Use of a cxcl14 inhibitor in the manufacture of a medicament for preventing or treating abdominal aortic aneurysm in a patient

Abstract

The application belongs to the technical field of biological medicine, and relates to application of a CXCL14 inhibitor in preparation of a drug for preventing or treating abdominal aortic aneurysm of a patient. The inhibitor effectively eliminates the gender difference of abdominal aortic aneurysm by specifically blocking the combination of CXCL14 and its receptor CXCR4, significantly reduces aortic macrophage infiltration and PDGFRA + macrophage accumulation in the progress of abdominal aortic aneurysm, reduces the degree of collagen degradation and elastic fiber damage, and thus inhibits the pathological progress of abdominal aortic aneurysm. The application provides a new target and drug combination for precise treatment of male abdominal aortic aneurysm, and has clear clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of CXCL14 inhibitors in the preparation of drugs for the prevention or treatment of abdominal aortic aneurysms in patients. Background Technology

[0002] Abdominal aortic aneurysm (AAA) is a fatal vascular disease characterized by progressive dilation and weakening of the abdominal aortic wall. Rupture of AAA results in a mortality rate exceeding 80%, posing a significant public health problem. Clinical and epidemiological studies have clearly shown a significant sex difference in the incidence of abdominal aortic aneurysm, with a much higher incidence in men than women. However, the molecular mechanisms underlying this sex difference are not yet fully understood.

[0003] In existing technologies, estrogen is known to have a certain protective effect against abdominal aortic aneurysms in women. However, estrogen replacement therapy carries risks such as thrombosis and tumor development, and its clinical application lacks sufficient evidence to meet current clinical treatment needs. Furthermore, there is a lack of sex-specific targeted drugs for the treatment of abdominal aortic aneurysms, and existing interventions are insufficient to effectively inhibit the progression of male abdominal aortic aneurysms. In addition, although some studies have shown a correlation between macrophage infiltration and abdominal aortic aneurysms, the key molecular targets regulating macrophage-mediated sex differences in abdominal aortic aneurysms have not been identified, and no specific therapeutic drugs targeting these targets have been reported. There is an urgent need to explore new mechanisms of action and targeted treatment strategies. Summary of the Invention

[0004] This invention provides the application of CXCL14 inhibitors in the preparation of drugs for the prevention or treatment of abdominal aortic aneurysms in patients, in order to solve the problem that the existing technology of using estrogen to treat abdominal aortic aneurysms is difficult to effectively inhibit the progression of male abdominal aortic aneurysms.

[0005] The inventors discovered that CXCL14 neutralizing antibodies or CXCR4 inhibitors, such as AMD3100, can inhibit CXCL14-mediated macrophage recruitment and PDGFRA. + Macrophage accumulation can block the progression of abdominal aortic aneurysms, making it particularly suitable for the treatment of abdominal aortic aneurysms in male patients.

[0006] Based on the above findings, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides the use of CXCL14 inhibitors in the preparation of medicaments for the prevention or treatment of abdominal aortic aneurysms in patients.

[0008] In conjunction with the first aspect of the present invention, in some embodiments, the CXCL14 inhibitor is a substance that inhibits CXCL14 expression or inhibits CXCL14 function. Further, the CXCL14 inhibitor is a CXCL14 neutralizing antibody.

[0009] In conjunction with the first aspect of the invention, in some embodiments, the patient is a male patient.

[0010] In conjunction with the first aspect of the present invention, in some embodiments, the pathological manifestations of the abdominal aortic aneurysm include one or more of the following: aortic wall dilation, collagen degradation, elastic fiber destruction, and macrophage infiltration.

[0011] In conjunction with the first aspect of the invention, in some embodiments, the drug is used in at least one of the following applications:

[0012] (1) Reduce aortic macrophage infiltration during the progression of abdominal aortic aneurysm;

[0013] (2) Reduces collagen degradation and elastic fiber damage during the progression of abdominal aortic aneurysm;

[0014] (3) Reduce aortic dilation during the progression of abdominal aortic aneurysm;

[0015] (4) Eliminate gender differences in abdominal aortic aneurysm.

[0016] In conjunction with the first aspect of the present invention, in some embodiments, the drug is an injectable preparation.

[0017] Secondly, the present invention provides the use of a combination of CXCL14 inhibitors and CXCR4 inhibitors in the preparation of a medicament for the prevention or treatment of abdominal aortic aneurysms in patients.

[0018] In conjunction with the second aspect of the present invention, in some embodiments, the CXCL14 inhibitor is a CXCL14 neutralizing antibody; the CXCR4 inhibitor is AMD3100.

[0019] In conjunction with a second aspect of the invention, in some embodiments, the medicament comprises a pharmaceutically acceptable carrier and is formulated as an injection, tablet, or capsule.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] The abdominal aortic aneurysm treatment drug prepared using the CXCL14 inhibitor of this invention is particularly suitable for the treatment of abdominal aortic aneurysms in male patients, and solves the problem that the existing technology of using estrogen to treat abdominal aortic aneurysms is difficult to effectively inhibit the progression of male abdominal aortic aneurysms. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This study examines the sex-specific expression of CXCL14 and its receptor in human and mouse abdominal aortic aneurysm tissues provided in Example 1. In the figures, a represents transcriptome sequencing analysis of human abdominal aortic aneurysm tissue; b represents transcriptome sequencing analysis of mouse abdominal aortic aneurysm tissue; and c represents single-cell sequencing analysis of human abdominal aortic aneurysm tissue.

[0024] Figure 2 This is a diagram showing the difference in CXCL14 expression and cell origin in the male and female mouse abdominal aortic aneurysm models provided in Example 2. In the diagram, a represents the CXCL14 mRNA expression level in male and female mouse abdominal aortic aneurysm tissues; b represents the CXCL14 protein expression level; and c represents the primary origin of CXCL14 from CD45. - PDGFRA + Fibroblasts.

[0025] Figure 3 Example 3 shows the promoting effect of recombinant CXCL14 protein on the progression of abdominal aortic aneurysm; where a is the statistical analysis of the appearance and diameter of the aorta in mice of different treatment groups; b is the results of pathological staining and immunohistochemical staining of MMP-9 and IL-1β; and c is the results of flow cytometry.

[0026] Figure 4 It is the PDGFRA provided in Example 4 + Sex differences in macrophages and their role in promoting abdominal aortic aneurysm development; where a) lineage tracing shows sex differences in macrophages acquiring the PDGFRA phenotype; b and c) sequencing reveals PDGFRA... + Macrophages possess both pro-inflammatory and pro-fibrotic properties; de is for adoptive transfer of PDGFRA. + Macrophages exacerbate the progression of abdominal aortic aneurysms in male rats.

[0027] Figure 5 This is a verification of the CXCL14-CXCR4 axis mechanism provided in Example 5. Where 'a' represents the addition of PDGFRA to the recombinant CXCL14 protein. + Macrophage ratio; bc indicates Transwell assay confirms CXCR4-mediated macrophage migration; de indicates AMD3100 blocks the pro-abdominal aortic aneurysm effect of CXCL14.

[0028] Figure 6 This describes the therapeutic effect of the targeted intervention provided in Example 6. Specifically, ac represents the CXCL14 neutralizing antibody significantly reducing macrophage infiltration and shrinking aneurysm diameter; df represents the AMD3100 intervention producing a similar protective effect, eliminating gender differences. Detailed Implementation

[0029] Abdominal aortic aneurysm is a fatal vascular disease, accounting for approximately 95% of aortic aneurysms. Its core pathology involves chronic inflammatory infiltration of the abdominal aortic wall, imbalance in extracellular matrix (ECM) degradation, and dysfunction of vascular smooth muscle cells (VSMCs). Early onset is often insidious, and rupture can lead to extremely high mortality rates. The regulatory roles of chemokines and their receptors in vascular diseases have attracted considerable attention. Among them, CXC chemokine ligand 14 (CXCL14) and its receptor CXCR4 are involved in the development and progression of abdominal aortic aneurysms, providing potential targets for diagnosis and treatment. CXCL14 is a secreted protein of the CXC chemokine family, also known as BRAK, MIP-2γ, etc., and its function is tissue- and microenvironment-dependent; its specific receptor is not fully understood. CXCR4 belongs to the GPCR superfamily and is a high-affinity receptor for CXCL12 and a candidate receptor for CXCL14, participating in processes such as immune regulation, hematopoiesis, and embryonic development. It is widely expressed in immune cells, endothelial cells, and VSMCs, and its expression is upregulated under pathological conditions. It functions primarily through the CXCL12 / CXCR4 axis, mediating hematopoietic stem cell homing, regulating inflammatory cell infiltration and tumor progression. Simultaneously, it can mediate some functions of CXCL14, enhancing monocyte / macrophage activation and participating in inflammation regulation. Based on these two regulatory roles, it shows great promise in the diagnosis and treatment of abdominal aortic aneurysms.

[0030] This invention is the first to discover that CXCL14 is a core molecular target mediating sex differences in abdominal aortic aneurysms, with significantly higher expression in aortic tissues of male patients and male animal models compared to females. CXCL14, through specific binding to its receptor CXCR4, recruits macrophages and directs them towards PDGFRA. + Phenotypic transformation of this cell subset, exhibiting both pro-inflammatory and pro-fibrotic properties, accelerates collagen degradation, elastic fiber destruction, and smooth muscle cell loss in the aortic wall, thereby promoting the progression of abdominal aortic aneurysms. In vivo drug intervention experiments in mice have demonstrated that CXCL14 neutralizing antibodies or CXCR4 inhibitors can reverse this progression, providing a novel targeted therapeutic strategy for the prevention and treatment of sex-specific abdominal aortic aneurysms, avoiding the potential risks of estrogen replacement therapy, and increasing treatment safety.

[0031] This invention provides the use of CXCL14 inhibitors in the preparation of medicaments for the prevention or treatment of abdominal aortic aneurysms.

[0032] In some embodiments, the CXCL14 inhibitor is a substance that inhibits CXCL14 expression or function. Further, the sex differences in the abdominal aortic aneurysm include: significantly higher CXCL14 expression levels in male abdominal aortic aneurysm tissue than in females. The CXCL14 inhibitor is a CXCL14 neutralizing antibody. In Example 6, direct use of a CXCL14 neutralizing antibody significantly reduced macrophage infiltration in the aortic wall of model mice. Figure 6 ab, 6d-e), and effectively reduced the diameter of the aneurysm ( Figure 6 c and 6f) demonstrate the effectiveness of CXCL14 neutralizing antibody therapy for abdominal aortic aneurysm.

[0033] In some embodiments, the patient is a male patient. As shown in Example 1, common sequencing data from humans and mice indicate sex differences in CXCL14 expression, and this difference is conserved across different species. Figure 1 The animal model in Example 2 directly confirmed that the expression level of CXCL14 in the abdominal aortic aneurysm tissue of male mice was significantly higher than that of female mice by detecting mRNA and protein levels. Figure 2 Example 3 demonstrates the promoting effect of CXCL14 in the pathological process of abdominal aortic aneurysm. Based on the gender difference of CXCL14 in patients with abdominal aortic aneurysm, the CXCL14 neutralizing antibody provided by this invention is particularly suitable for the prevention or treatment of male patients.

[0034] In some embodiments, the pathological features of the abdominal aortic aneurysm include one or more of the following: aortic wall dilation, collagen degradation, elastic fiber destruction, and macrophage infiltration. These pathological changes persist and gradually worsen during the progression of the abdominal aortic aneurysm: (The following text appears to be a separate, unrelated section: "mouse aortic appearance and diameter measurements after PPE-induced modeling (...") Figure 3 a) The abdominal aortic aneurysm model group showed aneurysm-like dilation of blood vessels; histopathological staining results ( Figure 3 b) A decrease in the area positive for Sirius Red staining indicates collagen degradation, while an increased EVG staining score indicates more severe damage to elastic fibers; such as Figure 3 Flow cytometry results showed that the abdominal aortic aneurysm model group had CD45 + CD11b + F4 / 80 + The proportion of macrophages increased significantly; Example 4 further confirmed the adoption of PDGFRA. + Macrophages can exacerbate collagen degradation and elastic fiber damage; the results of the above examples indicate that the pathological manifestations of abdominal aortic aneurysm include one or more of the following: aortic wall dilation, collagen degradation, elastic fiber damage, and macrophage infiltration.

[0035] In some embodiments, the drug is used in at least one of the following applications:

[0036] (1) Reduce aortic macrophage infiltration during the progression of abdominal aortic aneurysm:

[0037] like Figure 3 Flow cytometry results showed that treatment with recombinant CXCL14 protein significantly increased CD45 levels in the aorta of male mice with abdominal aortic aneurysms. + CD11b + F4 / 80 + Macrophage ratio; and such Figure 6 a and Figure 6 As shown in d, after intervention with CXCL14 neutralizing antibody or AMD3100, the proportion of aortic macrophages in male abdominal aortic aneurysm mice was significantly reduced, indicating that the drug can be used to reduce macrophage infiltration during the progression of abdominal aortic aneurysm.

[0038] (2) Reduces collagen degradation and elastic fiber damage during the progression of abdominal aortic aneurysm:

[0039] like Figure 3 Histopathological staining results showed that treatment with recombinant CXCL14 protein reduced collagen content as shown by Sirius Red staining and aggravated elastic fiber damage as shown by EVG staining; however, targeted intervention on the CXCL14-CXCR4 axis could alleviate these pathological changes, indicating that the drug can be used to reduce collagen degradation and elastic fiber damage during the progression of abdominal aortic aneurysms.

[0040] (3) Reduce aortic dilation during the progression of abdominal aortic aneurysm:

[0041] like Figure 3 As shown in a, treatment with recombinant CXCL14 protein significantly increased the aortic diameter in male mice with abdominal aortic aneurysms; treatment with the CXCL4 inhibitor AMD3100 completely blocked the increase in aortic diameter induced by recombinant CXCL14 protein. Figure 5 e); Figure 6 c and Figure 6 Further evidence showed that treatment with CXCL14 neutralizing antibody or AMD3100 significantly reduced the aortic diameter in male mice with abdominal aortic aneurysms, indicating that the drug could directly alleviate aortic dilation during the progression of abdominal aortic aneurysms.

[0042] (4) Eliminate gender differences in abdominal aortic aneurysms:

[0043] like Figure 6 c and Figure 6 As shown in f, after treatment with CXCL14 neutralizing antibody or AMD3100, the aneurysm diameter in male abdominal aortic aneurysm mice was significantly reduced, with no statistically significant difference compared to female mice. Figure 6 ab and Figure 6 de shows macrophages and PDGFRA + The sex difference in macrophage infiltration was also eliminated, indicating that the drug can be used to eliminate the sex difference in abdominal aortic aneurysms.

[0044] In some embodiments, the drug is an injectable preparation. In Example 6, the administration methods to mice were: 1) intraperitoneal injection, with a dose of AMD3100 injection solution of 240 μg / mouse, and the solvent being physiological saline containing 20% ​​sulfobutyl ether-β-cyclodextrin; 2) intravenous injection, with a dose of CXCL14 neutralizing antibody injection solution of 100 ng / mouse, and the solvent being physiological saline.

[0045] This invention provides the use of a combination of CXCL14 inhibitors and CXCR4 inhibitors in the preparation of a medicament for the prevention or treatment of abdominal aortic aneurysms in patients.

[0046] In some embodiments, the CXCL14 inhibitor is a CXCL14 neutralizing antibody; the CXCR4 inhibitor is AMD3100. Final treatment experiments showed that direct use of either the CXCL14 neutralizing antibody or AMD3100 significantly reduced macrophage infiltration in the aortic wall of male model rats. Figure 6 ab, 6d-e), and effectively reduced the diameter of the aneurysm ( Figure 6 (c and 6f), thus demonstrating that the combination of CXCL14 inhibitors and CXCR4 inhibitors can be used for the prevention or treatment of abdominal aortic aneurysms.

[0047] In some embodiments, the drug comprises a pharmaceutically acceptable carrier and is formulated as an injection, tablet, or capsule. In Example 6, the mice were administered the drug via: 1) intraperitoneal injection at a dose of 240 μg / mouse of AMD3100 injection solution in physiological saline containing 20% ​​sulfobutyl ether-β-cyclodextrin; 2) intravenous injection at a dose of 100 ng / mouse of CXCL14 neutralizing antibody injection solution in physiological saline.

[0048] Unless otherwise specified, the experimental procedures described in the following examples are all conventional techniques in the art, including but not limited to cell culture, tissue culture, total protein extraction, protein sample preparation, molecular biology detection (such as qPCR, Western blot, immunohistochemistry), cell biology techniques (such as flow cytometry, cell migration assays), H&E staining, Sirius Red staining, Victoria Blue (EVG) staining, animal model construction, data collection, bioinformatics analysis, and statistical methods. All experimental reagents used were purchased commercially and met the generally accepted quality standards in the art. Experimental animals were purchased commercially and then fed and modeled according to standard operating procedures in the art. The implementation methods of the above-mentioned conventional techniques are common knowledge to those skilled in the art, and specific operations can be referred to relevant authoritative literature. Experimental groups and control groups were set up according to the grouping methods commonly used in the art.

[0049] The C57BL / 6 mice and LysM-Cre / Rosa26-tdTomato transgenic mice used in the following examples were obtained from Beijing Vital River Laboratory Animal Co., Ltd., and were housed in an SPF-grade environment. All animal experiments have been approved by the Ethics Committee of the Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Approval No.:

[2022] 3779).

[0050] In this invention, all data collection and processing in the statistical analysis section were performed using a blinded method, and the quantitative data are expressed as mean ± standard deviation. All data were analyzed using GraphPad Prism software. A p-value < 0.05 was considered statistically significant.

[0051] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0052] Example 1: Verification of sex differences in CXCL14 and its receptor expression in human and mouse abdominal aortic aneurysm tissues

[0053] 1. Purpose

[0054] This embodiment aims to verify, using sequencing data from public databases, whether there are sex differences in the expression of CXCL14 and its receptors (CXCR4 and CCR1) in human and mouse abdominal aortic aneurysm tissues, providing preclinical evidence for CXCL14 as a sex-specific target.

[0055] 2. Experimental Materials

[0056] Data source:

[0057] 1) Transcriptome sequencing data of human abdominal aortic aneurysm tissue: GEO database GSE183464 dataset;

[0058] 2) Transcriptome data of mouse abdominal aortic aneurysm tissue: GEO database samples GSM5929409-5929412 and 5929418-5929424;

[0059] 3) Human abdominal aortic aneurysm tissue single-cell sequencing data: GEO database GSE166676 dataset.

[0060] Data analysis tools: GEO2R online analysis platform (genetic sex difference analysis), Seurat toolkit (single-cell data analysis), GraphPad Prism 8.0.2 (plotting and statistics).

[0061] 3. Experimental Methods

[0062] 3.1 Analysis of Human Transcriptome Data

[0063] Gene expression matrices were extracted from male and female patients with abdominal aortic aneurysms from the GSE183464 dataset. The expression levels of the CXCL14 gene were compared using GEO2R, ​​with log2 Fold Change (FC) and corrected P-value (adj. P<0.05) as significance criteria.

[0064] 3.2 Analysis of mouse transcriptome data

[0065] Differentially expressed genes were extracted from the mouse dataset (GSM5929409-5929412 were male mice, and 5929418-5929424 were female mice), the expression level of CXCL14 was calculated using GEO2R, ​​and a heatmap was generated using GraphPad Prism.

[0066] 3.3 Human Single-Cell Sequencing Analysis

[0067] The GSE166676 data were preprocessed using the Seurat toolkit (standardization, dimensionality reduction, clustering) to extract macrophage subsets and compare the expression levels of CXCR4 and CCR1 in males and females.

[0068] 3.4 Statistical Methods

[0069] Differences between genders were assessed using t-tests or Mann-Whitney U tests. Data are expressed as mean ± standard deviation, and P < 0.05 was considered significant.

[0070] 4. Experimental Results

[0071] Transcriptome analysis showed that the mRNA expression level of CXCL14 was significantly higher in male patients with abdominal aortic aneurysms than in female patients, confirming a gender difference in CXCL14 expression. Figure 1 a); CXCL14 expression in male mice was significantly higher than in female mice ( Figure 1 (b) Consistent with trends in human data, this supports interspecies conservation. Single-cell sequencing revealed significantly higher expression levels of CXCR4 and CCR1 in macrophages of male patients with abdominal aortic aneurysms compared to females, suggesting that the CXCL14-CXCR4 / CCR1 axis may drive sex differences.

[0072] 5. Conclusion

[0073] This embodiment demonstrates through multi-omics data that CXCL14 and its receptor show a high expression trend in males / male mice in both human and mouse abdominal aortic aneurysm tissues, providing key preclinical evidence for CXCL14 as a sex-specific diagnostic biomarker and therapeutic target.

[0074] Example 2: Differences in CXCL14 expression and verification of cell origin in male and female mouse abdominal aortic aneurysm models

[0075] 1. Purpose

[0076] To verify the differential expression of CXCL14 in male and female mouse abdominal aortic aneurysm models, clarify its cellular origin in aortic tissue, and provide experimental evidence for CXCL14 as a sex-differentiated target.

[0077] 2. Experimental Materials

[0078] Experimental animal grouping: 52 male C57BL / 6 mice weighing 20-22g and 8 weeks old and 40 female mice were divided into 8 groups after 1 week of acclimatization feeding (male and female mice were sampled on days 0, 3, 7 and 10 respectively).

[0079] Main reagents: porcine pancreatic elastase (PPE, Sigma-Aldrich, E1250), RNAisoPlus reagent (Takara, 9109), RIPA lysis buffer (containing protease inhibitor, Roche, 04693159001), CXCL14 primary antibody (Abcam, ab189119), PrimeScript RT kit (Takara, RR037A), SYBR Premix Ex Taq II (Takara, RR820A), BCA protein quantification kit (Thermo). Fisher, 23225), ECL chemiluminescence kit (ThermoFisher, 32106), collagenase IV (2 mg / mL, Sigma, C5138), hyaluronidase (1 mg / mL, Sigma, H3506), BV510-FVD (BioLegend, 423102), APC / CY7-anti-CD45 (BioLegend, 103116), PE-Cy7-anti-PDGFRA (BioLegend, 323508), APC-anti-αSMA (Abcam, ab225143), PE-anti-CD31 (BioLegend, 160203).

[0080] Main instruments: ABI 7500 real-time quantitative PCR instrument, ChemiDoc XRS+ imaging system (Bio-Rad), BDFACSAria III flow cytometer (BD Biosciences).

[0081] 3. Experimental Methods

[0082] 3.1 Construction of an abdominal aortic aneurysm model

[0083] The PPE (porcine pancreatic elastase) induction method was used: mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), the abdominal aorta below the renal artery was separated, cotton pads soaked in PPE solution were placed on the surface of the aorta, and after incubation for 40 minutes, the incision was rinsed with sterile saline and the incision was sutured layer by layer.

[0084] 3.2 Sample Collection and Processing

[0085] Mice were euthanized by cervical dislocation at 0, 3, 7, and 10 days after modeling. Abdominal aortic tissue was collected and divided into two parts: one part was immediately placed in RNAiso Plus reagent for cryopreservation for mRNA extraction, and the other part was placed in RIPA lysis buffer containing protease inhibitors for protein extraction.

[0086] 3.3 qPCR detection of CXCL14 mRNA expression

[0087] Total RNA was extracted using the Trizol method, and RNA purity was determined using Nanodrop D-1000. cDNA was reverse transcribed according to the PrimeScript RT kit instructions and stored at 4°C.

[0088] qPCR primers: CXCL14 upstream 5'-AGTGTAAGTGTTCCCGGAAGG-3' (SEQ ID NO.1), downstream 5'-GCAGTGTGGGTACTTTGGCTT-3' (SEQ ID NO.2), internal control GAPDH, using 2 -△△Ct The relative expression level is calculated using this method.

[0089] 3.4 Western Blot detection of CXCL14 protein expression

[0090] After homogenization, the tissue was centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The protein concentration was determined using a BCA protein quantification kit. 40 μg of protein sample was subjected to 12% SDS-PAGE electrophoresis and transferred to a 0.45 μm PVDF membrane (Millipore, IPVH00010) at a constant current of 200 mA for 90 minutes. The membrane was blocked with 5% skim milk at room temperature for 1.5 hours, then CXCL14 primary antibody (diluted 1:800) was added, and the membrane was incubated overnight at 4°C. The membrane was washed three times with TBST (10 minutes each time). HRP-labeled goat anti-rabbit secondary antibody (CellSignaling Technology, 7074) was added at a dilution of 1:5000, and the membrane was incubated at room temperature for 1 hour. The membrane was washed three times with TBST. The membrane was developed using an ECL chemiluminescence kit, and images were taken using a ChemiDoc XRS+ imaging system (Bio-Rad). The grayscale values ​​of the bands were quantified using ImageJ 1.53e software. The results were analyzed using GAPDH (Cell Signaling) assay. Technology, 5174) is used to calculate the relative expression level of internal references.

[0091] 3.5 Identification of Cell Origin

[0092] Flow cytometry sorting of aortic tissue single-cell suspension: Tissue was cut into 1 mm³ pieces, added to DMEM medium containing collagenase IV and hyaluronidase, and digested at 37°C with shaking for 45 minutes. Single-cell suspension was obtained by passing through a 70 μm filter, and fluorescently labeled antibodies BV510-FVD, APC / CY7-anti-CD45, PE-Cy7-anti-PDGFRA, APC-anti-αSMA, and PE-anti-CD31 were added. The mixture was incubated at 4°C for 30 minutes, and CD45 cells were sorted using a BD FACSAria III flow cytometer. - Non-immune cells and CD45 + Immune cells. CD45 - The cells were further sorted into fibroblasts (CD45). - PDGFRA + ), smooth muscle cells (CD45) - α-SMA + ), endothelial cells (CD45) - CD31 + ) and other CD45 - Cells; CD45 + Cells were sorted into macrophages (CD45) + CD11b + F4 / 80 + RNA was extracted and qPCR was performed on each sample.

[0093] 4. Experimental Results

[0094] 4.1 Sex differences in CXCL14 mRNA expression

[0095] After modeling, the CXCL14 mRNA level in the abdominal aortic aneurysm tissue of male mice was significantly higher than that of female mice (days 3, 7, and 10, p < 0.0001). Over time, CXCL14 expression in male mice significantly increased on day 3, remained at a high level on day 7, and fluctuated slightly on day 10 but remained at a high level overall. In contrast, the CXCL14 expression level in female mice remained at a low level at all time points, with no significant changes. Figure 2 a)

[0096] 4.2 Sex differences in CXCL14 protein expression

[0097] Western blot analysis showed that starting on the third day after modeling, the expression level of CXCL14 protein in male mice was significantly increased compared to that in female mice, with the most significant difference observed on the seventh day after modeling. Figure 2 b)

[0098] 4.3 Cellular origin localization of CXCL14

[0099] Flow sorting ( Figure 2 c) Shows that CXCL14 is primarily expressed on CD45. - PDGFRA + Fibroblasts showed higher expression levels than other CD45 cells. - Cells (such as smooth muscle cells and endothelial cells), and male mice are CD45 - CXCL14 expression in cells was significantly higher than in female mice (p<0.0008).

[0100] 5. Conclusion

[0101] This embodiment confirms that CXCL14 is expressed differently by sex in an abdominal aortic aneurysm model, with male mice showing significantly higher expression levels than female mice. Furthermore, CXCL14 is mainly derived from aortic fibroblasts, which provides important experimental evidence for understanding the cellular and molecular mechanisms underlying sex differences in abdominal aortic aneurysms.

[0102] Example 3: Effects of recombinant CXCL14 protein on abdominal aortic aneurysm progression and macrophage infiltration

[0103] 1. Purpose

[0104] To verify the promoting effect of exogenous recombinant CXCL14 protein on the progression of abdominal aortic aneurysm and to clarify its influence on macrophage infiltration and vascular pathological changes, so as to provide functional evidence for CXCL14 as a therapeutic target.

[0105] 2. Experimental Materials

[0106] Experimental animals: 20 male C57BL / 6 mice, 8 weeks old, weighing 20-22g;

[0107] Main reagents: recombinant mouse CXCL14 protein (MCE, catalog number HY-P71887A), Sirius Red staining reagent (Sigma, 365548), EVG staining kit (Sigma, HT25A), MMP-9 antibody (Thermo Fisher Scientific, MA5-15886), IL-1β antibody (Thermo Fisher Scientific, P420B).

[0108] 3. Experimental Methods

[0109] 3.1 Construction of the abdominal aortic aneurysm model:

[0110] An abdominal aortic aneurysm model was established using the PPE induction method, the same as in Example 2.

[0111] 3.2 Drug intervention and experimental animal grouping:

[0112] Experimental group: Recombinant CXCL14 protein (50 μg / kg) was injected intraperitoneally once a day for 10 consecutive days;

[0113] Control group: Administered an equal volume of isotype control antibody.

[0114] 3.3 Aneurysm diameter measurement:

[0115] Ten days after modeling, the aorta was harvested and the maximum diameter of the abdominal aorta was measured 2 mm below the renal artery. Each sample was measured three times and the average value was taken.

[0116] 3.4 Histopathology and detection of pro-inflammatory factors:

[0117] Sirius Red staining: Paraffin sections were dewaxed to water, stained with 0.1% Sirius Red solution for 45 minutes, and the percentage of collagen-positive area was quantified;

[0118] EVG staining: Stain with Verhoeff stain for 8 minutes, counterstain with eosin after differentiation, and score the elastic fiber breakage (0-3 points).

[0119] Immunohistochemistry: Detection of αSMA (1:400), MMP-9 (1:400) and IL-1β (1:300), and quantification of the percentage of positive cells.

[0120] 3.5 Macrophage infiltration detection:

[0121] A single-cell suspension was prepared from aortic tissue, and BV510-FVD, APC / CY7-anti-CD45, and BV421-anti-F4 / 80 were added. The suspension was incubated at 4°C for 30 minutes. CD45 levels were detected by flow cytometry. + CD11b + F4 / 80 + The proportion of macrophages in the total number of cells.

[0122] 4. Experimental Results

[0123] 4.1 Recombinant CXCL14 significantly increased aneurysm diameter:

[0124] The diameter of aneurysms in the CXCL14 treatment group was significantly increased compared to the control group, and the aorta showed more pronounced vascular dilation in the CXCL14 group. Figure 3 a)

[0125] 4.2 Worsening of vascular pathological damage:

[0126] Collagen content: Sirius Red staining showed that the collagen-positive area in the CXCL14 treatment group was significantly reduced compared with the control group;

[0127] Elastic fibers: EVG staining scores showed that the degree of elastic fiber breakage in the CXCL14 treatment group was significantly more severe than that in the control group;

[0128] Smooth muscle cell damage: αSMA staining showed that, compared with the control group, the smooth muscle cells in the vascular media of the CXCL14 treatment group were disordered and reduced in number;

[0129] Pro-inflammatory factors: Compared with the control group, the proportion of positive cells for pro-inflammatory factors MMP-9 and IL-1β was significantly increased in the CXCL14 treatment group.

[0130] 4.3 Macrophage infiltration significantly increased:

[0131] Flow cytometry analysis shows that the CXCL14 processing group CD45 + CD11b + F4 / 80 + The proportion of macrophages was higher than that in the control group. Figure 3 c) indicates increased macrophage infiltration.

[0132] 5. Conclusion

[0133] Exogenous recombinant CXCL14 protein significantly promoted the progression of abdominal aortic aneurysms, manifested as increased aneurysm diameter, collagen degradation in the vessel wall, aggravated elastic fiber damage, upregulation of pro-inflammatory factor expression, and increased macrophage infiltration. This result confirms the promoting role of CXCL14 in the pathological process of abdominal aortic aneurysms, providing direct functional evidence for its potential as a therapeutic target.

[0134] Example 4: PDGFRA + Sex differences in macrophages and their role in the progression of abdominal aortic aneurysms

[0135] 1. Purpose

[0136] Verification of the sex difference in PDGFRA phenotype acquired by macrophages in abdominal aortic aneurysms, clarifying the role of PDGFRA. + The pro-inflammatory and pro-fibrotic properties of macrophages and the impact of adoptive transfer on the progression of abdominal aortic aneurysms.

[0137] 2. Experimental Materials

[0138] Experimental animals: 8-week-old male and female LysM-Cre / Rosa26-tdTomato transgenic mice, and 8-week-old male C57BL / 6 mice;

[0139] Main reagents: PE-Cy7-anti-PDGFRA antibody (BioLegend, 323508), RNeasy Mini kit (Qiagen, 74104), TruSeq chain-specific mRNA library preparation kit (Illumina).

[0140] Main instruments: Agilent 2100 Bioanalyzer, Illumina NovaSeq 6000 sequencing platform.

[0141] 3. Experimental Methods

[0142] 3.1 Grouping of experimental animals:

[0143] Lineage Tracing Group: 28-35 male and female LysM-Cre / Rosa26-tdTomato transgenic mice (macrophages specifically express tdTomato red fluorescence) were selected at 8 weeks of age to construct an abdominal aortic aneurysm model. Samples were collected at 0, 3, 7, and 10 days.

[0144] Adoptive transfer group: 21 eight-week-old male C57BL / 6 mice were randomly divided into a control group and a PDGFRA group. + Macrophage transfer group, constructing an abdominal aortic aneurysm model, and collecting samples on day 7.

[0145] 3.2 Lineage Tracing Experiment:

[0146] Days 0, 3, 7, and 10 after modeling, aortic tissue from transgenic mice was isolated, single-cell suspensions were prepared, and PE-Cy7-anti-PDGFRA was added and incubated at 4°C for 30 minutes; tdTomato was detected by flow cytometry.+ (Macrophage-derived) PDGFRA + The proportion of cells was compared between male and female mice.

[0147] 3.3 Cell sorting and RNA sequencing:

[0148] CD45 was isolated from the aortic tissue of male C57BL / 6 mice 7 days after abdominal aortic aneurysm modeling. + CD11b + F4 / 80 + PDGFRA - Macrophages, PDGFRA + Macrophages and CD45 - PDGFRA + Fibroblasts. Total RNA was extracted using the RNeasy Mini kit. RNA quality was assessed using an Agilent 2100 bioanalyzer, requiring an RNA integrity index (RIN) ≥ 7.0. RNA sequencing (RNA-seq) libraries were constructed using the TruSeq chain-specific mRNA library preparation kit and sequenced on an Illumina NovaSeq 6000 sequencing platform, with a sequencing depth ≥ 6 Gb for each sample. The raw sequencing data was filtered to remove low-quality reads and adapter sequences; clean reads were aligned to the mouse reference genome (mm10) using HISAT2 software, and gene expression levels were quantified using HTSeq-count software; differentially expressed genes (DEGs) were screened using DESeq2 software, with the screening criteria being |log2FC| ≥ 1 and a corrected P-value < 0.05.

[0149] 3.4 Adoptive Transfer Experiment:

[0150] CD45 was isolated from the aortic tissue of male C57BL / 6 mice 7 days after abdominal aortic aneurysm modeling. + CD11b + F4 / 80 + PDGFRA + and PDGFRA - Macrophages; On day 7 after adoptive transfer to mice, 1×10⁻⁶ macrophages were injected into the adventitia of the aorta. 6 One corresponding macrophage was injected, and the control group was injected with an equal volume of PBS.

[0151] 3.5 Histopathological examination:

[0152] Ten days after transfer, the aortic diameter was measured, and histopathological examination and pro-inflammatory factor detection were performed (same as in Example 3).

[0153] 4. Experimental Results

[0154] 4.1 PDGFRA+ Sex differences in macrophage transformation

[0155] After modeling, starting from day three, PDGFRA macrophages in the aortic tissue of transgenic mice... + The proportion of cells begins to increase, peaks on day seven, and then begins to decline. PDGFRA in male mice... + Cells in tdTomato + The proportion of cells and fluorescence intensity were significantly higher in female mice than in female mice. Figure 4 a).

[0156] 4.2 PDGFRA + Unique characteristics of macrophages:

[0157] RNA sequencing and differential expression analysis Figure 4 bc) indicates that, with PDGFRA - Compared to macrophages and fibroblasts, PDGFRA + The gene expression profiles of macrophages showed significant differences, characterized by high expression of pro-inflammatory and pro-fibrotic genes (high expression of MMP-9 compared to fibroblasts), suggesting the presence of PDGFRA. + Macrophages possess both pro-inflammatory and pro-fibrotic properties in the progression of abdominal aortic aneurysms.

[0158] 4.3 Adoptive transfer promotes the progression of abdominal aortic aneurysm:

[0159] Compared with the control group injected with an equal volume of PBS, PDGFRA + The aneurysm diameter was significantly increased in the macrophage transfer group. Figure 4 d); Sirius Red staining showed a significant reduction in collagen-positive area; EVG staining score showed a significant increase in the degree of elastic fiber breakage; αSMA staining showed disordered smooth muscle cell arrangement and reduced number; the proportion of cells positive for pro-inflammatory factors MMP-9 and IL-1β was significantly increased. Figure 4 e).

[0160] 5. Conclusion

[0161] This embodiment confirms that macrophages convert to PDGFRA. + Phenotypic transformation showed significant gender differences, and PDGFRA + Macrophages promote the progression of abdominal aortic aneurysms by possessing both pro-inflammatory and pro-fibrotic properties. This discovery provides a new perspective on understanding the cellular mechanisms underlying sex differences in abdominal aortic aneurysms and offers insights for targeting PDGFRA. + The treatment strategy using macrophages provides a theoretical basis.

[0162] Example 5: CXCL14-CXCR4 axis mediates macrophage and PDGFRA +Mechanism verification of macrophage infiltration

[0163] 1. Purpose

[0164] Verify that CXCL14 mediates macrophage and PDGFRA through the CXCR4 receptor. + The molecular mechanism of macrophage migration was investigated, and the blocking effect of the CXCR4 inhibitor AMD3100 was clarified.

[0165] 2. Experimental Materials

[0166] 2.1 In vitro experiments

[0167] Cell source: Bone marrow-derived macrophages (BMDM) from 6-8 week old C57BL / 6 mice;

[0168] Main reagents: CXCL14 recombinant protein (0.1 μg / mL), CXCR4 inhibitor AMD3100 (10 μM), CCR1 inhibitor BX471 (1 μM).

[0169] 2.2 In vivo experiments

[0170] Experimental animals: 44 male C57BL / 6 mice, 8 weeks old, weighing 20-22g;

[0171] Main reagents: CXCL14 recombinant protein, CXCR4 inhibitor AMD3100.

[0172] 3. Experimental Methods

[0173] 3.1 Transwell transfer experiment

[0174] Add 5×10 to the upper chamber 4 One BMDM was prepared, and culture medium containing CXCL14 (0.1 μg / mL) was added to the lower chamber. CXCL14 alone, CXCL14 + AMD3100 (10 μM), CXCL14 + BX471 (1 μM), CXCL14 + AMD3100 + BX471, and a blank control group were also prepared. After 24 hours of culture, the cells in the lower chamber were fixed, stained with crystal violet, and the number of migrating cells was counted by flow cytometry.

[0175] 3.2 In vivo intervention experiment

[0176] Mice with abdominal aortic aneurysm were intraperitoneally injected with the CXCR4 inhibitor (AMD3100) at a dose of 240 μg per mouse, once daily for 10 consecutive days. The control group received an equal volume of phosphate-buffered saline (PBS) via the same route. When used in combination with recombinant CXCL14 protein, the administration method and dosage were the same, with a 12-hour interval between the two drugs. Ten days after administration, aneurysm diameter was measured, and macrophages and PDGFRA were detected by flow cytometry.+ Macrophage ratio.

[0177] 4. Experimental Results

[0178] like Figure 5 As shown in ab, treatment with CXCL14 recombinant protein significantly increased the proportion of aortic macrophages in male mice with abdominal aortic aneurysms; crystal violet staining and flow cytometry results both showed that the addition of AMD3100 completely blocked the migration effect, while the addition of BX471 had no significant effect. Figure 5 (bc) indicates that CXCL14 mediates macrophage migration through CXCR4.

[0179] In vivo intervention experiments showed that AMD3100 significantly inhibited CXCL14-induced PDGFRA. + Macrophage infiltration ( Figure 5 (a and 5d), the aneurysm diameter increase effect was completely reversed (5e); after AMD3100 treatment, recombinant CXCL14 protein did not increase aortic macrophages and PDGFRA in male abdominal aortic aneurysm mice. + The loss of macrophage ratio and aortic diameter in male mice with abdominal aortic aneurysms, indicating the disappearance of the aortic progression-promoting properties of recombinant CXCL14 protein, further confirms that CXCL14 mediates macrophage and PDGFRA through the CXCL14-CXCR4 axis. + Macrophage infiltration promotes the progression of abdominal aortic aneurysm.

[0180] 5. Conclusion

[0181] CXCL14 specifically mediates macrophage and PDGFRA through the CXCR4 receptor. + The CCR1 pathway is not involved in macrophage migration. The CXCR4 inhibitor AMD3100 can effectively block the pathological effects of CXCL14, providing a precise molecular target for targeted intervention.

[0182] Example 6: Therapeutic effects of CXCL14 neutralizing antibody and AMD3100 on abdominal aortic aneurysm and elimination of gender differences.

[0183] 1. Purpose

[0184] To verify the therapeutic effects of CXCL14 neutralizing antibody and CXCR4 inhibitor AMD3100 on abdominal aortic aneurysm, and to evaluate their inhibitory effect on macrophage infiltration and their ability to eliminate gender differences.

[0185] 2. Experimental Materials

[0186] Experimental animals: 38 male and 38 female C57BL / 6 mice, each 8 weeks old, weighing 20-22g;

[0187] Main reagents: Anti-mouse CXCL14 neutralizing antibody (Abcam, ab264467), CXCR4 inhibitor AMD3100.

[0188] 3. Experimental Methods

[0189] Drug intervention: In the CXCL14 neutralization intervention experiment, mice with abdominal aortic aneurysms were intravenously injected with 100 ng of anti-mouse CXCL14 neutralizing antibody (Abcam, ab264467) daily for 10 consecutive days; the control group mice were given an equal volume of isotype control antibody via the same route, and the AMD3100 intervention method was the same as in Example 5.

[0190] Group details:

[0191] CXCL14 neutralizing antibody group: 100ng antibody was injected intravenously daily for 10 consecutive days;

[0192] AMD3100 group: 240 μg intraperitoneally daily for 10 consecutive days;

[0193] Control group: Administered an equal volume of isotype control antibody or PBS for 10 consecutive days.

[0194] Flow cytometry analysis: PDGFRA + The changes in the proportion of macrophage subsets are the same as in Example 5.

[0195] Aneurysm diameter measurement: Same as in Example 5.

[0196] 4. Experimental Results

[0197] Flow cytometry results showed that after treatment with CXCL14 neutralizing antibody and AMD3100, male mice showed increased levels of aortic macrophages and PDGFRA. + The proportion of macrophages was significantly reduced, and there was no significant difference compared with female mice. Figure 6 ab and Figure 6 de); Statistical analysis of aortic diameter in each group of mice showed that CXCL14 neutralizing antibody and AMD3100 reduced aortic diameter, and there was no significant difference compared with female mice (de); Figure 6 c and Figure 6 f).

[0198] 5. Conclusion

[0199] Both the CXCL14 neutralizing antibody and AMD3100 can effectively inhibit macrophage infiltration, reduce aneurysm diameter, and eliminate gender differences, providing experimental evidence for the clinical development of gender-specific abdominal aortic aneurysm treatment regimens.

[0200] The results above demonstrate that CXCL14 is a key molecule mediating sex differences in abdominal aortic aneurysms. Targeting this pathway can significantly inhibit the progression of abdominal aortic aneurysms and eliminate sex differences, providing a novel solution for the diagnosis and treatment of sex-specific vascular diseases.

[0201] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0202] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0203] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. The use of CXCL14 inhibitors in the preparation of drugs for treating abdominal aortic aneurysms, characterized in that, The CXCL14 inhibitor is a CXCL14 neutralizing antibody; the CXCL14 inhibitor inhibits CXCL14-mediated macrophage recruitment and PDGFRA. + Macrophages accumulate to block the progression of abdominal aortic aneurysms in patients.

2. The application according to claim 1, characterized in that: The patient is male.

3. The application according to claim 1, characterized in that: The pathological manifestations of abdominal aortic aneurysm include one or more of the following: aortic wall dilation, collagen degradation, elastic fiber destruction, and macrophage infiltration.

4. The application according to claim 1, characterized in that: The drug is used in at least one of the following applications: (1) Reduce aortic macrophage infiltration during the progression of abdominal aortic aneurysm; (2) Reduces collagen degradation and elastic fiber damage during the progression of abdominal aortic aneurysm; (3) Reduce aortic dilation during the progression of abdominal aortic aneurysm; (4) Eliminate gender differences in abdominal aortic aneurysm.

5. The application according to claim 1, characterized in that, The drug is an injectable form.

6. The use of a combination of CXCL14 inhibitors and CXCR4 inhibitors in the preparation of a medicament for treating abdominal aortic aneurysms, characterized in that: The CXCL14 inhibitor is a CXCL14 neutralizing antibody; the CXCR4 inhibitor is AMD3100.

7. The application according to claim 6, characterized in that: The drug comprises a pharmaceutically acceptable carrier and is formulated as an injection, tablet, or capsule.

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