expressed in ARDS lung tissue and co-localizes with macrophages.

[0067] like Figure 2Figure 1 shows: (A) Quantitative real-time polymerase chain reaction (qRT-PCR) results of TARM1 mRNA in lung tissues of control and ARDS mice (n=3 mice per group). (B) Western blot analysis of TARM1 protein in lung tissues of control and ARDS mice, with βActin as a loading control (n=3 mice per group). (C) Immunohistochemical staining results of TARM1 in lung tissues of control and ARDS mice (n=3 mice per group). Scale bar = 50 μm. (D) Immunofluorescence staining results of TARM1 (yellow) and CD68 (red) in lung tissues of control and ARDS mice, with cell nuclei labeled with DAPI (blue); the merged image shows the colocalization of TARM1 and CD68 (n=3 mice per group). Each sample was measured in triplicate, and data are expressed as mean ± standard deviation. *p<0.05, ****p<0.0001 (compared to the control group).

[0068] In an ARDS mouse model, changes in TARM1 expression were verified: both qRT-PCR and Western blot experiments showed significantly increased mRNA and protein levels of TARM1. Figure 2 A, B); Immunohistochemical analysis showed a significant increase in the number of TARM1-positive cells in ARDS lung tissue (A, B); Figure 2 C); Immunofluorescence staining confirmed that TARM1 co-localized with the macrophage marker CD68 in ARDS lung tissue ( Figure 2 D), indicating that TARM1 is mainly expressed in lung macrophages.

[0069] 2.3 Knocking down TARM1 can inhibit the polarization of M1 macrophages and the expression of inflammatory cytokines.

[0070] like Figure 3In the figure: (A) Western blot results of TARM1 and iNOS proteins in RAW264.7 cells stimulated by LPS (24 hours) (n=3); (B) Morphological changes of RAW264.7 cells before and after LPS stimulation (n=3), scale bar is 50μm; (C) Western blot screening experiment of TARM1 siRNA: cells were transfected with NC or TARM1 siRNA, and the expression level of TARM1 protein was detected after 48 hours. βActin was used as a loading control (n=3); (D) s (e) qRT-PCR results of TARM1 mRNA after iRNA transfection (n=3); (f) Western blot results of TARM1 and iNOS proteins in cells transfected with NC or TARM1 siRNA and stimulated with LPS for 24 hours, with βActin as a loading control (n=3); (f) qRT-PCR results of IL1β and TNFα mRNA in the control group, LPS group, LPS+NC siRNA group, and LPS+TARM1 siRNA group (n=3), with three replicates for each sample. Data are expressed as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (compared to the control group).

[0071] To assess the function of TARM1 in macrophages, in vitro experiments were performed in RAW264.7 cells. After 24 hours of LPS stimulation, both TARM1 and the M1 marker iNOS were significantly upregulated. Figure 3 A). Morphological observation showed that after LPS stimulation, RAW264.7 cells changed from round to irregular shapes and developed more pseudopodia, exhibiting a typical activated M1 phenotype. Figure 3 B). To screen for effective siRNAs targeting TARM1, three siRNAs and one negative control (NCsiRNA) were designed and synthesized. Western blot results showed that all three siRNAs reduced TARM1 expression levels to varying degrees, with TARM1 siRNA #3 showing the most significant inhibitory effect. Figure 3 C); qRT-PCR further validated this result ( Figure 3 Therefore, TARM1 siRNA#3 was used in subsequent experiments. Based on this, the effect of TARM1 knockdown on M1 polarization was evaluated: Western blot confirmed that under LPS stimulation, TARM1 knockdown significantly reduced TARM1 protein levels, accompanied by a significant inhibition of iNOS protein expression (D). Figure 3 E); qRT-PCR showed that TARM1 silencing significantly reduced LPS-induced IL1β and TNFα mRNA levels (E); Figure 3F). Given that single-cell sequencing results indicate that TARM1 is specifically and highly expressed in M1 macrophages ( Figure 1 This study focuses on the regulatory role of F and G in M1 polarization, without further evaluating changes in M2 polarization markers.

[0072] 2.4 Macrophage-specific TARM1 gene knockdown was successfully achieved using AAVshRNA.

[0073] like Figure 4 Figure 1 shows: (A) Western blot analysis of TARM1 protein in lung tissue: TARM1 was almost undetectable in the control group; its expression increased significantly after LPS stimulation; and the AAVTARM1#3 vector most effectively inhibited its expression (n=3 mice per group). (B) Immunofluorescence staining results of TARM1 in macrophages: The TARM1 signal intensity in the LPS+AAVTARM1 group was lower than that in the LPS+AAVNC group; Pearson correlation coefficient is shown on the right (n=3 mice per group). Scale bar = 50 μm. Each sample was measured three times in duplicate, and the data are expressed as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (compared to the control group).

[0074] To verify the function of TARM1 in vivo, macrophage-specific knockdown was achieved using AAV6-shRNA virus driven by the CD68 promoter. Given the extremely low expression level of TARM1 in normal lung tissue, the knockdown efficiency was tested after establishing an LPS-induced ARDS model. Western blot analysis showed that the AAV-TARM1#3 vector had the most significant knockdown effect. Figure 4 A). Immunofluorescence staining showed that compared with the AAVNC group, the TARM1 fluorescence signal in macrophages (CD68+) of the AAVTARM1 group was significantly weakened, and the co-localization coefficient was also reduced. Figure 4 B) confirmed the effectiveness of the macrophage-specific knockdown protocol.

[0075] 2.5 Specific knockdown of TARM1 in macrophages alleviates lung injury in ARDS mice.

[0076] like Figure 5The figures show: (A) Body weight change curve after LPS stimulation (n=6 mice per group). (B) Lung wet weight / dry weight ratio (n=6 mice per group). (C) H&E staining and pathological score of lung tissue (n=3 mice per group). Scale bar = 50 μm. (D) TUNEL fluorescence staining results of lung tissue sections (n=3 mice per group). Scale bar = 50 μm. (E) IL-1β and TNF-α levels in BALF (n=3 mice per group). (FG) Total protein concentration, total cell count, macrophage count, and neutrophil count in BALF (n=3 mice per group). All samples were tested in triplicate. Data are expressed as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 are all compared with the control group.

[0077] During the 48-hour observation period following LPS stimulation, ARDS model mice exhibited typical clinical symptoms (including prickle hair, lethargy, arched back posture, and rapid breathing), which were significantly improved by TARM1 gene knockdown. No deaths occurred in any of the experimental groups prior to euthanasia. ARDS mice gradually lost weight, reaching a minimum at 48 hours; TARM1 gene knockdown significantly reduced this weight loss. Figure 5 A). From a functional perspective, TARM1 knockdown significantly alleviated lung injury in ARDS mice: the lung wet weight / dry weight ratio decreased ( Figure 5 B), H&E staining of lung tissue showed improved alveolar structural damage and inflammatory cell infiltration. Figure 5 C); In addition, the levels of IL-1β and TNF-α in BALF were also significantly reduced ( Figure 5 E). Simultaneously, ARDS mice showed significantly elevated BALF total protein concentration, total cell count, macrophage count, and neutrophil count, while TARM1 knockdown significantly reduced these indicators (E). Figure 5 F, G). TUNEL staining of lung tissue showed that LPS stimulation increased the number of apoptotic cells, while TARM1 knockdown significantly reduced the apoptosis index (F, G). Figure 5 D).

[0078] By integrating multi-omics analysis and functional validation experiments, this invention demonstrates that TARM1 expression is significantly upregulated in the lung tissue of ARDS mice. Furthermore, data suggest that TARM1 may exacerbate lung injury by promoting macrophage differentiation into the pro-inflammatory M1 phenotype. These findings provide new mechanistic insights into the pathogenesis of ARDS and significantly expand our current understanding of the functional role of TARM1 in inflammatory diseases.

[0079] This invention employs a stepwise focusing screening strategy: First, three independent ARDS transcriptome datasets were compared, identifying 81 common DEGS; simultaneously, single-cell RNA sequencing (scRNA-seq) data were used to analyze DEGS between M1 and M2 macrophages; then, intersection analysis of these two gene sets was performed, ultimately identifying 15 candidate genes; finally, quantitative PCR (qPCR) confirmed TARM1 as the research target. This strategy effectively reduces the subjectivity of candidate gene screening and enhances the correlation between the identified genes and the pathogenesis of ARDS.

[0080] This invention reveals that TARM1 is upregulated in the lung tissue of ARDS mice and co-localizes with the macrophage marker CD68. Single-cell data show that TARM1 is highly expressed in both M1 macrophages and neutrophils, consistent with the pathological features of macrophage polarization and neutrophil infiltration in ARDS. To further investigate the function of TARM1 in macrophages, in vitro experiments were conducted in RAW264.7 cells. The results show that LPS stimulation induces upregulation of TARM1 expression; silencing TARM1 significantly reduces the expression level of the M1 macrophage marker iNOS and decreases the mRNA levels of IL1β and TNFα.

[0081] This invention extends the function of TARM1 to the field of ARDS for the first time and reveals its regulatory role in M1 macrophage polarization, providing a new perspective for understanding the mechanism of TARM1 in inflammatory diseases. Mechanistically, TARM1 is a receptor containing the ITAM domain, which can bind to the FcRγ linker chain to activate downstream signaling pathways.

[0082] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of TARM1 inhibitors in the preparation of drugs for treating acute respiratory distress syndrome, characterized in that, The TARM1 inhibitor is a short hairpin RNA that targets the TARM1 gene. The short hairpin RNA is carried by a recombinant adeno-associated virus serotype 6 vector. The recombinant adeno-associated virus vector contains a macrophage-specific CD68 promoter to drive the expression of the short hairpin RNA in alveolar macrophages.

2. The application according to claim 1, characterized in that, The recombinant adeno-associated virus vector also contains a reporter gene ZsGreen or an enhanced green fluorescent protein gene, wherein the reporter gene is linked to the short hairpin RNA via a self-cleaving peptide for monitoring macrophage transduction efficiency.

3. The application according to claim 1, characterized in that, The TARM1 inhibitor is a small interfering RNA that targets the TARM1 gene and is used to knock down TARM1 expression in an in vitro macrophage model. The small interfering RNA is transfected with liposome Lipofectamine 3000, and the macrophage confluence is 70% at the time of transfection. The knockdown effect is detected 48 hours after transfection.

4. The application according to claim 1, characterized in that, The in vitro macrophage model was the mouse RAW264.7 macrophage line, which was stimulated with 1 μg / mL lipopolysaccharide for 24 hours to induce M1 polarization in order to simulate the activation state of macrophages in acute respiratory distress syndrome.

5. The application according to claim 1, characterized in that, The anti-inflammatory effect of the drug is achieved by inhibiting macrophage M1 polarization, specifically manifested as follows: in lipopolysaccharide-stimulated RAW264.7 cells, knockdown of TARM1 leads to a decrease in the expression level of inducible nitric oxide synthase protein, as well as a decrease in the mRNA expression levels of interleukin-1β and tumor necrosis factor-α.

6. The application according to claim 1, characterized in that, The acute respiratory distress syndrome (ARDS) model in mice was induced by intratracheal instillation of lipopolysaccharide (LPS). The LPS dose was 5 mg / kg body weight, administered once intratracheally, and lung injury was assessed 48 hours after administration.

7. A pharmaceutical composition, characterized in that, The recombinant adeno-associated virus vector of claim 2, comprising a therapeutically effective amount, and a pharmaceutically acceptable nebulized carrier, said nebulized carrier being composed of one or more of mannitol, sucrose, and polysorbate 80, in combination with sterile saline or phosphate buffer, for local airway delivery.

8. The pharmaceutical composition according to claim 7, characterized in that, It also includes a second therapeutic agent selected from one or more of glucocorticoids, IL-6 receptor antagonists, NF-κB pathway inhibitors, pulmonary surfactant, or antibacterial drugs.

9. An in vitro method for screening candidate drugs for the treatment of acute respiratory distress syndrome, characterized in that, Includes the following steps: Step (1) Culturing mouse RAW264.7 macrophages and stimulating them with 1 μg / mL lipopolysaccharide for 24 hours to construct an M1 polarization model; Step (2) Add the test compound to the model and incubate; Step (3) The mRNA expression levels of TARM1, IL-1β, and TNF-α were detected by qRT-PCR, and the iNOS protein expression level was detected by Western blot, with β-actin as an internal reference. Step (4) If the test compound can downregulate the expression level of TARM1 mRNA compared with the model group, and at least one of iNOS, IL-1β, and TNF-α is simultaneously downregulated, then the compound is determined to be a candidate drug for the treatment of acute respiratory distress syndrome.

10. The use of TARM1 according to claim 9 in the preparation of a drug for treating acute respiratory distress syndrome, characterized in that, In step (3), qRT-PCR was normalized with the internal reference gene Gapdh; Western blot used β-actin as the loading control, and the primary antibody dilution ratios were TARM1 1:500, iNOS 1:1000, and β-actin 1:20000.

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