Use of atypical chemokine receptor 1 in the preparation of a medicament for treating aortic dissection type a

Abstract

The application discloses application of atypical chemokine receptor 1 in preparation of a drug for treating type A aortic dissection and belongs to the technical field of biotechnology.The aforementioned application comprises application in preparation of a drug or product for inhibiting type A aortic dissection.Experiments prove that knocking out ACKR1 effectively reduces development and incidence of TAAD.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of atypical chemokine receptor 1 in the preparation of drugs for treating type A aortic dissection. Background Technology

[0002] Type A aortic dissection (TAAD) is one of the major challenges facing cardiovascular medicine. This disease not only threatens lives but also often leads to serious long-term consequences. Although the causes of this disease involve multiple factors such as genetics, inflammation, and environment, the specific mechanisms of TAAD at the cellular and molecular levels are not fully understood, which greatly hinders the clinical treatment of this serious disease.

[0003] Recently, researchers have increasingly focused on the dynamic changes of macrophages in aortic aortic lesions (TAAD), particularly their polarization and infiltration patterns. Observations have revealed a significant influx of inflammation-associated immune cells, such as neutrophils, monocytes, macrophages, and T cells, as TAAD progresses. Notably, macrophage infiltration plays a central role in the pathogenesis of TAAD. The differentiation of macrophages into pro-inflammatory M1 and anti-inflammatory M2 types, and their different roles in TAAD, highlight the complexity of the immune response during disease progression. M1 macrophages exacerbate aortic lesions by secreting metalloproteinases, interleukins, and vascular endothelial growth factor, while M2 macrophages appear to participate in post-disease repair processes.

[0004] However, the macrophage population is complex and diverse, and cannot be simply categorized into two groups. Single-cell RNA sequencing (scRNA-seq) technology has revealed previously unknown cellular heterogeneity and provided a more detailed understanding of macrophage subsets in TAAD. For example, some researchers have used scRNA-seq technology to propose that IL1RN... + / TREM1 + Macrophage subsets may be an effective treatment for aortic dissection. This discovery not only provides a new perspective for TAAD treatment but also demonstrates the enormous potential of single-cell technology in elucidating the pathological mechanisms of cardiovascular diseases.

[0005] Despite these advances, the exact mechanisms of macrophage infiltration and polarization in TAADs, as well as the role of endothelial cells in regulating inflammatory cell migration, remain unclear. Local stimuli such as chemokines induce adhesion between circulating leukocytes and endothelial cells, thereby promoting leukocyte capture, adhesion, and migration across the vessel wall to the site of inflammation. However, these complex processes are not yet fully elucidated and require further investigation. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide genes / proteins that regulate type A aortic dissection. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0008] This invention provides an application of atypical chemokine receptor 1 or a substance that regulates the expression of the gene encoding said atypical chemokine receptor 1 or a substance that regulates the activity and / or content of said atypical chemokine receptor 1, in any of the following ways:

[0009] A1) Application in the preparation of drugs for regulating type A aortic dissection;

[0010] A2) Application in the preparation and development of products for regulating type A aortic dissection;

[0011] A3) Application in the preparation of drugs that inhibit type A aortic dissection;

[0012] A4) Application in the preparation and development of drugs to inhibit type A aortic dissection.

[0013] In the above application of red, the atypical chemokine receptor 1 is any one of the following:

[0014] A1) The amino acid sequence of this protein is that of SEQ ID No. 1;

[0015] A2) A protein that has more than 55% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1).

[0016] A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acids shown in A1) or A2).

[0017] The tagged proteins include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein. Those skilled in the art can readily mutate the nucleotide sequences of the proteins encoding the proteins of this invention using known methods, such as directed evolution or point mutation. Nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequences of the proteins isolated by this invention, as long as they encode and have the functions of the aforementioned proteins, are derived from and equivalent to the nucleotide sequences of this invention. The aforementioned 75% or more identity can be 80%, 85%, 90%, or 95% or more. In this document, identity refers to the identity of the amino acid sequence or nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained. In this document, the 55% or more of identity can be at least 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0018] The dosage form of the drug is an injection. The pharmaceutical excipients include physiologically or pharmaceutically acceptable carriers or excipients. In this document, "physiologically or pharmaceutically acceptable carriers" refers to carriers and diluents that do not cause significant irritation to the organism and do not impair the biological activity and properties of the reagents in the administered pharmaceutical composition. Carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.) and poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.). Water-soluble carrier materials are preferred. To formulate unit-dose dosage forms into injectable preparations, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable preparations. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added.

[0019] In the above applications, the atypical chemokine receptor 1 can be either human atypical chemokine receptor 1 or mouse atypical chemokine receptor 1.

[0020] In the above applications, the substance that regulates the expression of the gene encoding atypical chemokine receptor 1 or the substance that regulates the content of atypical chemokine receptor 1 is a substance that inhibits the expression of the atypical chemokine receptor 1 gene in cells.

[0021] In the above applications, the substances are all any of the following biological materials:

[0022] B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein.

[0023] B2) The gene encoding the RNA molecule described in B1);

[0024] B3), an expression cassette containing the gene described in B2);

[0025] B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3);

[0026] B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4);

[0027] B6) A transgenic animal cell line containing the gene described in B2), or a transgenic animal cell line containing the expression cassette described in B3), or a transgenic animal cell line containing the recombinant vector described in B4);

[0028] B7), transgenic animal tissue containing the gene described in B2), or transgenic animal tissue containing the expression cassette described in B3), or transgenic animal tissue containing the recombinant vector described in B4);

[0029] B8) Transgenic animal organs containing the gene described in B2), or transgenic animal organs containing the expression cassette described in B3), or transgenic animal organs containing the recombinant vector described in B4).

[0030] In the above applications, the expression cassette containing nucleic acid molecules described in B2) refers to DNA capable of expressing the gene encoding B2) in a host cell. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all the regulatory sequences necessary for expressing the gene encoding B2) described above. The regulatory sequences, under compatible conditions, guide the expression of the RNA molecule in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. Regulatory sequences can also be suitable transcription termination sequences, i.e., sequences that can be recognized by host cells and thus terminate transcription. The termination sequence is operatively attached to the 3' end of a nucleic acid sequence encoding a protein. Any terminator that can function in a selected host cell can be used in this invention. Regulatory sequences can also be suitable leader sequences, i.e., untranslated regions of mRNA that are crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of a nucleic acid sequence encoding a protein. Any leader sequence that can function in a selected host cell can be used in this invention. Examples of regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that enable gene amplification.

[0031] In the above applications, the RNA molecule is siRNA, and one strand of the siRNA is SEQ ID No. 2 and the other is SEQ ID No. 3, or one strand is SEQ ID No. 5 and the other is SEQ ID No. 6.

[0032] In the above application, the RNA molecule is shRNA, and the nucleotide sequence of the shRNA is SEQ ID No. 4.

[0033] The present invention also provides a method for constructing recombinant cells, the method comprising silencing the encoding gene of the aforementioned atypical chemokine receptor 1 contained in receptor cells to obtain recombinant cells.

[0034] In the above method, the recipient cell is a mammalian cell. The mammalian cell may not include germ cells, fertilized eggs, and human embryonic stem cells; it may be a somatic cell or cell line. The animal organ may not include an embryo.

[0035] The present invention also provides substances that target the aforementioned atypical chemokine receptor 1 (i.e. substances that can bind to atypical chemokine receptor 1).

[0036] In the above application, the substance is Akamicin.

[0037] In the above applications, the atypical chemokine receptor 1 inhibits or reduces or downregulates the activity or content of nuclear factor κB and secretory phosphoprotein 1.

[0038] The present invention also provides any of the following applications of biomarkers and / or substances for detecting said biomarkers:

[0039] A1) Application in identifying or assisting in the identification of whether a person has type A aortic dissection;

[0040] A2) Application in the preparation of products for identifying or assisting in the identification of whether a human body has type A aortic dissection;

[0041] A3) Application in evaluating or assisting in the evaluation of whether a person has type A aortic dissection;

[0042] A4) Application in the preparation of products for evaluating or assisting in the evaluation of whether a human body has type A aortic dissection;

[0043] The biomarkers are atypical chemokine receptor 1 and / or secretory phosphoprotein 1.

[0044] The amino acid sequence of the atypical chemokine receptor 1 is SEQ ID No. 1, and its nucleotide sequence is the nucleotide sequence of human ACKR1.

[0045] This invention demonstrates through experiments that knocking out ACKR1 effectively reduces the development and incidence of TAAD. Attached Figure Description

[0046] Figure 1This document presents the results of single-cell RNA sequencing (scRNA-seq) for identifying cellular heterogeneity and differential gene expression during TAAD progression. (A) is a schematic diagram of the scRNA-seq workflow. (B) shows the single-cell RNA sequencing data atlas, depicting eight distinct lineages: T cells (IMMT), NK cells (NK), B cells (IMMB), leukocytes (LEU), mast cells (Mast), endothelial cells (ECs, LECs), fibroblasts (FB, myoFB), and smooth muscle cells (SMC1, SMC2). (C) is a dot plot representing typical markers for 11 major cell types, where the size of the dots indicates the proportion of cells expressing each gene in a given lineage, and the color intensity reflects the gene expression level. (D) shows the results of differential proportion analysis (DPA) of the relative cell proportions of various cell types, where *P<0.05 and **P<0.01 indicate significance; ns indicates no significance.

[0047] Figure 2 Leukocyte heterogeneity and IL1B in TAAD + The effect of Mac. (A) Results of 19,694 white blood cells visualized by tSNE into different subsets. (B) IL1B in TAAD and non-TAAD. + Relative dominance of Mac subsets. (C) DEG counts between TAAD and non-TAAD subsets using Wilcoxon rank-sum test and Bonferroni correction. Purple and green bars represent upregulated and downregulated genes, respectively, with avg_log2fc>0.5 and p.adjust<0.05 as criteria. (D) Comparative immunofluorescence of aortic sections from control and BAPN-induced TAAD mice, nuclei (blue), macrophages (green), and IL-1β (red). The merged image highlights the colocalization of macrophages with IL-1β. The graph shows a significant increase in IL-1β in the TAAD group compared to the control group (***p<0.001; n=3). Scale: 50 μm. (E) Compared with IL1B + The top 10 GO terms related to Mac biological processes were used to calculate FDR using the Benjamini-Hochberg method. Color gradients represent adjusted p-values, and circle size represents gene counts. (F) Comparative KEGG pathway analysis within cell clusters; color and size reflect p-adjustment and gene count, respectively; FDR calculated using the Benjamini-Hochberg method. (G) Pathway enrichment map of selected pathways (top) and IL1B. + Mac's cutting-edge gene expression heatmap (bottom), including NES. Vertical lines in the graph mark the location of gene set members; the heatmap shows the average expression for each group.

[0048] Figure 3 Endothelial cell diversity and ACKR1 in TAAD hi Expansion of ECs. (A) tSNE analysis revealed subsets within 7,089 endothelial cells. (B) ACKR1 was assessed in TAAD and non-TAAD cells using DPA. hi (C) Comparison of serum ACKR1 levels in clinical TAAD and non-TAAD samples (n=3), assessed using a two-tailed unpaired t-test. (D) Comparative immunofluorescence of aortic sections from control and BAPN-induced TAAD mice, showing cell nuclei (blue), ECs (green), and ACKR1 (red). The merged image highlights the colocalization of ECs and ACKR1. The graph shows a significant increase in ACKR1 in the TAAD group compared to the control group (n=3). Scale: 50 μm. (E) GO analysis of biological processes associated with each endothelial cell subset, with FDR calculated using the Benjamini-Hochberg method; marker colors and sizes represent adjusted p-values ​​and gene counts. (F) Volcano plot showing ACKR1. hi The top 20 DEGs in ECs are sorted by avg_log2fc; red indicates upregulated genes and blue indicates downregulated genes. (G) Enrichment maps and heatmaps of selected pathways, including NES. The vertical lines in the figures and the average gene expression in the heatmaps depict pathway components and expression levels. Statistical significance is indicated by *P<0.05, **P<0.01, and ***P<0.001.

[0049] Figure 4 To investigate macrophage-endothelial interactions and ACKR1-mediated signaling in TAAD (Tymothermal Adenocarcinoma), (A) scatter plots were used to compare ingress and egress signal in 2D space to identify cell populations with significant signal changes between TAAD and non-TAAD samples. (B) Circos plots showed IL1B levels in TAAD samples. + The interaction density of Mac cells with other subsets is shown, with line width representing interaction strength and dot size representing subpopulation communication. (CD) Transwell migration assays assessed macrophage chemotaxis induced by ACKR1 overexpression (ACKR1-OE) or knockout (ACKR1si) in ECs. Control groups consisted of ECs transfected with an empty vector (Vector) or a non-targeting siRNA plasmid (NCsi). Scale: 200 μm. Results are presented in accompanying statistical charts. (EF) Flow cytometry analyzed the pro-inflammatory polarization status of macrophages after co-culturing with ACKR1-OE or ACKR1si ECs. Pro-inflammatory macrophages were labeled with specific antibodies CD68 and IL-1β. Control groups consisted of ECs containing Vector or NCsi. Statistical charts show CD68 concentrations in each group. + IL-1β +The proportion of Mac cells differed. (GH) Western blot analysis was used to analyze ACKR1 and IL-1β in ECs treated with Vector or ACKR1-OE, and with NCsi or ACKR1si. The accompanying figures depict relative cell numbers or protein expression levels, analyzed using a two-tailed unpaired t-test, with significance defined as *P<0.05, **P<0.01, ***P<0.001, n=3 for each experimental group.

[0050] Figure 5 For SPP1 to ACKR1 hi Mediated effects of ECs and macrophages. (A) Cellchat analysis identified SPP1 as the most enriched gene in TAAD, with red indicating differentially activated signaling pathways in TAAD and green indicating those in non-TAAD. (B) Comparative analysis of SPP1 levels in the serum of TAAD patients and non-TAAD individuals. (C) Assessment of SPP1 secretion in ECs after ACKR1 overexpression (ACKR1-OE) or knockout (ACKR1si). (D) Transwell assay assessed the effect of SPP1 neutralizing antibody on macrophage migration in co-culture of ACKR1-OE ECs and macrophages. Scale: 200 μm. (E) Flow cytometry analysis and quantification of CD68 levels after SPP1 intervention in co-culture of ACKR1-OE ECs and macrophages. + IL-1β + (F) Proportion of macrophages. Western blot quantified changes in IL-1β expression regulated by SPP1 in co-culture of ACKR1-OE ECs and macrophages. (G) JASPAR network diagrams highlight the molecular interactions between ACKR1, NFκB1, and SPP1. (H) Detection of changes in SPP1 secretion levels in ECs after overexpression (p65-OE) or knockout (p65si) of p65 compared to their respective controls. (I, J) Western blot detected the regulation of p65 phosphorylation by ACKR1 in ECs with decreased or increased ACKR1 expression. Statistical significance between experiments was compared between two groups using a two-tailed unpaired t-test, and between three or more groups using one-way ANOVA and Tukey's test. *P<0.05, **P<0.01, ***P<0.001, n=3 for each experimental group.

[0051] Figure 6The effect of ACKR1 deficiency on the incidence and progression of TAAD. (A) The effect of ACKR1 knockout on the incidence of TAAD was assessed in BAPN-treated mice using AAV9-shACKR1 knockout and AAV9-control as a control. The incidence and appearance of aortic dissection are shown in each group, n = 9 mice per group. (B) Histological analysis using HE and VG elastography showed reduced elastic fiber degradation in the ACKR1-decreased group during TAAD progression. Elastic fiber degradation was graded from 0 (intact) to 3 (severely damaged). (CE) Representative immunofluorescence co-staining images of ACKR1, SPP1, and IL-1β in aortic sections from control and BAPN-treated mice. Ruler: All panels are 100 μm. Statistical analysis of these panels was performed using two-way ANOVA and Sidak's test. Significance levels are expressed as ns (not significant), P < 0.05, P < 0.01, and P < 0.001, n = 3 per experimental group.

[0052] Figure 7 To evaluate amikacin as a targeted molecular agent against ACKR1 in TAAD therapy. (A) Molecular docking simulations demonstrated the potential binding interaction between amikacin and ACKR1. (B) ACKR1 was incubated with a specified concentration of amikacin in a SPR assay. (C) Transwell migration assays assessed macrophage chemotaxis in ACKR1-OE EC and macrophage co-culture with or without amikacin treatment (10 μM, 24 h). Scale: 100 μm. (D) Flow cytometry quantified CD68 after the same treatment. + IL-1β + The proportion of macrophages was determined using specific antibodies CD68 and IL-1β to label pro-inflammatory macrophages. (E) Western blot showed IL-1β expression, while (F) ELISA detected SPP1 secretion levels under the same treatment conditions. (G) Western blot analysis compared phosphorylated p65 and total p65 levels in ACKR1-OE EC with or without amikacin treatment to their respective controls. Statistical analysis of panel CF was performed using one-way ANOVA and Tukey's test for multiple group comparisons. For panel G, a two-tailed unpaired t-test was used to characterize the significance of changes in protein expression and phosphorylation levels. Significance levels are expressed as P < 0.05, P < 0.01, and P < 0.001. Beta-tubulin was used as a loading control to ensure consistency in protein quantification. Data are presented as mean ± standard deviation.

[0053] Figure 8To evaluate the therapeutic effects of amikacin in a BAPN-induced mouse model. (A) The effect of amikacin on the incidence of aortic dissection (TAAD) in BAPN-treated and control mice was examined, showing the occurrence of aortic dissection and the incidence in specific groups, n=9 per group. (B) Histopathological analysis by HE and VG elastography showed the slowing of elastic fiber degradation in the development of TAAD under amikacin treatment. Elastic fiber integrity was quantitatively assessed, and degradation scores are shown in the accompanying chart. (CE) Immunofluorescence assays assessed the expression levels of IL-1β, SPP1, and ACKR1 in the treatment group, indicating that amikacin modulates inflammatory markers and structural proteins within the aortic wall. The relative expression levels of each marker were statistically analyzed to reflect the effects of treatment on cell infiltration and molecular expression patterns. Statistical analysis of BE was performed using one-way ANOVA and Tukey's test, with significance levels marked as ns (not significant), P<0.01, and *P<0.001. Ruler: 100 μm.

[0054] Figure 9 For supplementary illustrations. (A) Marker expression analysis in leukocyte subsets. The dot plot shows the classic markers for nine leukocyte subsets. The size of the dots represents the proportion of cells expressing each gene within the subset, and the color of the dots is related to their expression level. (B) Marker expression in endothelial cell subsets. The violin plot shows the expression levels of key genes in the subsets. (C) Comparison of serum ACKR1 levels in mouse models, analyzed using a two-tailed unpaired t-test, ***P<0.001. (D) Comparison of DEG counts between TAAD and non-TAAD samples using the Wilcoxon rank-sum test and Bonferroni correction. Criteria: avg_log2fc>0.25 and p.adjust<0.01. Upregulated genes in TAAD are indicated by purple bars, and downregulated genes by green bars. (E) BAPN-induced mouse TAAD model. Control mice show normal aortic anatomy. The aortas of BAPN-treated mice show significant congestion and dilation. The bar chart shows the incidence of TAAD in the control group (0 / 10) and the BAPN treatment group (6 / 10). Black bars represent mice that did not develop TAAD, and red bars represent mice that developed TAAD. (F) is a map of the vector pAV-ICAM2-GFP-mir30. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] Ethical Statement: All animal and human experiments described below have been approved by the Medical Ethics Committee of Shenzhen People's Hospital, and all patients have signed informed consent forms before joining the study.

[0058] Patient Samples: The human tissue samples from TAAD patients used in the following examples were obtained from five male patients who underwent total aortic arch replacement and stent implantation. Aortic samples from non-TAAD patients were obtained from three male patients with aortic valve disease. One non-TAAD sample (VS002) was further excluded due to its sequencing quality being below standard. Sample information and characteristics are referenced below.

[0059] Table 1.

[0060]

[0061]

[0062] Statistical analysis: All experiments were repeated at least three times. Data are expressed as mean ± standard deviation. Unless otherwise specified, two-tailed, unpaired Student's t-test or Wilcoxon rank-sum test was used to compare differences between groups. Multiple comparisons were performed using one-way ANOVA and Tukey's test or two-way ANOVA and Sidak's test. A p-value <0.05 was considered statistically significant.

[0063] Example 1: Single-cell RNA sequencing analysis reveals the potential role of leukocytes and endothelial cells in the progression of thoracic aortic dissection (TAAD).

[0064] I. Experimental Methods

[0065] Tissue dissociation: Aortic samples collected from both TAAD and non-TAAD patients were washed twice in phosphate-buffered saline (PBS). The intima, media, and adventitia were then finely minced and dissociated in a lysin mixture at 37°C for 30 minutes. The mixture was filtered through a 40-micron filter to obtain a single-cell suspension. Red blood cells were removed using lysis buffer (Thermo Fisher Scientific, product number 1966634). Cell viability was assessed using trypsin blue staining (Thermo Fisher Scientific, product number T10282). If cell viability was below 80%, viable cells were further purified using a dead cell removal kit (Miltenyi Biotec, product number 130-090-101).

[0066] Single-cell library preparation and sequencing: Single-cell gel bead-in-emulsion (GEM) generation, barcode labeling, sample cleaning, cDNA amplification, and cDNA library construction were performed using the Chromium Next GEM Single Cell 3′ kit v3.1 (PN-1000121). After single-cell library preparation, final library quality testing was performed using an Agilent 2100 bioanalyzer. Sequencing was performed on a HiSeq-PE150 platform (Novogene Bioinformatics Technology Co., Ltd., Beijing, China).

[0067] Single-cell data preprocessing: Raw reads were preprocessed using Cell Ranger software (version 5.0.1, V5.0.1). Reads were mapped to the human genome (pre-built GRCh38 reference assembly, V3.0.0, 10×Genomics). Unique molecular identifiers (UMIs) were identified and counted for each sample using Cell Ranger count. The UMI count matrix was then output and downstream analysis was performed using the R package Seurat (V4.2.1). Strict quality control was performed, screening for low-quality cells in each sample: mitochondrial genes <10%, erythrocytes <1%, gene counts ranging from 500 to 6000. Diomysomes were filtered using DoubletFinder, and cells identified as “high-confidence diomysomes” were removed. UMI counts were normalized using the Seurat function NormalizeData(parameters: normalization.method="LogNormalize", scale.factor=10000) to reduce batch effects between samples. The top 2000 highly variable genes (HVGs) were selected using the Seurat function FindVariableFeatures. All samples were then integrated using the standard Seurat V5 ensemble algorithm (FindIntegrationAnchors and IntegrateData functions) for technical batch correction.

[0068] Dimensionality reduction and clustering: Principal component analysis (PCA) was performed using the Seurat function RunPCA for dimensionality reduction, and the Seurat function ElbowPlot was used to identify important dimensions. The top 30 PCs were selected, and a shared nearest neighbor (SNN) graph was constructed based on Euclidean distance in the PCA space using the Seurat function FindNeighbors. Cell clustering was then performed using the Seurat function FindClusters according to the Louvain algorithm, with a resolution of 1.5, and visualized on an unsupervised uniform manifold approximation and projection (UMAP) graph. The Seurat function FindAllMarkers(logfc.threshold=0.25,test.use="wilcox") and previously reported typical marker genes were used to select the marker genes for each cluster.

[0069] Subpopulation Analysis: After identifying the major cell types, the inventors selected specific types with high heterogeneity for detailed annotation. To perform more complex subpopulation analysis, a UMI count matrix corresponding to each major cell population was extracted from the comprehensive matrix, with a resolution of 1 for leukocytes and 0.5 for ECs. This data allows for detailed analysis of the characteristics of each cell population.

[0070] Cell-cell interaction network analysis: The R package CellChat (V1.1.3) was used to analyze potential interactions among various cell populations. The CellChatDB.human database and secretion signaling methods were used to analyze ligand-receptor interactions and intercellular communication.

[0071] Differential Proportion Analysis: To determine whether the change in cell proportion compared to the control was accidental, a permutation-based statistical analysis called Differential Proportion Analysis (DPA) was performed, with all parameters set to their default values. The threshold for statistical significance was set to P < 0.05.

[0072] II. Experimental Results

[0073] In this embodiment, live cells were isolated from human aortic tissue and single-cell RNA sequencing was performed. Figure 1 After sequence data preprocessing and rigorous quality control, 42,442 TAAD sample cells and 28,192 non-TAAD sample cells were obtained for further analysis. tSNE analysis identified eight major cell lineages, primarily including immune cells and stromal cells. These lineages included T cells (IMMT), natural killer cells (NK), B cells (IMMB), leukocytes (LEU), mast cells (Mast), endothelial cells (ECs, LECs), fibroblasts (FB, myoFB), and smooth muscle cells (SMC1, SMC2), etc. Figure 1 (Middle B). Typical marker genes for these cell types include CD3D and IL7R for IMMT, NKG7 for NK cells, CD37 and CD79A for IMMB, CD68 and IL1B for LEU, PECAM1 and LYVE1 for ECs, COL1A2 and DCN for fibroblasts, and ACTA2, MYL9, and TAGLN for SMCs, etc. Figure 1 (C)

[0074] Furthermore, differential proportion analysis (DPA) was used to quantify the relative abundance of cells. Compared with non-TAAD samples, the proportions of smooth muscle cells and fibroblasts were significantly decreased in TAAD samples, while the proportions of leukocytes and endothelial cells were significantly increased. Figure 1 (Middle D). These changes in cell composition suggest that leukocytes and endothelial cells may play a key role in the pathogenesis of TAAD.

[0075] Example 2: In TAAD, IL1B + Elevated levels of macrophages are associated with the progression of TAAD.

[0076] I. Experimental Methods

[0077] Functional analysis (GO analysis): Gene ontology (GO) functional enrichment analysis of differentially expressed genes (DEGs) was performed using the R package clusterProfiler V4.6.220. False discovery rate (FDR) was calculated using the Benjamini-Hochberg procedure as a metric for GO terms in resolving multiple testing problems. GO terms significantly enriched in different cell types were visualized using dot plots.

[0078] KEGG analysis: Enrichment plot analysis was performed on the significant KEGG (Kyoto Encyclopedia for Genes and Genomes) gene set for each cell population. The statistical significance threshold was set to FDR q value < 0.05, and was calculated using the compareCluster function in the R package clusterProfiler V4.6.220.

[0079] Gene set enrichment analysis (GSEA): Gene sets for the KEGG pathway and GO were obtained from the REACTOME pathway pre-compiled in MSigDB21. The statistical significance threshold was set to FDR q value < 0.05. The results were visualized using the gseaplot2 function in the R package enrichplot V1.18.422.

[0080] Immunofluorescence assay: Freshly harvested tissues were fixed in cold 4% PFA. Paraffin-embedded or frozen tissues were embedded in OCT compounds and sectioned to 10 μm. After blocking with 10% horse serum and infiltrating with 0.25% Triton X-100, frozen sections were incubated with primary antibodies against human or mouse antigens, including anti-CD68 (ab303565, Abcam) and anti-IL-1β (ab254360, Abcam). Incubation was performed overnight at 4°C to ensure optimal antibody-antigen binding. Sections were then washed with PBS and stained with secondary antibodies conjugated to Alexa Fluor 488 (SA00013-2, Proteintech) or Alexa Fluor Cy3 (SA00009-3, Proteintech). Nuclear staining was performed using DAPI (C1002, Beyotime). Finally, the prepared specimens were observed under a Zeiss fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).

[0081] II. Experimental Results

[0082] The results showed that these white blood cells were classified into four main subsets: monocytes (Mono), neutrophils (NTP1, NTP2), dendritic cells (DC), and macrophages (Mac). Figure 2 (A). Furthermore, experimental results showed that macrophages had the highest abundance in TAAD, and these macrophages could be further distinguished into IL-1B and IL-1B with elevated CCL20 levels. + Macrophages (IL1B) + Mac), C1QA, C1QB, and C1QC level improvement C1Q + Increased APOE levels in Mac, FABP5, and APOE + HSP with elevated levels of Mac, HSPA6, and HSP B1 + Mac, and proliferating macrophages MacCycl ( Figure 9 (A). Furthermore, by Figure 2 As can be seen from B, macrophages, especially IL1β, are present in TAAD samples. + Significant enrichment of the Mac subgroup ( Figure 2 (B)

[0083] Furthermore, immunofluorescence experiments showed that IL1B expression in TAAD samples was significantly higher than in non-TAAD samples, further confirming the presence of a pro-inflammatory macrophage subset (IL1B) in TAAD samples. + The significant increase in Mac) Figure 2 (D).

[0084] To further clarify IL1B + The inventor of Mac conducted GO enrichment analysis on biological processes and discovered IL1B. + Mac primarily involves pro-inflammatory responses, including pathways such as "cytokine-mediated signaling" and "leukocyte migration." Figure 2 (E). Comparative analysis of TAAD and non-TAAD samples showed that IL1B + Mac differentially expressed genes (DEGs) have the largest number ( Figure 2 These DEGs are mainly enriched in the IL-17 and TNF signaling pathways. Figure 2 (F). In addition, IL1B + Mac DEGs' GSEA results further confirm IL1B. + Mac plays a central role in promoting pro-inflammatory responses, particularly the role of TNFA through the NF-κB signaling pathway. Figure 2 (G).

[0085] This suggests that macrophages, especially the pro-inflammatory subtype IL-1B, are involved in TAAD. +The significant infiltration of Mac suggests that it may be a key factor driving the inflammatory cascade response in the progression of TAAD.

[0086] Example 3: In TAAD, endothelial cells that highly express ACKR1 (ACKR1) hi The number of ECs increased, and they exhibited unique inflammation-related gene expression characteristics.

[0087] I. Experimental Methods

[0088] The analyses for GO, KEGG, and GSEA are the same as above.

[0089] Immunofluorescence assay: The method is the same as above, and the antibodies used are anti-CD31 (MA1-26196, ThermoFisher) and anti-ACKR1 (PA5-112940, ThermoFisher).

[0090] Enzyme-linked immunosorbent assay (ELISA): Human blood samples were collected from TAAD and non-TAAD sources. ACKR1 levels in human serum were measured using an ACKR1 detection kit (LS-F6811, LSBio) according to the manufacturer's instructions. Mice were fasted for 16 hours before blood samples were collected. Blood samples were obtained immediately after euthanasia using carbon dioxide via enucleation. Blood samples were allowed to coagulate at room temperature for 1 hour, followed by centrifugation at 1500×g for 20 minutes. The supernatant was then collected for analysis. ACKR1 levels in mouse serum were measured according to the instructions provided with the detection kit (KBR-hlk8158, Shanghai Keruibo Biotechnology).

[0091] II. Experimental Results

[0092] By isolating and re-clustering cells identified as endothelial cells and lymphoendothelial cells, four distinct subpopulations based on their gene expression characteristics were revealed, including cells with high ACKR1 expression (ACKR1...). hi ECs, cells with low ACKR1 expression (ACKR1) lo ECs, lymphoendothelial cells (LECs), and a minority group with immune properties are called EC-like IMMs (ECs, lymphoendothelial cells, and lymphoendothelial cells). Figure 3 China A, Figure 9 (B) Compared to non-TAAD samples, ACKR1 in TAAD samples hi ECs subgroups increased significantly ( Figure 3 (B). Furthermore, ELISA analysis of ACKR1 in the serum of TAAD and non-TAAD patients ( Figure 3 (C) and the assessment of immunofluorescence intensity of ACKR1 in TAAD tissue samples ( Figure 3The study further confirmed the elevated ACKR1 level in TAAD. Upregulation of ACKR1 was also clearly observed in the BAPN-induced mouse model. Figure 9 (C). This shows that ACKR1 overexpression is closely related to the onset and progression of TAAD.

[0093] GO analysis results show that ACKR1 hi ECs are primarily involved in the chemotaxis and migration of inflammatory cells, particularly in processes such as leukocyte chemotaxis and leukocyte migration. Figure 3 (E). Further comparison of differentially expressed genes (DEGs) of ECs in TAAD and non-TAAD samples revealed that ACKR1 was present in TAAD samples. hi ECs have the most DEGs ( Figure 9 In TAAD, pro-inflammatory factors such as CCL2, IL6, and secreted pyroprotein 1 (SPP1) were upregulated most significantly by DEGs. Figure 3 (F). Furthermore, GO and GSEA results indicate that the aforementioned upregulated DEGs are involved in the "inflammatory response," "TNFA via the NF-κB signaling pathway," and "interferon-γ response." Figure 3 (G), thus ACKR1 hi The crucial role of ECs in the inflammatory process.

[0094] The aforementioned experimental data highlight the complex heterogeneity of ECs in TAAD, particularly emphasizing ACKR1. hi The central role of EC subsets. These cells appear to play a complex role in the pathogenesis of TAAD, particularly by mediating leukocyte migration and amplifying the inflammatory response, which are key processes in the development of TAAD.

[0095] Example 4, ACKR1 hi ECs promote IL1B in the microenvironment of TAAD. + Mac Immersion

[0096] I. Cell populations with significant signal changes in TAAD and non-TAAD cells

[0097] 1. Experimental Methods

[0098] Cell-cell interaction network analysis: The method is the same as in Example 1.

[0099] 2. Experimental Results

[0100] Cell-cell interaction network analysis results showed that ACKR1 hi ECs and IL1B +Macrophages (MACs) play a central role in the TAAD-specific signaling network. In TAAD samples, macrophages were identified as important signal transducers. Figure 4 (A) indicates that macrophages are more actively involved in the pathological process of TAAD compared to other cell types. Furthermore, ACKR1... hi ECs play a dual role in TAAD, acting as both receivers and transmitters of signals, communicating with various cells within the TAAD network. Specifically, ACKR1 hi ECs were identified as major signal receivers, highlighting their crucial role in intercellular communication and their potential importance in the pathogenesis of TAAD. Figure 4 (A)

[0101] II. IL1B in TAAD samples + Interaction density of Mac cells with other subpopulations

[0102] 1. Experimental Methods

[0103] Cell-cell interaction network analysis: The method is the same as in Example 1.

[0104] 2. Experimental Results

[0105] The results show that ACKR1 hi ECs and IL1B + The close interaction between Macs suggests a high degree of synergistic effect in TAAD pathology. Figure 4 (B)

[0106] III. Macrophage chemotaxis induced by ACKR1 overexpression (ACKR1-OE) or knockout (ACKR1si) in ECs

[0107] 1. Experimental Methods

[0108] (1) ACKR1-OE group: ACKR1-OE endothelial cells (preparation method see Example 7) were seeded in the lower chamber of a Transwell apparatus (Corning, #3422) in DME M containing 10% FBS and 50 μg / mL primosin. Meanwhile, THP-1 macrophages were placed in serum-free medium in the upper chamber. After co-culturing at 37°C and 5% CO2 for 24 hours, the Transwell inserts were removed, and unmigrated cells on the inner side were wiped away with a cotton swab. Cell migration, flow cytometry analysis, and Western blot experiments were then performed according to Example 7.

[0109] (2) Vector group: The main difference between this group and the ACKR1 overexpression group (ACKR1-OE group) is that an empty vector (Vector) is used during transfection, instead of an expression vector containing the ACKR1 gene. Please refer to Example 7 for the preparation method of the empty vector. Apart from the difference in transfection vector, all other operation steps of the Vector group are consistent with those of the ACKR1-OE group.

[0110] (3) ACKR1si group: ACKR1 siRNA (ACKR1si) was transfected into human umbilical vein endothelial cells (HUVECs) using HiPerFect transfection reagent (Qiagen, Hilden, Germany, 301705). This siRNA (Jin Kairui (Wuhan, China)) targets bases 1510-1529 of the nucleotide sequence of human ACKR1 (GenBank: MK813892.1, November 4, 2019, https: / / www.ncbi.nlm.nih.gov / nucleotide / MK813892.1?report=genbank&log$=nucltop&blast_rank=1&RID=6J8U9G09013). See the instruction manual for transfection procedures. The positive strand of ACKR1si is 5'-C UUCCUAUGGUGUGAAUGAUUTT-3' (SEQ ID No. 2), and the anti strand is 5'-AAUCAUUCACACCAUAGGAAGTT-3' (SEQ ID No. 3). The ACKR1 silencing effect was subsequently evaluated by Western blot analysis. Cells successfully silenced by ACKR1 were used as ACKR1si cells. Cell migration, flow cytometry, and Western blot experiments were also performed.

[0111] (4) NCsi group: The significant difference between this group and the ACKR1si group is that the negative control siRNA (NCsi) was used instead of the siRNA targeting the ACKR1 gene. The positive strand of NCsi is 5'-CGUGACACGUUCGGAGAAdTd T-3', and the reverse strand is 5'-TTCTCCGAACGTGTCACGTdTdT-3' (Jin Kairui (Wuhan, China)). All other operations were the same as those in the ACKR1si group.

[0112] 2. Experimental Results

[0113] Cell migration results showed that overexpression of ACKR1 significantly increased the relative migration of macrophages, while knockdown of ACKR1 significantly decreased the relative migration of macrophages. Figure 4 (C and D). Meanwhile, flow cytometry ( Figure 4E and F) and Western blot technique ( Figure 4 (G and H) showed that ECs overexpressing ACKR1 were associated with increased macrophage recruitment compared to those with ACKR1 knockout. Furthermore, IL1B... + The proportion of Mac and the expression level of IL-1β also increased. These experimental results confirm the presence of ACKR1. hi ECs significantly promoted macrophage infiltration and inflammatory differentiation.

[0114] Example 5, ACKR1 hi ECs regulate macrophage differentiation in TAAD via the ACKR1 / NF-κB / SPP1 signaling pathway.

[0115] I. Cellchat analysis identified SPP1 as the most enriched gene in TAAD.

[0116] 1. Experimental Methods

[0117] Cell-cell interaction network analysis: The method is the same as in Example 1.

[0118] 2. Experimental Results

[0119] The inventors' research revealed significant overexpression of SPP1 in TAAD, particularly in ACKR1. hi Significantly increased in ECs ( Figure 3 Cellchat analysis further confirmed that SPP1 was the most upregulated gene in TAAD tissues compared to non-TAAD tissues. Figure 5 (A)

[0120] II. Comparative analysis of serum SPP1 levels in TAAD patients and non-TAAD individuals

[0121] 1. Experimental Methods

[0122] Human blood samples were collected from TAAD and non-TAAD individuals. Blood samples were allowed to clot at room temperature for 1 hour, followed by centrifugation at 1500×g for 20 minutes. The supernatant was then collected for analysis. SPP1 levels in human serum were measured using the SPP1 assay kit (LS-F171, LSBio) according to the manufacturer's instructions. SPP1 levels in mouse serum were measured using the SPP1 assay kit (LS-F4158, LSBio) according to the manufacturer's instructions, with blood collection methods as described above.

[0123] 2. Experimental Results

[0124] SPP1 levels were significantly elevated in patients with TAAD ( Figure 5 The same applies to BAPN-induced mouse TAAD model (B). Figure 9 (D).

[0125] III. Regulatory Role of ACKR1 in SPP1 Expression

[0126] 1. Experimental Methods

[0127] Overexpression of ACKR1 (ACKR1-OE) or knockout (ACKR1si) was performed in human umbilical vein endothelial cells (HUVECs). A control group (untreated HUVECs) and a negative control group (transfected with non-specific siRNA or empty vector) were established. The experimental methods were the same as above. Cells from each group were collected, and the SPP1 level in each group was detected according to the manufacturer's instructions using an SPP1 assay kit (KE00233, Proteintech).

[0128] 2. Experimental Results

[0129] Regulation of ACKR1 expression in endothelial cells showed that ACKR1 overexpression significantly upregulated SPP1 levels, while ACKR1 downregulation led to a decrease in SPP1 expression. Figure 5 (C). This finding highlights the role of ACKR1 in regulating SPP1 expression, further reinforcing the possibility that SPP1 is a key factor in the pathogenesis of TAAD and a potential diagnostic biomarker.

[0130] IV. SPP1 in ACKR1 hi The role of ECs in regulating macrophage migration and infiltration

[0131] 1. Experimental Methods

[0132] (1) ACKR1-OE + anti-SPP1 group: ACKR1-overexpressing (ACKR1-OE) endothelial cells were seeded in the lower chamber (Corning, #3422) of a Transwell culture apparatus (see Example 7). Cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 50 μg / mL Primocin. The system was cultured at 37°C and 5% CO2 for 24 hours. Subsequently, a neutralizing antibody against SPP1 (10 μg / mL, R&D Systems, Inc., product number AF1433) was added to the downstream chamber, while THP-1 macrophages were suspended in serum-free medium and placed in the upper chamber of the Transwell apparatus for further co-culture for 24 hours. After treatment, the upper chamber of the Transwell was carefully removed (intercalated), and unmigrated cells were gently wiped away from the inside of the upper chamber using a cotton swab. The expression level of SPP1 was measured, cell migration ability was assessed, flow cytometry analysis was performed, and Western blot experiments were conducted to assess changes in protein expression, following the method described in Example 7.

[0133] (2) ACKR1-OE+IgG group: The difference between this group and the ACKR1-OE+anti-SPP1 group is that IgG (10μg / mL, R&D Systems, Inc. 1-001-A) is used instead of the neutralizing antibody for SPP1. The rest of the operation is the same as the ACKR1-OE+anti-SPP1 group.

[0134] (3) Vector+anti-SPP1 group: The main difference between this group and the ACKR1-OE+anti-SPP1 group is that an empty vector (Vector) is used during transfection, instead of an expression vector containing the ACKR1 gene. Please refer to Example 7 for the preparation method of the empty vector. Apart from the difference in the transfection vector, all other operation steps of the Vector group are consistent with those of the +anti-SPP1 group.

[0135] (4) Vector+IgG group: The difference between this group and the ACKR1-OE+anti-SPP1 group is that IgG is used instead of SPP1 neutralizing antibody. The rest of the operation is the same as the Vector+anti-SPP1 group.

[0136] 2. Experimental Results

[0137] Compared to the control group, introducing SPP1 neutralizing antibody into a co-culture system of ACKR1-overexpressing endothelial cells and macrophages inhibited SPP1 secretion levels. Furthermore, macrophage migration was significantly inhibited in the group treated with SPP1 neutralizing antibody (ACKR1-OE + anti-SPP1 group). Figure 5 (D). This indicates that SPP1 actively regulates macrophage dynamics. This hypothesis was supported by flow cytometry ( Figure 5 (E) and Western blot analysis ( Figure 5 Further support from the analysis of SPP1 showed that neutralization of SPP1 significantly reduced pro-inflammatory IL-1β. + The cell population of Mac and the expression of IL-1β indicate that SPP1 plays a role in establishing the inflammatory environment characteristic of TAAD.

[0138] V. Mechanisms by which ACKR1 regulates NF-κB activity and SPP1 expression in ECs

[0139] 1. Experimental Methods

[0140] (1) ACKR1-OE group: ACKR1-OE endothelial cells (preparation method see Example 7) were seeded in the lower chamber of a Transwell apparatus (Corning, #3422) in DMEM containing 10% FBS and 50 μg / mL primosin. Meanwhile, THP-1 macrophages were placed in serum-free medium in the upper chamber. After co-culturing at 37°C and 5% CO2 for 24 hours, the Transwell inserts were removed, and unmigrated cells on the inner side were wiped away with cotton swabs. Cell migration, flow cytometry analysis, and Western blot experiments were then performed according to Example 7.

[0141] (2) Vector group: The difference between this group and the ACKR1-OE group is that the empty vector is used instead of the expression vector containing the ACKR1 gene (see Example 7 for preparation method). The rest of the operation is the same as the ACKR1-OE group.

[0142] (3) ACKR1si group: The difference between this group and the ACKR1-OE group is that ACKR1si cells (see Example 4) are used instead of ACKR1-OE endothelial cells. The rest of the operation is the same as the ACKR1-OE group.

[0143] (4) NCsi group: The difference between this group and the ACKR1si group is that the negative control siRNA (NCsi) is used instead of ACKR1si, and the rest of the operation is the same as the ACKR1si group.

[0144] 2. Experimental Results

[0145] The interaction network between ACKR1 and SPP1 was analyzed using the JASPAR transcription factor binding site database in the Network Analyst platform. The results showed that NFKB1 is a potential key mediator connecting ACKR1 and SPP1, indicating that ACKR1 may regulate SPP1 expression through the NF-κB signaling pathway. Figure 5 (G).

[0146] For ACKR1 hi GSEA analysis of genes with increased expression in ECs showed that these cells were significantly enriched in "TNFA signaling via the NF-κB pathway". Figure 3 The study further confirmed that the NF-κB signaling pathway may play a central role in ACKR1 regulation of SPP1 expression, thereby producing a synergistic effect in the inflammatory process within the endothelial environment. Simultaneously, compared with the control group, p65 overexpression significantly increased SPP1 expression levels; conversely, p65 reduction led to a significant decrease in SPP1 expression. Figure 5 The study further confirmed the key role of the NF-κB signaling pathway in regulating SPP1 expression.

[0147] Western blot results showed that ACKR1 knockout reduced p65 expression and its phosphorylation level at Ser536. Figure 5 (I). Conversely, overexpression of ACKR1 produced the opposite result (I). Figure 5 (J). This indicates that ACKR1 can regulate NF-κB expression and activity. These results further demonstrate the complex interactions between ACKR1, the NF-κB signaling pathway, and SPP1.

[0148] In summary, the aforementioned data indicates that ACKR1 hi Endothelial cells (ECs) regulate macrophage differentiation in TAAD via the ACKR1 / NF-κB / SPP1 signaling pathway. ACKR1 regulates NF-κB activity, thereby affecting SPP1 transcription, indicating that endothelial cells participate in TAAD-related inflammatory processes.

[0149] Example 6: Knockout of ACKR1 in vivo effectively reduced the development and incidence of TAAD.

[0150] I. Experimental Methods

[0151] 1. Construction of TAAD mouse model

[0152] Experimental group: Three-week-old C57BL / 6J mice (Beijing Huafukang Biotechnology Co., Ltd.) were given β-aminopropionitrile (BAPN, A3134, Sigma-Aldrich) dissolved in drinking water at a concentration of 0.25% for 4 weeks. Samples from the ascending aorta and aortic arch were then collected. Immediate necropsy was performed to confirm whether any mice died from aortic complications (such as aortic rupture). Serum samples were also collected from the mice, and the ACKR1 level in the serum was measured according to the instructions provided by the detection kit (KBR-hlk8158, Shanghai Keruibo Biotechnology).

[0153] Control group: Only the same amount of drinking water was given, and the rest of the procedures were the same as those for the experimental group.

[0154] 2. Carrier Construction

[0155] The amino acid sequence of the protein encoded by the nucleotide sequence of ACKR1 (NCBI Reference Sequence: NM_010045.3, June 2, 2024, https: / / www.ncbi.nlm.nih.gov / nucleotide / NM_010045.3?report=genbank&log$=nuclalign&blast_rank=1&RID=6J9AWKNZ016) is shown in NCBI Reference Sequence: NP_034175.2 (June 2, 2024, https: / / www.ncbi.nlm.nih.gov / protein / NP_034175.2?report=genbank&log$=protalign&blast_rank=1&RID=6J9BKP50013).

[0156] Construction of shACKR1 adeno-associated virus vector: A shRNA sequence targeting the ACKR1 gene (SEQ ID No. 4: ggguuagugucuuguggaucuuuCAAGAGAAGAUCCACAAGACACUAACCCUUUUUU, nucleotides 821 to 841 of the nucleotide sequence targeting mouse ACKR1) was designed and chemically synthesized. The adenovirus vector pAV-ICAM2-GFP-mir30 (catalog number PAV100016-KD, WZ biosciences inc., etc.) specifically targeting endothelial cells was selected. Figure 9 As shown in Figure F, this vector contains a built-in U6 promoter. The shRNA sequence was cloned into the multiple cloning site Bpi I of the vector via restriction enzyme digestion and ligase reaction. Colony PCR and restriction enzyme digestion analysis confirmed correct insertion, and DNA sequencing verified the vector's accuracy. The resulting shACKR1 adeno-associated virus vector produces siRNA that interferes with mouse ACKR1. One strand of this siRNA is 5'-ggguuagugucuuguggaucu-3' (SEQ ID No. 5), and the other strand is 5'-agauccacaagacacuaaccc-3' (SEQ ID No. 6).

[0157] Construction of the control shRNA adeno-associated virus vector: The control shRNA adeno-associated virus vector pAV-ICAM2-GFP-mir30 (catalog number PAV100016-KD, WZ biosciences inc) was used, and its structure is as follows. Figure 9 As shown in Figure F. This control vector was used to provide a non-targeted negative control in the experiment to evaluate the effect of shRNA-mediated gene silencing.

[0158] 3. The effect of ACKR1 knockdown in the BAPN-induced TAAD model

[0159] (1) BAPN-AAV9-shACKR1 group: 3-week-old C57BL / 6J mice (Beijing Huafukang Biotechnology Co., Ltd.) received a single intravenous injection of shACKR1 adeno-associated virus vector, with an injection volume of 10 × 10^ 11 VG / mouse. Three weeks later, β-aminopropionitrile (BAPN, A3134, Sigma-Aldrich) was dissolved in drinking water at a concentration of 0.25% (mass percentage) for 4 weeks, and samples of the ascending aorta and aortic arch were collected. Immediate necropsy was performed to confirm whether any mice died from aortic complications (such as aortic rupture). Nine mice were in each group. The following experiments were then conducted:

[0160] ①Incidence of TAAD

[0161] Methods for calculating the incidence of TAAD: First, a detailed phenotypic analysis of the aorta was performed on each group of mice to identify individuals exhibiting TAAD characteristics. Then, the proportion of mice displaying the TAAD phenotype to the total number of mice in the group was calculated. Specifically, the number of mice diagnosed with TAAD was divided by the initial total number of mice in the group, and the resulting proportion was multiplied by 100% to obtain the incidence of TAAD in that group.

[0162] ② Histological analysis

[0163] Aortas from the aforementioned mice were collected. The entire aorta was fixed with 4% PFA at room temperature for 24 hours, then embedded in paraffin and sectioned. Sections were 8 μm thick and stained using hematoxylin-eosin (H&E) or Vangeliscan (VG) staining. Degradation of the medial elastic membrane was analyzed by VG staining (Sigma, HT-25A), and scored according to the degree of elastic fiber degradation. All images were captured using an Olympus microscope. Elastic fiber degradation was graded according to the characteristics of the elastic fibers: Grade 0 indicates intact fibers with normal curvature; Grade 1 indicates fiber stretching without normal curvature; Grade 2 indicates slight fiber fragmentation; Grade 3 indicates severe fiber damage.

[0164] ③ Immunofluorescence co-staining

[0165] Freshly harvested aortic tissue was fixed in cold 4% PFA. Paraffin-embedded or frozen tissue was embedded in OCT compounds and sectioned to 10 μm. After blocking with 10% horse serum and infiltrating with 0.25% Triton X-100, frozen sections were incubated with primary antibodies against human or mouse antigens, including anti-CD31 (MA1-26196, ThermoFisher), anti-ACKR1 (PA5-112940, ThermoFisher), anti-F4 / 80 (Cat No: 29414-1-AP, proteintech), anti-SPP1 (Cat No: 22952-1-AP, proteintech), and anti-IL-1β (ab254360, Abcam). Incubation was performed overnight at 4°C to ensure optimal antibody-antigen binding. Then, sections were washed with PBS and stained with secondary antibodies conjugated to Alexa Fluor 488 (SA00013-2, Proteintech) or Alexa Fluor Cy3 (SA00009-3, Proteintech). Nuclear staining was performed using DAPI (C1002, Beyotime). Finally, the prepared specimens were observed under a Zeiss fluorescence microscope (Carl Zeiss AG, Oberkochen, German). The expression levels of ACKR1, IL-1β, and SPP1 in different experimental groups were characterized by measuring relative fluorescence intensity.

[0166] (2) BAPN-AAV9-control group: The difference between this group and the BAPN-AAV9-shACKR1 group is that the control shRNA adeno-associated virus vector is used instead of the shACKR1 adeno-associated virus vector. The rest of the operation is the same as the BAPN-AAV9-shACKR1 group.

[0167] (3) Control-AAV9-shACKR1 group: The difference between this group and the BAPN-AAV9-shACKR1 group is that the β-BAPN solution is replaced with an equal amount of drinking water. The rest of the operation is the same as the BAPN-AAV9-shACKR1 group.

[0168] (4) Control-AAV9-control group: The difference between this group and the Control-AAV9-shACKR1 group is that the shACK R1 adeno-associated virus vector is replaced by the shACK R1 adeno-associated virus vector. The rest of the operation is the same as the Control-AAV9-shACKR1 group.

[0169] II. Experimental Results

[0170] Compared with the control group ( Figure 9In the BAPN group of mice (E), the aorta showed obvious congestion and dilation, which led to the formation of dissection. Figure 9 The bar chart in the middle section shows the number of layers in each group, indicating that the success rate of establishing the BAPN model reached 60%. Therefore, the TAAD mouse model was successfully constructed.

[0171] In a mouse model of acute aortic dissection (TAAD) induced by β-aminopropionitrile (BAPN), the expression of atypical chemokine receptor 1 (ACKR1) was knocked down using RNA interference. The results were as follows: Compared with the control group, mice with knocked-down ACKR1 showed significantly reduced aortic congestion and dilation (see...). Figure 6 (Figure 2 in A). Knocking down ACKR1 significantly reduced the incidence of TAAD; specific statistics are shown in [the figure]. Figure 6 The third image in section A. Tissue staining results show that knockout of ACKR1 significantly reduced the degradation of aortic elastin in a BAPN-induced mouse TAAD model. Figure 6 (See the right image in section B), and the statistical results of elastin degradation were also confirmed accordingly. Immunofluorescence co-staining results revealed that knocking out ACKR1 significantly reduced ACKR1 levels in the aorta of the BAPN-induced mouse TAAD model. Figure 6 C), IL-1β ( Figure 6 (middle D) and SPP1 ( Figure 6 The expression levels of ACKR1 were significantly reduced. The substantial decrease in IL-1β expression indicated that ACKR1 knockout reduced macrophage infiltration in the BAPN-induced mouse TAAD model. Simultaneously, the decrease in SPP1 expression further confirmed that ACKR1 knockout alleviated the inflammatory response and improved vascular remodeling.

[0172] The above results indicate that the absence of ACKR1 not only plays a role in regulating macrophage differentiation, but also significantly reduces the occurrence of TAAD and inhibits its progression.

[0173] Example 7: Screening and evaluation of amikacin as an ACKR1-targeting molecular drug for the treatment of TAAD

[0174] I. Molecular simulation of potential binding sites between AKAR1 and AQAR1 molecules

[0175] Virtual screening of small molecule drugs targeting ACKR1: The crystal structure of the ACKR1 protein was obtained from the AlphaFold protein structure database (AlphaFoldDB: Q16570). The structure was prepared using Discovery Studio (DS) v4.5, including adding hydrogen atoms, removing heteroatoms, water molecules, and repairing incomplete amino acids. Virtual screening was performed on three computing platforms using a library containing 1436 FDA-approved compounds: DS's LibDock module, Pyrx's VinaWizard module, and... The XP module. The selection of potential small molecule inhibitors is based on each platform's unique scoring metrics: DS's LibDockScore, Pyrx's binding energy, and... The docking score.

[0176] Molecular docking of ACKR1: The structure of the ACKR1 protein was obtained from the AlphaFold protein structure database and refined using Discovery Studio v4.5 to remove foreign molecules. The molecular structure (SDF format) of amikacin (CAS:37517-28-5) was obtained from PubChem, converted to a mol2 file using OpenBabel v3.1.1, and then docking was performed using AutoDockTool-1.5.6. The docking process involved semi-flexible docking using the Lamarckian genetic algorithm, spanning 50 iterations. Gridbox parameters were defined based on active site data provided by POCASA 1.1 (http: / / altair.sci.hokudai.ac.jp / g6 / service / pocasa / ), and all other parameters used default settings. Discovery Studio v4.5 was used for visualization of the docking results. Further molecular docking was performed using AutoDock to evaluate the binding affinity and interaction mode between the aforementioned potential drugs and ACKR1, with results as follows: Figure 7 As shown in Figure A, the top-ranked small molecules formed conventional hydrogen bonds with amino acid residues GLU290, LEU197, and ARG267, and also formed π-π conjugated interactions with TRP136. Furthermore, these small molecules interact with various amino acids in the protein's active site through van der Waals forces and hydrophobic interactions. The docking process generated a binding energy of -7.5 kcal / mol, indicating a significant binding affinity between amikacin and ACKR1, suggesting that amikacin has strong potential as a small-molecule drug targeting ACKR1.

[0177] Surface plasmon resonance (SPR) technology: The interaction between ACKR1 (catalog number TMPH-00990, TargetMol) and amikacin (HY-B0509A, MCE) was investigated using a BIAcore T200 and CM5 sensor chip, conducted at a constant temperature. Proteins were immobilized on the chip in 1.0×PBS-P+ buffer at pH 7.4 using an amine coupling method, while the interaction buffer was PBS-P+ containing 5% DMSO. The CM5 chip was loaded, and the flow cell was activated with EDC and NHS. Ligand proteins were immobilized on flow cell 4 and subsequently blocked with ethylenediamine. A reference flow cell 3 was also prepared. For interaction assays, the buffer was switched to 1×PBS-P+ containing 5% DMSO, and the analytes were passed through the chip at different concentrations. After each cycle, the chip was regenerated with glycine-HCl solution. Data collection and analysis were performed using BIAcore T200 evaluation software (version 2.0, GE Healthcare), with binding and dissociation constants determined by fitting a 1:1 Langmuir binding model. The data was then exported to GraphPad Prism software (version 8.0.2) to generate the final graphs.

[0178] II. Evaluation of Amikacin as a Targeted Molecular Drug Against ACKR1 in TAAD Treatment

[0179] 1. Experimental Materials

[0180] THP-1 cells, derived from a human monocytic cell line from a patient with acute monocytic leukemia, were provided by the American Type Culture Collection Center (ATCC, TIB-202). These cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, Sigma-Aldrich, F8687), 100 U / mL penicillin, and 100 U / mL streptomycin (Sigma-Aldrich, P4333). To induce differentiation into macrophages, THP-1 cells were stimulated with 100 ng / mL dodecyl palmitate (PMA, MedChemexpress, HY-18739) for 48 hours; these treated cells were termed THP-1 macrophages.

[0181] Human umbilical vein endothelial cells (HUVECs) were provided by the American Type Culture Collection Center (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., DFSC-EC-01). HUVECs were typically cultured in endothelial cell culture medium (ECM, #1001, ScienCell) enriched with 5% FBS (Sigma-Aldrich, F8687), 100 U / mL penicillin, 100 U / mL streptomycin, and 30 μg / mL endothelial cell growth supplement (#1052, ScienCell). Cells were cultured at 37°C with 5% CO2, and cells used for experiments were typically from passage 2 to 7.

[0182] 2. Preparation of ACKR1-OE endothelial cells

[0183] (1) Construction of ACKR1 overexpression vector: By replacing the sequence between the BsrGI and XhoI restriction sites in the pCDNA3.1-EGFP vector, the DNA fragment containing the human ACKR1 gene was integrated into the vector to form a recombinant expression vector, while the rest of the vector sequence remained unchanged.

[0184] (2) Cell transfection and validation: The ACKR1 expression vector was transfected into human umbilical vein endothelial cells (HUVECs) using Attractene transfection reagent (Qiagen, product number 301005), strictly following the manufacturer's instructions. After transfection, the expression level of ACKR1 protein was detected by Western blot to confirm whether ACKR1 was overexpressed.

[0185] (3) Cell nomenclature and control group setup: HUVECs overexpressing ACKR1 were named ACKR1-OE cells. Meanwhile, HUVECs transfected with the empty vector pCDNA3.1-EGFP served as a negative control group to analyze the effect of ACKR1 overexpression on cell function.

[0186] 3. Cell co-culture

[0187] (1) ACKR1-OE + Amikacin group: ACKR1-overexpressing (ACKR1-OE) endothelial cells were seeded in the lower chamber (Corning, #3422) of a Transwell culture apparatus. Cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 50 μg / mL Primocin. The system was cultured at 37°C and 5% CO2 for 24 hours. Subsequently, 10 μM Amikacin solution was added to the downstream chamber, while THP-1 macrophages were suspended in serum-free medium and placed in the upper chamber of the Transwell apparatus for further co-culture for 24 hours. After treatment, the upper chamber of the Transwell was carefully removed (intercalated), and unmigrated cells were gently wiped away from the inside of the upper chamber with a cotton swab.

[0188] ① Cell migration assay: THP-1 macrophages migrating in the lower part of the above co-culture system were fixed with 4% PFA, stained with crystal violet (C0121, Beyotime), and observed and counted using a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany). The intensity of cell migration was assessed and characterized by comparing the relative cell numbers of the experimental group and the control group.

[0189] ② Flow cytometry analysis: THP-1 macrophages in the above co-culture system were digested with trypsin, then stained with CD68 antibody (anti-CD68-PE, MA5-23572, ThermoFisher) and IL-1β antibody (anti-IL-1β-APC, MA5-23597, ThermoFisher), and incubated at room temperature in the dark for 30 minutes. Cells were washed to remove excess antibody and resuspended in PBS, then analyzed by flow cytometry (BD, Biosciences).

[0190] ③Western blot: Proteins were extracted by lysing ACK R1-OE endothelial cells or THP-1 macrophages in the co-culture system with RIPA buffer (FNN0021, Invitrogen). An equal amount of total protein obtained from the cell treatment was subjected to 10% SDS-PAGE electrophoresis and then transferred to a polyvinylidene fluoride (PVDF, Millipore) membrane. The following primary antibodies were incubated on the membrane: anti-ACKR1 (1:1000 dilution, ab137044, Abcam), anti-IL-1β (1:500 dilution, ab315084, Abcam), anti-p65 (1:1000 dilution, #8242, Cell Signaling Technology), anti-phosphorylated p65 (Ser536) (1:500 dilution, #3033, Cell Signaling Technology), anti-β-Tubulin (1:1000 dilution, ab15568, Abcam), and an HRP-conjugated secondary antibody (SA00001-2, Proteintech). Protein signals were captured using an enhanced chemiluminescence system (EC L, Thermo, 32109).

[0191] ④ Enzyme-linked immunosorbent assay (ELISA): Collect the culture supernatant of ACKR1-OE endothelial cells in the above co-culture system and use the SPP1 detection kit (KE00233, proteintech) to determine the SPP1 level. For specific operation, please refer to the instruction manual.

[0192] (2) ACKR1-OE+Vehicle group: The difference between this group and ACKR1-OE+Amikacin is that an equal amount of deionized water is used instead of the acaricin solution. The rest of the operation is the same as the ACKR1-OE+Amikacin group.

[0193] (3) Vector+Amikacin group: The difference between this group and the ACKR1-OE+Amikacin group is that the empty vector pCDNA3.1-EGFP vector is used instead of the ACKR1 gene expression vector in "2. Preparation of ACKR1-OE endothelial cells" in this example. The rest of the operation is the same as the ACKR1-OE+Amikacin group.

[0194] (4) Vector+Vehicle group: The difference between this group and the Vector+Amikacin group is that the same amount of deionized water is used instead of the acaricin solution. The rest of the operation is the same as the Vector+Amikacin group.

[0195] 4. Experimental Results

[0196] This indicates that surface plasmon resonance (SPR) technology accurately determined the binding affinity between ACKR1 and amikacin. SPR measurements further provided an estimated dissociation constant (KD) of 0.913 μM and a correlation rate constant (ka) of 6 x 10⁵ M⁻¹ s⁻¹. Figure 7 (B). This indicates that ACKR1 has a strong binding affinity for amikacin, and the binding process is relatively fast.

[0197] Cell migration assays showed that, compared with the control group, overexpression of ACKR1 significantly promoted macrophage migration (***, P < 0.001), while the addition of amikacin significantly inhibited macrophage migration (***, P < 0.001). Figure 7 (C). Flow cytometry and Western blot analysis confirmed the cell migration results, showing that CD68 was significantly higher than the control group. + IL-1β + The number of macrophages and the expression level of IL-1β were both significantly decreased. Figure 7 (D and E). Enzyme-linked immunosorbent assay (ELISA) revealed that, compared with the control group, amikacin treatment significantly reduced the expression level of SPP1 (D and E). Figure 7 (Middle F). Western blot results showed reduced phosphorylation of p65 at Ser536 ( Figure 7 The presence of G indicates that amikacin may weaken the NF-κB signal.

[0198] The results above demonstrate that amikacin inhibits macrophage function and inflammatory signaling through multiple mechanisms. By reducing macrophage migration, lowering pro-inflammatory cytokine levels, and interfering with the key NF-κB signaling pathway, amikacin exhibits strong therapeutic potential.

[0199] III. Evaluation of the therapeutic effects of amikacin in a BAPN-induced mouse model

[0200] 1. Experimental Grouping

[0201] (1) BAPN-Amikacin group: β-aminopropionitrile (BAPN, A3134, Sigma-Aldrich) was dissolved in drinking water at a concentration of 0.25%, and 3-week-old C57BL / 6J mice (Beijing Huafukang Biotechnology Co., Ltd.) were orally administered the β-BAPN solution. Simultaneously, amikacin (injection) 0.675 mg / day (HY-B0509A, MedChemExpress) was subcutaneously injected for 4 weeks. Then, samples from the ascending aorta and aortic arch were collected. Immediate necropsy was performed to confirm whether any mice died due to aortic complications (such as aortic rupture). The incidence of TAAD was then calculated according to Example 6, and histological analysis and immunofluorescence co-staining were performed.

[0202] (2) BAPN-Vehicle group: The difference between this group and the BAPN-Amikacin group is that the same amount of physiological saline is used instead of acaricin (injection), and the rest of the operation is the same as the BAPN-Amikacin group.

[0203] (3) Control-Amikacin group: The difference between this group and the BAPN-Amikacin group is that the β-aminopropionitrile solution is replaced with an equal amount of drinking water. The rest of the operation is the same as the BAPN-Amikacin group.

[0204] (4) Control-Vehicle group: The difference between this group and the Control-Amikacin group is that an equal volume of physiological saline is used instead of acaricin (injection), and the rest of the operation is the same as the Control-Amikacin group.

[0205] 2. Experimental Results

[0206] In a BAPN-induced mouse TAAD model, subcutaneous injection of amikacin significantly reduced the incidence of TAAD under the same BAPN conditions. Figure 8 (A), and significantly reduced arterial wall thickness and elastin degradation (HE and VG staining, Figure 8 (Middle B). Simultaneously, the proportion of macrophages and the expression level of IL-1β were significantly reduced in the amikacin-treated group, indicating that amikacin effectively inhibited macrophage infiltration, especially inflammatory macrophages (B). Figure 8 (C). Furthermore, amikacin treatment significantly reduced SPP1 expression levels (C). Figure 8 It affects D), but has no effect on ACKR1 expression. Figure 8 These results indicate that amikacin intervenes in the development of TAAD by affecting downstream signaling pathways of ACKR1, thereby influencing macrophage infiltration and differentiation.

[0207] In summary, the experimental results of this invention show that, compared with non-TAAD samples, IL1B in TAAD... + Mac and ACKR1 hi The interactions between ECs are significantly enhanced. This interaction highlights the ACKR1 hi ECs play a crucial role in promoting pro-inflammatory signaling and macrophage migration, highlighting their significant contribution to the progression of TAAD. ACKR1, primarily expressed on ECs, is unique in its ability to bind to a series of major inflammation-associated chemokines, aiding in their clearance or transcellular transport. This activity is essential for leukocyte migration across the blood vessel wall, directly linking ACKR1 to the inflammatory response. Unlike traditional chemokine receptors, ACKR1 lacks the specific sequence required for G protein binding, thus disrupting standard signaling pathways. This unique characteristic of ACKR1 makes understanding its signaling pathways and mechanisms crucial for elucidating the underlying inflammatory processes in TAAD. To gain a deeper understanding of ACKR1... hi How do ECs promote the pathological changes in TAAD by regulating macrophage infiltration and inflammatory polarization? Differential gene expression analysis revealed a significant increase in SPP1 expression in TAAD tissue samples. This finding was further confirmed by subsequent clinical sample analysis and a BAPN-induced mouse TAAD model study, where both TAAD patients and model mice exhibited significantly elevated serum SPP1 levels. Furthermore, ACKR1 regulation can affect SPP1 expression. SPP1, or osteopontin, plays a crucial role in various biological processes, including immune regulation and tissue remodeling. Increased expression in cardiovascular disease enhances macrophage adhesion and migration, highlighting its importance in pathological states. Experiments further confirmed that neutralizing SPP1 significantly reduced macrophage migration and decreased IL1B levels in inflammatory macrophages. + The reduction in Mac ratio and IL-1β expression suggests that ACKR1 may influence macrophage polarization via SPP1. Furthermore, gene interaction network analysis identified NFKB1 as a potential key mediator connecting ACKR1 and SPP1, indicating that ACKR1 may regulate SPP1 expression through the NF-κB signaling pathway. Subsequent co-immunoprecipitation experiments not only confirmed the interaction between ACKR1 and the NF-κB subunit p65 but also demonstrated how ACKR1 regulates NF-κB activity and subsequent SPP1 transcription.

[0208] In summary, the experimental results of this invention clearly demonstrate that ACKR1 hiECs regulate macrophage migration and inflammatory polarization in TAAD through the ACKR1 / NF-κB / SPP1 signaling pathway, further highlighting how ACKR1 affects SPP1 transcription by modulating NF-κB activity. These findings not only provide new insights into the pathological mechanisms of TAAD but also highlight potential targets for future therapeutic strategies targeting the inflammatory pathways of TAAD. Surgical repair remains the primary treatment option for TAAD patients. Despite its effectiveness, the significant surgery-related mortality and complication risk underscore the limitations of existing treatments. Therefore, developing drugs targeting ACKR1 has the potential to reduce short-term mortality and alleviate the severity of TAAD, and also offers valuable therapeutic opportunities, heralding new directions in TAAD management. Through molecular docking and comprehensive evaluation, amikacin was identified as a promising small molecule targeting ACKR1. Amikacin, traditionally an aminoglycoside antibiotic for treating bacterial infections, works by inhibiting bacterial protein synthesis—a function previously not linked to ACKR1 interaction. Extensive in vitro and in vivo studies by the inventors have demonstrated that amikacin effectively reduces macrophage infiltration and inflammatory polarization. Furthermore, it significantly slowed the progression of TAAD, as evidenced by a reduction in TAAD incidence, decreased levels of IL-1β and SPP1, and minimized elastin degradation. These findings highlight the potential of amikacin as a novel regulator of ACKR1, marking a potential advance in targeted therapy for TAAD. However, the inventors discovered that amikacin does not directly alter ACKR1 expression; it may exert a therapeutic effect on TAAD progression by modulating macrophage infiltration and activation through downstream ACKR1 signaling pathways, the specific molecular mechanisms involved requiring further investigation. While amikacin shows promise as an ACKR1 regulator in these models, further studies are needed to establish its efficacy and safety in humans. The inventors' in vitro and in vivo findings underscore the therapeutic potential of targeting ACKR1 in TAAD, highlighting the necessity for extensive testing and validation of amikacin and similar compounds prior to clinical application. In conclusion, the experimental results of this invention mark a significant step forward in elucidating the complex cellular and molecular landscape of TAAD, opening new avenues for targeted therapy.

[0209] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of substances that regulate the expression of the gene encoding mouse atypical chemokine receptor 1 in the preparation of drugs that inhibit type A aortic dissection in mice; The substance is siRNA or shRNA; One strand of the siRNA is SEQ ID No. 5, and the other strand is SEQ ID No. 6; The nucleotide sequence of the shRNA is SEQ ID No.

4.

2. Application of amikacin in the preparation of drugs for treating type A aortic dissection.

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