Application of macrophage specific E3 ubiquitin ligase TRIM31 in diagnosis and treatment of atherosclerosis

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of macrophage specific E3 ubiquitin ligase TRIM31 in diagnosis and treatment of atherosclerosis. According to the application disclosed by the invention, the action and molecular mechanism of the TRIM31 as a macrophage specific endogenous protection factor in atherosclerosis are defined for the first time, and the core position of the TRIM31-LOX-1 axis in regulation and control of macrophage lipid metabolism and inflammatory response is disclosed. The therapeutic drug developed based on the target spot has the advantages of high specificity and strong targeting property, can accurately intervene in the key pathological process of atherosclerosis, effectively reduces plaque formation, enhances plaque stability, and reduces the risk of cardiovascular events. Meanwhile, the TRIM31 can be used for disease risk assessment and treatment monitoring as a biomarker, and a related screening method provides an efficient tool for research and development of novel drugs, so that the TRIM31 has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine and molecular biology, and particularly relates to the application of macrophage-specific E3 ubiquitin ligase TRIM31 in the diagnosis and treatment of atherosclerosis. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

[0003] Atherosclerosis (AS) is the leading cause of cardiovascular disease death worldwide, and its core pathological features are lipid deposition in the intima, immune cell infiltration and chronic inflammatory response. Macrophages, as key effector cells, form foam cells by taking up oxidized low-density lipoprotein (ox-LDL) and release pro-inflammatory factors, promoting plaque formation and instability, and are the core driving force for the occurrence and development of the disease.

[0004] Post-translational modifications (PTMs) have become one of the important mechanisms for regulating immune and inflammatory responses in AS. Among the numerous PTMs, ubiquitination mediated by E3 ubiquitin ligase has attracted much attention due to its ability to finely regulate protein stability, intracellular localization and activity. E3 ubiquitin ligases in the TRIM (tripartite motif-containing) family have been extensively studied due to their role in maintaining immune homeostasis and cellular stress response. However, their functional significance in AS is still poorly understood, especially the specific regulatory mechanisms in macrophages and their contribution to atherosclerosis formation have not been fully elucidated.

[0005] Lectin-like oxidized LDL receptor-1 (LOX-1), also known as oxidized LDL receptor 1 (OLR1), is a key scavenger receptor in the development of atherosclerosis. LOX-1 can enhance the uptake of ox-LDL by macrophages, promote the formation of foam cells, and induce the release of pro-inflammatory cytokines, thereby collectively accelerating the progression of atherosclerosis. Clinical and epidemiological studies consistently show that the level of LOX-1 is elevated in patients with coronary heart disease and hyperlipidemia, suggesting its important clinical significance. Therefore, down-regulating LOX-1 is considered a promising strategy for treating atherosclerosis. However, transcription factors that regulate LOX-1 at the transcriptional level often simultaneously regulate multiple genes, thus lacking sufficient target specificity, which poses a challenge for the development of drugs that inhibit the transcription of LOX-1. In contrast, the ubiquitin-proteasome pathway has high specificity in targeting specific protein degradation. Finding an E3 ubiquitin ligase that can mediate the ubiquitination of LOX-1 and promote its proteasome degradation has important scientific and clinical significance. However, the fine regulation mechanisms of LOX-1, especially in post-translational modifications such as ubiquitination, are still unclear. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application provides the application of macrophage-specific E3 ubiquitin ligase TRIM31 in the diagnosis and treatment of atherosclerosis. Specifically, the present application first clarifies the role and molecular mechanism of TRIM31 as a macrophage-specific endogenous protective factor in atherosclerosis, and reveals the central role of the TRIM31-LOX-1 axis in regulating macrophage lipid metabolism and inflammatory response. The therapeutic drugs developed based on this target have the advantages of high specificity and strong targeting, which can precisely intervene in the key pathological processes of atherosclerosis, effectively reduce plaque formation and enhance plaque stability, and reduce the risk of cardiovascular events. At the same time, TRIM31 can be used as a biomarker for disease risk assessment and treatment monitoring, and the related screening method provides an efficient tool for the development of new drugs. Based on the above research results, the present application is completed.

[0007] Specifically, the present application relates to the following technical solutions: In a first aspect of the present application, the use of a reagent for detecting TRIM31 in the preparation of any one or more of the following products is provided: (a1) an atherosclerosis diagnosis or auxiliary diagnosis product; (a2) an atherosclerosis prognosis evaluation or auxiliary prognosis evaluation product.

[0008] The product may be a test kit, a test device, or a test equipment.

[0009] A second aspect of the present invention provides a system for diagnosing and / or assessing the prognosis of atherosclerosis, said system comprising at least: The acquisition module is configured to acquire the expression level of TRIM31 in the subject; The assessment module is configured to assess the subject's disease status based on the expression level of TRIM31 obtained by the acquisition module; The TRIM31 mentioned herein refers to the TRIM31 encoding gene and / or the TRIM31 encoding gene expression product, namely the E3 ubiquitin ligase TRIM31 protein.

[0010] The TRIM31 is derived from macrophages, and can further be derived from monocyte-derived macrophages.

[0011] This invention reveals that TRIM31 is selectively upregulated in both mouse and human macrophages within the lipid and inflammatory microenvironment associated with atherosclerosis, suggesting its potential involvement in macrophage-mediated lipid responses and inflammatory processes. Therefore, it could serve as a biomarker for the diagnosis or prognostic assessment of atherosclerosis. Furthermore, the expression of TRIM31 is positively correlated with disease progression (severity).

[0012] It should be noted that the system for diagnosing or assisting in the diagnosis of gliomas of the present invention can be a virtual device, as long as it can realize the functions of the analysis module and the evaluation module. The analysis module can include various detection reagents and / or detection instruments and equipment, etc.; the evaluation module can be any computing instrument, module, or virtual device that can analyze and process the detection results of the analysis module to obtain an assessment of the risk of atherosclerosis. For example, it can be a data chart that pre-defines various possible detection results and corresponding disease risk conditions, and the risk assessment result of atherosclerosis can be obtained by comparing the detection results of the detection module with the data chart.

[0013] A third aspect of the invention provides the use of TRIM31 as a target in screening drugs for the prevention and / or treatment of atherosclerosis.

[0014] The method for screening drugs for the prevention and / or treatment of atherosclerosis includes: 1) Treat the system expressing and / or containing TRIM31 with the candidate substance; set up a parallel control without the candidate substance treatment; 2) After completing step 1), detect the expression level of TRIM31 in the system; if the expression level of TRIM31 in the system treated with the candidate substance is significantly increased compared with the parallel control, the candidate substance can be used as a candidate glioma drug.

[0015] In another specific embodiment of the present invention, the system may be a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

[0016] A fourth aspect of the invention provides the use of a TRIM31 expression promoter in any one or more of the following: (a) To prepare products that inhibit lipid accumulation and / or foam cell formation; (b) To prepare products that inhibit inflammatory responses; (c) Prepare products that promote the degradation of LOX-1; (d) Prepare products for the prevention and / or treatment of atherosclerosis and related diseases.

[0017] The TRIM31 expression promoter includes substances capable of promoting the expression and / or activity of the TRIM31 gene and / or protein; that is, any substance that works through TRIM31 using any means and method falls within the scope of protection of this invention. Preferably, the TRIM31 gene and / or TRIM31 protein expression promoter includes at least one of the following: a recombinant expression vector containing the TRIM31 gene or a gene fragment having more than 90% homology with the TRIM31 gene; a polypeptide, protein, nucleic acid and nucleic acid aptamer, polysaccharide, natural active substance, or biological agent capable of upregulating the expression of the TRIM31 gene and / or TRIM31 protein.

[0018] The recombinant expression vector includes any one or more of the following: vector-based eukaryotic expression plasmids that specifically target the TRIM31 gene and / or TRIM31 protein, adenovirus, adeno-associated virus, lentivirus, retrovirus, LNP liposome, microinjection technology, gene editing system elements, and homologous recombinant vectors.

[0019] The product may be a drug or a general testing reagent for non-medical purposes, and the testing reagent may be used for basic research.

[0020] According to the present invention, the above-mentioned drug also includes at least one inactive pharmaceutical ingredient.

[0021] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0022] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0023] In application (b), the inhibition of the inflammatory response is specifically manifested in inhibiting the expression of inflammatory factors and reducing the phosphorylation level of key proteins in the inflammatory signaling pathway. The inflammatory factors include, but are not limited to, Tnf-α, Il-6 and Il-1β, and the key proteins in the inflammatory signaling pathway include, but are not limited to, p65, ERK, JNK and p38.

[0024] In the application (d), the atherosclerosis-related diseases include cardiovascular diseases mediated by atherosclerosis, such as coronary heart disease, stroke, peripheral artery disease, renal artery stenosis, and aortic aneurysm, etc., without specific limitations.

[0025] A fifth aspect of the invention provides the application of TRIM31 expression inhibitors in constructing atherosclerosis models.

[0026] The atherosclerosis model can be a cell model, tissue model, organ (or organoid) model, or non-human animal model; wherein, the non-human animal model can be a non-human mammal, and more specifically, a mouse.

[0027] The TRIM31 expression inhibitors include substances capable of inhibiting the expression and / or activity of the TRIM31 gene and / or protein, specifically including shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microRNA, antisense nucleic acid, or constructs (such as lentiviruses) capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microRNA, or antisense nucleic acid; and antibodies against the TRIM31 protein, and may also include compound inhibitors.

[0028] The beneficial technical effects of one or more of the above technical solutions: The above-mentioned technical solution is the first to report that TRIM31 can specifically target the lysine 12 site of LOX-1 through its E3 ubiquitin ligase activity, mediate K48 linker-type ubiquitination modification and promote its proteasome degradation, thereby inhibiting macrophage uptake of oxidized low-density lipoprotein (ox-LDL), foam cell formation and inflammatory factor release, ultimately reducing atherosclerotic plaque formation and enhancing plaque stability.

[0029] The above technical solution clarifies the mechanism of action of TRIM31 as an endogenous protective factor in atherosclerosis, provides a treatment strategy targeting the TRIM31-LOX-1 axis, and offers a new solution for the precision treatment of atherosclerosis. At the same time, TRIM31 can be used as a biomarker for disease risk assessment and treatment monitoring. Therefore, TRIM31 can also be used as a molecular biomarker, thus having good practical application value. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0031] Figure 1. Expression of TRIM31 in macrophages and atherosclerotic lesions. (A) Heatmap showing the differential expression of classical E3 ubiquitin ligase in RAW264.7 macrophages treated with oxidized LDL (ox-LDL) compared to the control group (n = 5). Genes with FDR-corrected (adj) p < 0.05 were screened; color scale represents normalized expression level (z-score; blue = low expression, red = high expression).

[0032] (B, C) Time-dependent changes in Trim31 mRNA (qRT-PCR, top) and TRIM31 protein levels (representative Western blot, bottom) in mouse peritoneal macrophages (PMs, B) and bone marrow-derived macrophages (BMDMs, C) after ox-LDL treatment for 0–24 h (n = 5).

[0033] (D) Apoe - / - Representative immunofluorescence staining of TRIM31 (red) and MOMA2 (green) in atherosclerotic plaques in mice at early (WD 8 weeks) and late (WD 24 weeks) stages. Scale bar = 200 μm.

[0034] (E) Representative immunofluorescence staining of TRIM31 and MOMA2 in human coronary artery plaques during the intimal thickening and fibro-atherosclerotic plaque stages. Scale bar = 100 μm.

[0035] (F) Representative Western blot (top, n = 3) and qRT-PCR (bottom, n = 5) analysis of TRIM31 expression in aortic macrophages isolated from the aorta of Apoe- / - mice after WD feeding.

[0036] (G) Experimental Flowchart: Demonstrates the process of collecting peripheral blood mononuclear cells (PBMCs) from healthy controls and patients with atherosclerotic cardiovascular disease (ASCVD), and obtaining human mononuclear cell-derived macrophages (hMDMs) from healthy donors.

[0037] (H) qRT-PCR analysis of Trim31 mRNA in ASCVD patients and age-matched healthy control PBMCs (n = 15).

[0038] (I) qRT-PCR analysis of Trim31 mRNA in healthy donor-derived hMDMs after ox-LDL treatment for 0–24 h (n = 5).

[0039] Data are expressed as mean ± standard deviation (mean ± SD); each point represents an independent biological replicate. Statistical analysis: time series data were analyzed using one-way ANOVA with Bonferroni post-hoc test; comparisons of human PBMCs were performed using the Kruskal-Wallis test with Bonferroni correction.

[0040] Figure 2. Macrophage-specific Trim31 deficiency exacerbates atherosclerosis progression in both male and female mice. (A) Male Apoe 16 weeks after WD - / - Trim31 fl / fl and Apoe - / - Trim31 fl / fl Lyz2 cre In situ image of the aortic arch in mice (left) and en-face Oil Red O staining image of the entire aorta (middle). Yellow arrows indicate representative atherosclerotic lesions at the aortic arch. The right side shows the quantitative results of the percentage of lesion area to the total aortic surface area (n = 8). (B) Representative images and quantitative results of female mice (n = 8). (CI, male) Male Apoe - / - Trim31 fl / fl and Apoe - / - Trim31 fl / fl Lyz2 cre Representative histological and molecular analyses of mouse aortic root sections and aortic tissue, scale bar = 200 µm (n = 8). (C) Representative hematoxylin-eosin (H&E) stained images (left) and quantitative analysis of the total lesion area and the percentage of necrotic core area to plaque (right); (D) Representative Oil Red O stained images and Oil Red O+ Quantification of the percentage of the area occupied by the region in the plaque; (E) Representative immunofluorescence images and quantification, showing macrophages (MOMA2). + (green) and smooth muscle cells (α-SMA) + (F) Representative Masson trichrome staining image (left) and quantification of collagen content as a percentage of plaque area (right); (G) Representative Sirius Red staining image and quantification of fibrous cap thickness (μm); (H) Vulnerable plaque index = (macrophage area percentage + necrotic core area percentage) / (smooth muscle cell area percentage + collagen area percentage); (I) qRT-PCR analysis of Tnf-α, Il-6, and Il-1β mRNA expression in aortic tissue. Data are expressed as mean ± SD. Statistical analysis: Except for the Welch t test for Il-6 and Il-1β in (F) and (I), and the Mann-Whitney test for (H), all other comparisons were performed using a two-sided unpaired Student t test.

[0041] Figure 3. Macrophage-specific Trim31 overexpression reduces atherosclerotic lesions and inflammation. (A) Male Apoe 16 weeks after WD - / - With Apoe - / - Trim31 Lyz2-KI In situ image of the mouse aortic arch (left) and en-face Oil Red O staining image of the entire aorta (middle). Yellow arrows indicate representative atherosclerotic lesions. The right side shows the quantitative results of the percentage of lesion area to the total aortic surface area (n = 8). (B) Representative images and quantitative results of female mice (n = 8). (CI, male) Male Apoe - / - With Apoe - / - Trim31 Lyz2-KI Representative histological and molecular analyses of mouse aortic root sections and aortic tissue, scale bar = 200 µm (n = 8). (C) Representative H&E staining image (left) and quantification of total lesion area and necrotic core area as a percentage of plaque (right); (D) Representative Oil Red O staining image and Oil Red O + Quantification of the percentage of the area occupied by the region in the plaque; (E) Representative immunofluorescence images and quantification, showing macrophages (MOMA2). + (green) and smooth muscle cells (α-SMA) +(A) Percentage of plaque area positive (red); (F) Representative Masson trichrome staining image (left) and quantification of collagen content as a percentage of plaque area (right); (G) Representative Sirius Red staining image and quantification of fibrous cap thickness (μm) (n = 8); (H) Vulnerable plaque index = (percentage of macrophage area + percentage of necrotic core area) / (percentage of smooth muscle cell area + percentage of collagen area); (I) qRT-PCR analysis of Tnf-α, Il-6, and Il-1β mRNA expression in aortic tissue. Data are expressed as mean ± SD; each point represents one mouse. Statistical analysis: Except for Tnf-α in (H and I), which was analyzed using the Welch t test, all other comparisons were performed using the two-sided unpaired Student t test.

[0042] Figure 4. TRIM31 regulates lipid uptake, foam cell formation, and inflammatory response in macrophages. (A) UMAP visualization of Apoe- / -Trim31 after WD 16 weeks fl / fl With Apoe - / - Trim31 fl / fl Lyz2 cre Single-cell RNA sequencing (scRNA-seq) data from mouse aortic tissue (n = 4). (B) GO enrichment analysis of differentially expressed genes (DEGs) between two genotypes in macrophage clusters. Input genes were significantly upregulated DEGs (|log2FC|>1, corrected p<0.05). The size and color of the dots represent the number of genes annotated to each GO entry; the color of the lines indicates the significance of enrichment (…). (log10 corrected p-value). GeneRatio (%) is displayed after each GO entry. (C) From WT and Trim31 - / - Representative images of mouse PMs stained with Oil Red O (n = 6), NBD-cholesterol (n = 5), and DiI-oxLDL (n = 5) after ox-LDL treatment. Scale bar = 50 μm. Below is the quantification of lipid accumulation (fold change relative to WT). (D) Trim31 - / - qRT-PCR analysis of Tnf-α, Il-6, and Il-1β mRNA expression in PMs treated with ox-LDL compared to WT (n = 5). (E) ELISA assay of WT and Trim31. - / -Concentrations of TNF-α and IL-6 in the supernatant of PMs after ox-LDL stimulation (n = 5). (F) Representative Oil Red O and DiI-oxLDL staining of hMDMs transfected with control siRNA (NC) or TRIM31 siRNA (si-Trim31) after ox-LDL treatment. Scale bar = 50 μm. Below is the quantification of lipid accumulation (fold change relative to NC, n = 6). (G) qRT-PCR quantification of Tnf-α, Il-6, and Il-1β mRNA levels in Trim31 knockdown and ox-LDL-stimulated hMDMs (n = 5). (H) ELISA quantification of TNF-α and IL-6 in the supernatant of Trim31 knockdown and NC control hMDMs after ox-LDL treatment (n = 5). Data are expressed as mean ± SD; each point represents a biological replicate. Statistical analysis: (C) Two-tailed unpaired Student t test was used; (F) Welch t test was used; other comparisons were performed using Welch ANOVA combined with Dunnett T3 multiple comparison test.

[0043] Figure 5. TRIM31 interacts with LOX-1 and promotes its degradation, thereby inhibiting macrophage lipid uptake and inflammation. (A) Schematic diagram illustrating two complementary mass spectrometry strategies for identifying TRIM31-related proteins. In IP-MS analysis, ox-LDL-treated WT mouse PMs were immunoprecipitated with an anti-TRIM31 antibody, followed by LC-MS / MS identification of TRIM31 interacting proteins (n ​​= 3). Quantitative proteomics analysis was performed on ox-LDL-treated WT and Trim31... - / - PMs (n = 3) underwent whole-proteome analysis to screen for differentially expressed proteins between two genotypes. The intersection of the two datasets (IP-MS × proteome) yielded 5 overlapping candidate proteins. The proteome data selection criteria were fold change > 1.5 and p < 0.05. (B) WT and Trim31 - / - Representative Western blot of lipid-related proteins in PMs after ox-LDL stimulation. (C, D) Apoe after 16 weeks of WD - / - Trim31 fl / fl Lyz2 cre and Trim31 Lyz2-KIRepresentative Western blot of LOX-1 in mouse aorta (n = 3). (E) Representative Western blot (left) and quantitative analysis (right) of protein stability assessed in cycloheximide (CHX) tracking assay after co-transfection of HEK-293T cells with Flag-TRIM31 and Myc-LOX-1. LOX-1 signal was normalized to tubulin and quantified (n = 3). (F) Representative Western blot of HEK-293T cells co-expressing Flag-LOX-1 and GFP-TRIM31 after administration of proteasome or lysosomal inhibitors or DMSO control. (G) Representative Western blot of LOX-1 and TRIM31 in hMDMs treated with ox-LDL after Trim31 siRNA-mediated knockdown. (H) Representative Co-IP images of GFP-TRIM31 and Myc-LOX-1 in HEK-293T cells, confirming their protein interaction. (I) Representative images of in vitro binding experiments using recombinant GST-TRIM31 and Myc-LOX-1 proteins obtained through in vitro translation. (J) Representative Co-IP images of endogenous TRIM31 binding to LOX-1 detected in PBMCs from healthy controls and ASCVD patients. (K) Representative confocal microscopy images of colocalization of TRIM31 (Flag, red) and LOX-1 (GFP, green) in HEK-293T cells. Scale bar = 10 μm. (L) From WT, Trim31 - / - Trim31 - / - + si-NC and Trim31 - / - + si-Lox-1 mouse PMs after ox-LDL treatment with representative Oil Red O and DiI-oxLDL staining images. The right side shows the quantification of staining intensity (n = 5). Scale bar = 50 μm. qRT-PCR analysis of Tnf-α, Il-6, and Il-1β mRNA levels in PMs from the same experimental group (M) and (L) (n = 5). Data are expressed as mean ± SD, with each point representing an independent biological replicate. Statistical analysis: Except for (E), which used Welch ANOVA combined with Dunnett T3 multiple comparisons, all other analyses used one-way ANOVA followed by Bonferroni post-hoc analysis.

[0044] Figure 6. TRIM31-mediated LOX-1 ubiquitination and its role in foam cell formation. (A) Western blot analysis of representative LOX-1 ubiquitination in HEK-293T cells co-transfected with Myc-LOX-1 and Flag-TRIM31 (wild-type or E3 ligase-inactivated mutants C53 / 56A and ΔRING) and simultaneously expressing HA-ubiquitin. (B) Western blot analysis of representative LOX-1 ubiquitination in HEK-293T cells expressing GFP-TRIM31 and different HA-ubiquitin variants (WT, K48-only, K63-only), showing that TRIM31 selectively promotes K48-linked ubiquitination of LOX-1, rather than K63-linked ubiquitination. (C) WT and Trim31 - / -Representative Western blots of LOX-1 ubiquitination in mouse PMs after ox-LDL stimulation. (D) Representative images of in vitro ubiquitination experiments using GST-TRIM31 and Myc-LOX-1 obtained through in vitro translation and the reconstructed ubiquitination system; protein analysis by anti-Myc and anti-GST immunoblotting. (E) LC-MS / MS spectra identifying LOX-1 lysine 12 (K12) as a K48-linked ubiquitination site. Specifically, HEK-293T cells were co-transfected with Flag-LOX-1, HA-K48-ubiquitin, and GFP-TRIM31 or control vectors, and the LOX-1 complex was analyzed by LC-MS / MS after anti-Flag immunoprecipitation. (F) Representative LOX-1 Western blots of HEK-293T cells co-expressing GFP-TRIM31 and different LOX-1 lysine mutants (K→R). (G) Representative Co-IP showing that the LOX-1 K12R mutant, but not other lysine mutants, loses K48 linked ubiquitination. (HI) Representative Oil Red O and DiI-oxLDL staining images of Lox-1- / - PMs reconstructed with Ad-Lox-1(WT)(H) or Ad-Lox-1(K12R)(I), then infected with Ad-Trim31 or the vector, and treated with ox-LDL. Staining intensity is quantified on the right (n = 5). Scale bar = 50 μm. (J) qRT-PCR analysis of Tnf-α, Il-6, and Il-1β mRNA expression in cells in the same group as (HI) (n = 5). Data are expressed as mean ± SD; each point represents a biological replicate. Statistical analysis: Quantitative analyses were performed using a two-tailed unpaired Student's t-test.

[0045] Figure 7. Lox-1 knockdown in vivo salvages atherosclerotic plaque instability caused by Trim31 deficiency. (A) WD Apoe 16 weeks later - / - Trim31 fl / fl Apoe - / - Trim31 fl / fl Lyz2 cre Apoe who received AAV-shLox-1 or as a control - / - Trim31 fl / fl Lyz2 creIn situ image of the mouse aortic arch (left) and Oil Red O staining image of the entire aortic face (middle). Yellow arrows indicate plaques. The right side shows the quantitative results of the area of ​​Oil Red O positive lesions as a percentage of the total aortic surface area (n = 8). (BG) Representative histological and molecular analyses of mouse aortic root sections and aortic tissue, scale bar = 200 µm (n = 8): (BC) Representative H&E staining image (top) and quantitative analysis of total lesion area and necrotic core area (percentage of plaque area) (bottom); (DE) Representative Oil Red O, MOMA-2, α-SMA, Masson tricolor and Sirius Red staining images (left), and the quantitative analysis on the right, including Oil Red O. + Area (percentage of patch area), MOMA-2 + Area (percentage of patch area, green), α-SMA + Area (percentage of patch area, red), collagen + Area (percentage of plaque area, blue) and fibrous cap thickness (μm); (F) Vulnerable plaque index = (percentage of macrophage area + percentage of necrotic core area) / (percentage of SMC area + percentage of collagen area); (G) qRT-PCR analysis of Tnf-α, Il-6, and Il-1β mRNA expression in aortic tissue. Data are expressed as mean ± SD; each point represents one mouse. Statistical analysis: Most comparisons were performed using one-way ANOVA with Bonferroni post-hoc test; (C) necrotic core and (F) vulnerable plaque index were compared using the Kruskal-Wallis test; (E) Oil Red O + (Area) Welch analysis of variance was used in conjunction with Bonferroni post-hoc tests.

[0046] Figure 8. LOX-1 as a target molecule of TRIM31 in atherosclerosis, macrophage lipid deposition, and inflammation. (AD, male Apoe) - / - Lox-1 - / - (A) Schematic diagram of experimental design (left) and Apoe in mice after 16 weeks of WD (reconstructed Aav-Lox-1 (WT) combined with Aav-Trim31 or vector treatment (WD). - / - Lox-1 - / -In situ images of the aortic arch and en-face Oil Red O staining images (middle) after mouse Aav-Lox-1 (WT) reconstruction and administration of Aav-Trim31 or vector control. Yellow arrows indicate plaques. The right side shows the quantitative results of the percentage of Oil Red O positive lesion area to the total aortic surface area (n = 8). (BD) Representative histological staining and analysis of aortic root sections, scale bar = 200µm (n = 8): (B) Representative H&E staining images (left) and quantification of total lesion area and necrotic core area as a percentage of plaque (right); (C) Representative Oil Red O, MOMA-2, α-SMA, Masson tricolor and SiriusRed staining images of the aortic root, with corresponding quantitative analysis of lipid area, macrophage content, smooth muscle coverage, collagen content and fibrous cap thickness (μm); (D) Vulnerable plaque index = (macrophage area percentage + necrotic core area percentage) / (SMC area percentage + collagen area percentage).

[0047] (EH, male Apoe) - / - Lox-1 - / - Mouse Aav-Lox-1 (K12R) reconstructed and treated with Aav-Trim31 or vector (E) Experimental design diagram (left) and in situ and en face Oil Red O staining images of AAV-vector and AAV-Trim31 groups (middle). Yellow arrows indicate typical plaques; the right side shows the quantification of lesion area (n = 8). (FH) Representative histological staining and analysis of aortic root sections, scale bar = 200 µm (n = 8): (F) Representative H&E staining and quantification of total lesion area and necrotic core area; (G) Representative Oil Red O, MOMA2, α-SMA, Masson tricolor and Sirius Red staining, and quantitative analysis according to the same indicators in (C); (H) Vulnerable plaque index calculated according to the above formula.

[0048] (IJ) qRT-PCR analysis of Tnf-α, Il-6, and Il-1β mRNA in the aorta of mice in two groups: Aav-Lox-1(WT) (I) and Aav-Lox-1(K12R) (J) (n = 8). Data are expressed as mean ± SD; each point represents one mouse. Statistical analysis: except for (C) MOMA-2 +Except for the Mann-Whitney test for area and the Welch t test for vulnerable plaque index in D, all other comparisons were performed using the two-sided unpaired Student t test. Detailed Implementation

[0049] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0050] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0051] The present invention will be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the selling company. Materials and reagents used in the examples, unless otherwise specified, are commercially available.

[0052] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0053] Example Materials and Methods Ethical Statement All animal experiments were conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and the relevant guidelines of the Laboratory Animal Evaluation and Accreditation Association, and were approved by the Laboratory Animal Ethics Committee of Qilu Hospital, Shandong University (Approval No.: KYLL 2019(KS)-021). All studies involving humans were conducted in accordance with the Declaration of Helsinki and were approved by the Research Ethics Committee of Qilu Hospital, Shandong University (same approval No.: KYLL 2019(KS)-021). All subjects signed written informed consent forms before enrollment in the studies.

[0054] Human samples, peripheral blood mononuclear cells (PBMCs), and human mononuclear cell-derived macrophages (hMDMs) Human coronary atherosclerotic plaques were obtained from autopsy specimens of male patients with coronary artery disease, while peripheral blood samples were collected from patients with coronary artery disease (CAD) and age-matched healthy individuals. Coronary artery segments were embedded in optimal cutting temperature (OCT) compounds and serially cut into 7 μm cross-sections for histopathological analysis. Human atherosclerotic plaque staging was determined according to the classification criteria proposed by Virmani et al. (ATVB 2000) and AHA (Stary). PBMCs were obtained by classical density gradient centrifugation. To obtain hMDMs, monocytes were induced to differentiate for 7 days in culture containing M-CSF before functional experiments. This study was reported strictly in accordance with the STROBE guidelines to ensure transparency and reproducibility.

[0055] Animal experiments All mice were C57BL / 6J background mice housed in a specific pathogen-negative (SPF) animal room at 22 ± 2°C and 50-60% relative humidity, using a 12-hour light / dark cycle, with free access to autoclaved drinking water and standard or Western diet (WD). Atherosclerosis was induced according to the American Heart Association's scientific statement on animal studies of atherosclerosis, by feeding mice WD for 16 weeks. Macrophage-specific Trim31 knockout mice (Trim31...) fl / fl Lyz 2cre Macrophage-specific Trim31 overexpression mice (Trim31) Lyz2-KI ) and Lox-1 knockout (Lox-1 - / - Mice, constructed using the CRISPR / Cas9 system, were subsequently coupled with Apoe - / - A mouse hybridization was used to establish an experimental model of atherosclerosis. In some experiments, AAV9-mediated TRIM31 overexpression or Lox-1 knockdown / replenishment were used to induce atherosclerosis in Apoe. - / - Or Apoe- / -Lox-1 - / -Functional rescue experiments were conducted in mice. All experiments used age- and sex-matched mice, and data from male and female animals were analyzed separately. Animal experiments were again conducted in accordance with NIH guidelines and AAALAC standards, and the research protocol was approved by the Laboratory Animal Ethics Committee of Qilu Hospital, Shandong University (Approval No.: KYLL 2019(KS)-021). Adenoviruses and adeno-associated viruses (AAVs) encoding Trim31, Trim31-ΔRing, Lox-1, and Lox-1(K12R) were constructed and prepared by BioSune Biotechnology (Shanghai, China). For RNA interference, double-stranded siRNA was transfected into macrophages using RNAiMAX reagent at a final concentration of 50 nM. Examples of siRNA target sequences are as follows: Mouse Lox-1: positive strand 5′-GGCAGACCTGCCAATCTTT-3′; Human Trim31: positive strand 5′-GGACCACAAAUCCCAUAAUTT-3′.

[0056] Analysis of atherosclerotic plaques in mice Mice were fed WD for 16 weeks, then fasted for 12 hours before being euthanized by injection of sodium pentobarbital at a dose of 150 mg / kg. Blood samples were collected and the systemic circulation was perfused with ice-cold physiological saline. Subsequently, the heart and intact aorta were removed, fixed in 4% paraformaldehyde, and the entire aorta was stained with En Face Oil Red O to evaluate the lipid deposition area.

[0057] To analyze aortic root plaques, tissues were embedded in OCT after excising the base of the heart and the root of the ascending aorta, and serially sectioned at a thickness of 7 μm, systematically harvested from the origin of the aortic valve to the ascending aorta. The aorta was then longitudinally cut and stained with Oil Red O for lipid analysis. Frozen sections were sequentially stained with hematoxylin-eosin (H&E), Oil Red O, Masson's Red, and Sirius Red, as well as immunofluorescence staining (e.g., MOMA-2, α-SMA). Quantitative indicators included: total plaque area, necrotic core percentage, collagen content, fibrous cap thickness, macrophage infiltration (MOMA-2 positive area), and smooth muscle cell coverage (α-SMA positive area). The fragile plaque index was calculated using the following formula: Fragile Plaque Index = (Macrophage Load Percentage + Necrotic Core Percentage) / (Smooth Muscle Cell Content Percentage + Collagen Content Percentage), used to comprehensively assess plaque stability. All quantitative analyses were performed under blinded conditions by researchers whose genotype grouping was unknown. The experimental design and reporting in this section strictly followed the ARRIVE 2.0 guidelines and AHA recommendations regarding atherosclerosis research.

[0058] Statistical analysis All data were statistically analyzed using GraphPad Prism 9 software and are expressed as mean ± standard deviation (mean ± SD) unless otherwise specified. First, the Shapiro-Wilk test was used to assess whether the data distribution conformed to normality. For comparisons between two normally distributed groups, the F-test was used to determine homogeneity of variance; if the variances were homogeneous, a two-tailed independent samples t-test was used; if the variances were unequal, a Welch t-test was used. For comparisons involving multiple groups where the data conformed to normality, the Brown-Forsythe test was used to assess homogeneity of variance; if the variances were homogeneous, one-way ANOVA was used for one-way comparisons, and two-way ANOVA was used for two-way comparisons, followed by a Bonferroni post-hoc test. If the variances were unequal, a Welch ANOVA was used. For data that do not conform to a normal distribution, the Mann-Whitney test was used for comparisons between two groups, and the Kruskal-Wallis test was used for comparisons among multiple groups, supplemented by Bonferroni post-hoc correction. In the analysis of population data, continuous variables were expressed as medians (25%–75% quartiles) and compared using the Mann-Whitney test; categorical variables were compared using the Fisher exact test. All time-point measurements were from independent biological replicates, not replicates from the same sample. A p-value < 0.05 was considered statistically significant.

[0059] result Upregulation of Trim31 in macrophages and atherosclerotic lesions suggests its involvement in the development and progression of atherosclerosis. To investigate the role of E3 ubiquitin ligases in atherosclerosis (AS), we performed transcriptome sequencing analysis on RAW264.7 macrophages treated with oxidized low-density lipoprotein (ox-LDL). A heatmap of differentially expressed E3 ubiquitin ligases (Figure 1A) showed that TRIM31 was significantly upregulated, suggesting a possible close relationship with the pathogenesis of atherosclerotic cardiovascular disease (ASCVD). This result was validated in mouse peritoneal macrophages (PMs), where both the mRNA and protein levels of TRIM31 increased in a time- and dose-dependent manner after ox-LDL treatment (Figure 1B). Similar trends were observed under LDL and cholesterol stimulation. Correspondingly, TRIM31 was also upregulated in bone marrow-derived macrophages (BMDMs) upon lipid stimulation (Figure 1C), while no significant changes were observed in either mRNA or protein levels in mouse smooth muscle cells (mSMCs) and mouse endothelial cells (mECs), suggesting that this response is macrophage-specific.

[0060] Immunofluorescence results showed that TRIM31 was mainly located in MOMA2-rich areas of atherosclerotic plaques. + The region of macrophages, in Apoe - / - Significant expression of TRIM31 was observed in both mouse and human coronary artery lesions (Figures 1D-E). In mice, TRIM31 was upregulated in early lesions and further increased in late plaques fed a Western diet (WD) for 24 weeks (Figure 1D). In human samples, TRIM31 expression gradually increased with disease progression from the pathological intimal thickening stage to the fibroatherosclerotic plaque stage, especially in the necrotic core region (Figure 1E). These results suggest that TRIM31 exhibits a consistent pattern of enrichment in diseased macrophages across different species and at different stages of atherosclerosis. - / - Time-series analysis of mouse aortic tissue showed that the mRNA and protein levels of Trim31 gradually increased with WD feeding at 8, 16, and 24 weeks. Furthermore, qRT-PCR and Western blot analysis of isolated aortic macrophages also confirmed the inducible upregulation of TRIM31 (Figure 1F).

[0061] To further assess its clinical relevance, we examined peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease (CAD) (Figure 1G). The results showed that, compared with healthy controls, the levels of Trim31 mRNA and protein in PBMCs from CAD patients were significantly elevated. Figure 1 H). Furthermore, human monocyte-derived macrophages (hMDMs) derived from healthy donors also showed a time-dependent increase in TRIM31 expression after ox-LDL stimulation. Figure 1 I). In summary, these results indicate that TRIM31 is selectively upregulated in both mouse and human macrophages within the atherosclerosis-associated lipid and inflammatory microenvironment, suggesting its potential involvement in macrophage-mediated lipid responses and inflammatory processes.

[0062] Macrophage-specific Trim31 deficiency exacerbates atherosclerotic lesions and promotes plaque instability. To clarify the functional role of TRIM31 in AS, we constructed macrophage-specific TRIM31 conditional knockout mice (Trim31) using the CRISPR-Cas9 system. fl / fl Lyz2 cre Genotyping and Western blot results confirmed successful TRIM31 knockout in macrophages. Subsequently, this strain was compared with Apoe... - / - Mouse hybridization was used to evaluate its effect on the development of atherosclerotic lesions. Apoe - / - Trim31 fl / fl Lyz2 cre Mice and control Apoe - / - Trim31 fl / fl Mice were compared after being fed WD for 16 weeks. We conducted simultaneous in vivo analyses in both males and females to systematically evaluate the effects of TRIM31 deficiency.

[0063] Serum lipid parameters (LDL-C, TC, TG, HDL-C) and body weight showed no significant differences among groups, suggesting that systemic metabolic factors are unlikely to explain the observed phenotypic differences. Compared with the control group, male and female Apoe - / - Trim31 fl / fl Lyz2 creMice showed a greater atherosclerotic plaque burden in both in situ observation of the aorta and en face Oil Red O staining (Fig. 2A-B). Quantitative analysis further confirmed that males (Fig. 2C) and female Apoe... - / - Trim31 fl / fl Lyz2 cre The area of ​​plaques and the proportion of necrotic cores in the aortic root of mice were significantly increased.

[0064] Histological analysis showed that TRIM31 deficiency significantly increased lipid deposition within plaques, accompanied by decreased collagen deposition and smooth muscle cell (α-SMA) loss. + The fibrous cap coverage is reduced, the fibrous cap is thinned, and macrophages (MOMA-2) + Infiltration was significantly enhanced (male: Fig. 2D-G). These structural parameters were synthesized into a vulnerable plaque index ((% macrophage area + % necrotic core area) / (% smooth muscle cell area + % collagen area)). It was then observed that, regardless of whether male (Fig. 2H) or female, Apoe... - / - Trim31 fl / fl Lyz2 cre The vulnerable plaque index was significantly elevated in all mice, suggesting that the overall plaques were more prone to rupture and instability. Consistent with this, the mRNA levels of inflammatory cytokines Tnf-α, Il-6, and Il-1β in the aortic tissue were significantly upregulated in both males (Figure 2I) and females, further indicating that macrophage-specific Trim31 deficiency drives focal inflammation amplification.

[0065] To further analyze the impact of Trim31 deficiency on plaque cell composition and intercellular communication, we performed single-cell RNA sequencing (scRNA-seq) on the aforementioned mouse aortic tissue under strict quality control. Combining previously reported cell marker genes and highly expressed genes in various cell types, we identified multiple cell populations, including smooth muscle cells (SMCs, Tagln, Acta2), endothelial cells (ECs, Pecam1, Cdh5), and neutrophils (S100a8, S100a9). Cell-cell interaction analysis showed that Trim31 deficiency... fl / flIn control mice, intraplaque signaling communication was dominated by stromal cells (SMCs); however, in Trim31-deficient mice, the communication network shifted to a macrophage-centric model. This shift from SMC-dominated to macrophage-dominated behavior reflects a change in plaque structure from relatively stable to inflammatory and vulnerable remodeling, which is highly consistent with the aforementioned histological findings of enlarged necrotic cores, reduced collagen, and increased inflammatory factors.

[0066] Further KEGG and gene set enrichment analysis (GSEA) revealed that multiple inflammation-related, extracellular matrix remodeling, and apoptosis-regulating pathways were significantly enriched in Trim31-deficient aortas. These results, combined with scRNA-seq evidence and histological findings, indicate that Trim31 deficiency in macrophages disrupts vascular wall homeostasis, driving plaque transformation from a "stable" phenotype primarily supported by smooth muscle to a "vulnerable" phenotype driven by macrophages and inflammation. This mechanistically explains phenomena such as enlarged plaque necrosis core, thinning of the fibrous cap, and enhanced inflammation, highlighting the crucial role of macrophage TRIM31 in maintaining plaque stability.

[0067] Macrophage-specific overexpression of Trim31 alleviates atherosclerotic lesions and enhances plaque stability. To further verify the protective effect of TRIM31 in macrophages from the opposite perspective, we constructed macrophage-specific Trim31 overexpressing mice using a targeted knock-in strategy. Lyz2-KI This strain was compared with Apoe. - / - Background mouse hybridization to obtain Apoe - / - Trim31 Lyz2-KI and Apoe - / - Control mice were used, and phenotypic assessments were performed on male and female mice after 16 weeks of WD feeding.

[0068] There were no significant differences in lipid profiles (LDL-C, TC, TG, HDL-C) and body weight between the two groups, suggesting that Trim31 overexpression did not cause systemic lipid metabolism or body weight changes. Gross observation and en face Oil Red O staining results showed that in both male (Fig. 3A) and female (Fig. 3B) groups, Apoe - / - Trim31 Lyz2-KI The atherosclerotic lesion area in mice was significantly lower than that in Apoe. - / - Control mice. Quantitative histological analysis further confirmed that Trim31 overexpressing mice had significantly reduced plaque area and necrotic core ratio in the aortic root, decreased lipid deposition and macrophage infiltration within the plaque, increased smooth muscle cell coverage, increased collagen deposition, and significantly thickened fibrous cap (male: Figure 3C-G).

[0069] When the above multiple indicators are integrated into the vulnerable plaque index, Apoe can be seen to... - / - Trim31 Lyz2-KI The vulnerable plaque index in mice was significantly decreased in both males (Fig. 3H) and females, indicating a significant enhancement in overall plaque stability. This protective effect was consistent in both sexes, highlighting the robustness of TRIM31's anti-atherosclerotic effect. Consistent with the morphological improvement, the mRNA expression of inflammatory cytokines Tnf-α, Il-6, and Il-1β in aortic tissue was significantly reduced in both male (Fig. 3I) and female Trim31-overexpressing mice, suggesting that TRIM31 slows plaque formation and progression by limiting local inflammatory responses.

[0070] In summary, macrophage-specific Trim31 overexpression can significantly inhibit atherosclerotic plaque formation and promote plaque stability. The mechanism is at least partly related to reducing inflammatory burden and promoting plaque microenvironment remodeling.

[0071] Trim31 deficiency promotes lipid accumulation and inflammatory response in macrophages. To elucidate the functional consequences of Trim31 deficiency at the cellular level, we again used scRNA-seq to analyze Apoe after WD feeding. - / - Trim31 fl / fl With Apoe - / - Trim31 fl / fl Lyz2 cre Analysis of mouse aortic tissue was performed. Data integration and unsupervised clustering showed a significant increase in the macrophage population in the Trim31-deficient group (Figure 4A). Gene Ontology (GO) enrichment analysis revealed that pathways related to proteasome-mediated protein catabolism and multiple pro-inflammatory signaling cascades were significantly upregulated in the Trim31-deficient aorta. Simultaneously, pathways related to lipid transport and lipid metabolism regulation were also significantly enriched (Figure 4B), suggesting that TRIM31 may simultaneously regulate macrophage immune activation and lipid processing.

[0072] Based on this, we utilize Trim31 whole-body knockout (Trim31 - / - Functional validation was performed using peritoneal macrophages from wild-type (WT) mice. Following ox-LDL stimulation, Trim31... - / -Lipid droplets were significantly increased in macrophages, and OilRed O, NBD-cholesterol, and DiI-oxLDL staining all showed enhanced lipid uptake and foaming (Figure 4C). Conversely, the efflux capacity of cholesterol into ApoA-I and HDL did not show significant changes, suggesting that TRIM31 mainly regulates intracellular lipid load by limiting lipid uptake rather than promoting efflux. Meanwhile, Trim31... - / - The mRNA levels of inflammatory factors Tnf-α, Il-6 and Il-1β in macrophages were significantly increased (Figure 4D-E), accompanied by enhanced phosphorylation of NF-κB and MAPK cascade-related proteins (p65, ERK, JNK, p38), thus establishing a link between TRIM31 deficiency and inflammatory signal amplification.

[0073] Conversely, in mice with macrophage-specific Trim31 overexpression (Trim31... Lyz2-KI In these cells, ox-LDL stimulation significantly reduced lipid accumulation and inflammatory cytokine expression, and Western blot analysis also confirmed weakened activation of the NF-κB and MAPK pathways. Notably, overexpression of the TRIM31 mutant lacking the RING domain (Ad-Trim31-ΔRing) significantly eliminated the inhibitory effect on foaming and inflammatory gene expression, suggesting that the E3 ubiquitin ligase activity of TRIM31 is essential for its protective effect.

[0074] To verify the transferability of these findings in human systems, we knocked down Trim31 in hMDMs. The results showed that Trim31 knockdown significantly enhanced macrophage foaming and lipid uptake (Fig. 4F), while also increasing inflammatory cytokine mRNA expression (Fig. 4G) and cytokine secretion levels (Fig. 4H), highly consistent with mouse data. In summary, TRIM31 is a protective factor enriched in macrophages and induced by atherosclerosis-related stimuli. It exerts its anti-atherosclerotic effect by restricting ox-LDL uptake and inhibiting downstream pro-inflammatory signaling, thereby suppressing foam cell formation and inflammatory responses, with E3 ligase activity as its core mechanism.

[0075] TRIM31 inhibits foam cell formation and inflammation by promoting LOX-1 degradation. To identify potential substrates for TRIM31, we employed two complementary strategies: first, screening for TRIM31-interacting proteins using immunoprecipitation-mass spectrometry (IP-MS); and second, comparing WT with Trim31. - / -Quantitative proteomic differences in macrophages after ox-LDL stimulation. Intersection analysis of the two datasets identified five candidate proteins, including upregulated LOX-1, TIAP, and KIF4, and downregulated TRIM31 and PDCL (Figure 5A). LOX-1 was highlighted as a key lipid uptake receptor and inflammatory regulator in the development of atherosclerosis. Combined with aortic single-cell transcriptome results, lipid metabolism and inflammation-related pathways were highly enriched in Trim31-deficient macrophages, further suggesting that LOX-1 is a key executor of TRIM31-mediated macrophage pathological effects.

[0076] Western blot results showed that Trim31 - / - LOX-1 protein levels were significantly elevated in macrophages, while other lipid processing-related proteins (ABCA1, ABCG1, SRA1) showed no significant changes (Figure 5B). Conversely, the mRNA levels of these genes (Abca1, Abcg1, Sra1, and Lox-1) did not differ statistically between the two groups, suggesting that TRIM31 regulation of LOX-1 mainly occurs at the post-transcriptional level. In vivo results also corroborated this. - / - Trim31 fl / fl Lyz2 cre LOX-1 protein expression was upregulated in mouse aortic macrophages, while it was downregulated in mice overexpressing Trim31 (Figures 5C-D), further supporting the negative regulation of LOX-1 by TRIM31.

[0077] In HEK-293T cells, TRIM31 overexpression dose-dependently reduced LOX-1 levels, while mutants lacking E3 ligase activity (TRIM31-C53 / C56A and TRIM31-ΔRing) failed to inhibit LOX-1 protein expression (Figure 6A), further confirming that TRIM31's E3 activity is essential for its regulation of LOX-1. Cycloheximide (CHX) tracking experiments showed that TRIM31 significantly accelerated LOX-1 protein degradation (Figure 5E), while proteasome inhibitors could reverse this effect (Figure 5F), suggesting that TRIM31 promotes LOX-1 degradation through a proteasome-dependent pathway. Knockdown of Trim31 in human hMDMs also led to an increase in LOX-1 protein levels (Figure 5G), further demonstrating that TRIM31 can also regulate LOX-1 protein homeostasis in human macrophages.

[0078] Co-IP and in vitro binding assays confirmed a direct interaction between TRIM31 and LOX-1: GFP-TRIM31 overexpressed in HEK-293T cells co-precipitated with Myc-LOX-1 (Fig. 5H), and the recombinant protein obtained through in vitro translation also directly bound (Fig. 5I). Furthermore, endogenous Co-IP results in mouse macrophages showed that ox-LDL stimulation enhanced their binding. Co-IP in human PBMCs further validated the physiological relevance of this interaction in clinical samples (Fig. 5J). Confocal microscopy results showed that TRIM31 and LOX-1 were colocalized in both HEK-293T and primary macrophages, with enhanced colocalization signals under ox-LDL conditions (Fig. 5K). Domain mapping analysis revealed that the coiled-coil midsection of TRIM31 is essential for binding with LOX-1.

[0079] At the functional level, Lox-1 will be integrated into Trim31. - / - Knockdown in macrophages significantly reversed the aforementioned enhanced foaming and pro-inflammatory cytokine expression (Figure 5 L-M), indicating that LOX-1 is a key downstream target mediating the pro-atherosclerotic effects of Trim31 deficiency. In summary, TRIM31 is a novel LOX-1 regulator that promotes proteasome-dependent degradation of LOX-1 through its E3 ubiquitin ligase activity, thereby regulating macrophage lipid uptake, foam cell formation, and inflammatory responses under atherosclerosis-related conditions.

[0080] TRIM31 mediates K48-linked ubiquitination of LOX-1 and localizes it to lysine 12, thereby limiting foam cell formation and inflammation. To further elucidate the molecular mechanism by which TRIM31 promotes LOX-1 degradation, we focused on its role in LOX-1 ubiquitination and its impact on foam cell formation. In HEK-293T cells, TRIM31 overexpression significantly enhanced the ubiquitination level of Myc-LOX-1, while the TRIM31-C53 / C56A and TRIM31-ΔRing mutants lacking E3 ligase activity could not induce this modification (Figure 6A), further confirming the essentiality of TRIM31's ubiquitination ligase activity. Further analysis revealed that TRIM31 selectively promotes K48-linked rather than K63-linked ubiquitination of LOX-1 (Figure 6B), and K48-linked ubiquitination is a classic proteasome degradation signal. Correspondingly, Trim31... - / - PMs and Apoe - / - Trim31 fl / fl Lyz2 cre K48-linked LOX-1 ubiquitination was significantly reduced in mouse aortic macrophages. Figure 6 C), similar results were observed in hMDMs with Trim31 knockdown, suggesting that this mechanism is species conserved. In vitro ubiquitination experiments using Promega's TNT in vitro transcription and translation system and Boston Biochem's ubiquitination reaction system (containing E1, UbcH5a and multiple ubiquitin variants: WT, K48-only, K63-only) further confirmed that TRIM31 can directly catalyze the K48-linked ubiquitination of LOX-1 (Figure 6D).

[0081] To identify key ubiquitination sites, we co-transfected Flag-LOX-1, GFP-TRIM31, and HA-K48 ubiquitin into HEK-293T cells. Subsequent liquid chromatography-mass spectrometry (LC-MS) analysis of the immunoprecipitated LOX-1 complex revealed that lysine 12 (K12) of LOX-1 was identified as a key residue in TRIM31-mediated ubiquitination (Figure 6E). Further analysis using a series of lysine→arginine mutants showed that only the K12R mutation significantly blocked TRIM31-mediated K48-linked ubiquitination and degradation of LOX-1, while other single-site or combined mutations (K7, K18, K22, K23, K25) had relatively small effects (Figures 6F-G), supporting the view that K12 is the main receptor site for TRIM31-catalyzed K48-linked ubiquitination of LOX-1. Further analysis of the Trim31... - / - Overexpression of WT-LOX-1 or K12R mutants in macrophages revealed that only WT-LOX-1 could restore TRIM31's inhibition of LOX-1 ubiquitination and protein levels, while the K12R mutant completely lost this regulation, further demonstrating that K12 site ubiquitination is crucial for TRIM31-mediated LOX-1 degradation.

[0082] To assess the pathophysiological significance of TRIM31-mediated LOX-1 K12 ubiquitination in atherosclerosis, we first constructed Lox-1 knockout mice using CRISPR-Cas9 and validated this finding using Western blot. Functional studies revealed that Lox-1... - / -Lipid accumulation and DiI-oxLDL uptake in macrophages were significantly reduced, while the expression of inflammatory factors was downregulated, and the phosphorylation levels of key proteins in inflammatory signaling pathways such as p65, ERK, JNK, and p38 were also significantly decreased, confirming that LOX-1 is the core receptor regulating macrophage lipid metabolism, foam cell formation, and inflammation amplification.

[0083] Subsequently, we visited Lox-1 - / - In macrophages, WT-LOX-1 (Ad-Lox-1(WT)) or the K12R mutant (Ad-Lox-1(K12R)) were reconstructed via adenovirus, and TRIM31 was overexpressed in conjunction with these reconstructed mutants. The results showed that in Lox-1 expressing WT-LOX-1... - / - In macrophages, TRIM31 overexpression significantly reduced ox-LDL-induced lipid accumulation and foam cell formation, while this protective effect was completely lost in cells expressing the K12R mutant LOX-1 (Fig. 6H-I). Meanwhile, in WT-LOX-1 reconstructed Lox-1... - / - In macrophages, TRIM31 effectively inhibited the expression of Tnf-α, Il-6, and Il-1β, while in cells reconstructed from the K12R mutant, its inhibitory effect on the expression of inflammatory factors was significantly weakened. Figure 6 J).

[0084] In summary, TRIM31 restricts macrophage foaming and inflammatory activation by promoting K48-linked ubiquitination of LOX-1 at lysine 12 and targeting proteasome degradation. This modification is crucial for regulating ox-LDL uptake and downstream pro-inflammatory signaling, making TRIM31 a key molecule for regulating macrophage function and preventing atherosclerosis.

[0085] Lox-1 downregulation reverses the worsening of atherosclerosis caused by macrophage-specific Trim31 deficiency in vivo. To verify the pathophysiological significance of the TRIM31-LOX-1 axis in the progression of atherosclerosis, we conducted an in vivo salvage experiment using AAV-mediated Lox-1 knockdown (Aav-shLox-1). Specifically, Apoe - / - Trim31 fl / fl Lyz2 cre Mice were randomly divided into Aav-shLox-1 and Aav-vector control groups and subjected to systemic drug intervention. There were no significant differences in blood lipid levels (TC, TG, LDL-C, HDL-C) among the four groups of mice (including the control genotype), excluding the interference of differences in systemic lipid metabolism.

[0086] Gross observation and en face Oil Red O staining results showed that macrophage-specific Trim31 deficiency significantly increased the burden of atherosclerotic plaques, while AAV-mediated Lox-1 knockdown significantly alleviated this phenotype. Figure 7 A). Cross-sectional analysis of the aortic root further confirmed Apoe - / - Trim31 fl / fl Lyz2 cre In mice, plaque area and the proportion of necrotic cores were significantly increased, while Aav-shLox-1 treatment significantly reduced these indicators (Figures 7B-C). Plaque composition analysis showed that TRIM31 deficiency was closely associated with unstable features such as increased lipid deposition, enhanced macrophage infiltration, reduced smooth muscle coverage, reduced collagen deposition, and thinning of the fibrous cap, leading to a higher vulnerable plaque index; while Lox-1 silencing significantly improved plaque stability in multiple dimensions (Figures 7D-F). In addition, the elevation of Tnf-α, Il-6, and Il-1β in aortic tissue was effectively inhibited by Aav-shLox-1 (Figure 7G). These results strongly demonstrate in vivo that LOX-1 is a key downstream mediator of the anti-atherosclerotic effect of TRIM31 in macrophages, and its knockdown can significantly alleviate the pro-atherosclerotic effect caused by TRIM31 deficiency.

[0087] LOX-1 K12 site-dependent ubiquitination and degradation are crucial for the in vivo anti-atherosclerotic effect of TRIM31. To further determine the functional importance of LOX-1 in the in vivo anti-atherosclerotic effect of TRIM31-mediated ubiquitination at lysine 12 (K12), we conducted experiments on Apoe. - / - Lox-1 - / - In mice, macrophage-specific overexpression of Trim31 mediated by AAV9 was used, and WT-LOX-1 (Lox-1-WT) or the ubiquitination-deficient mutant Lox-1-K12R were reconstructed to express the drug. The lipid levels (TC, TG, LDL-C, HDL-C) of mice in each group were similar, thus excluding the influence of differences in lipid levels.

[0088] In reconstructing the Apoe of Lox-1-WT - / - Lox-1 - / -In mice, Trim31 overexpression significantly reduced the burden of atherosclerosis, as evidenced by reduced plaque area in the face Oil Red O staining and aortic root cross-section (Fig. 8A-D). Simultaneously, it showed shrinkage of the necrotic core, increased collagen deposition, enhanced smooth muscle coverage, thickened fibrous cap, and a decrease in the vulnerable plaque index, indicating a significant improvement in plaque stability. Conversely, in mice reconstructed with the Lox-1-K12R mutant, Trim31 overexpression no longer significantly improved lesion area, plaque composition, or vulnerable plaque index; these indicators were essentially the same as those in the vector control group. Figure 8 EH) indicates that ubiquitination at the K12 site is a key prerequisite for TRIM31 to exert its protective effect in vivo.

[0089] Similarly, in reconstructing the Apoe of Lox-1-WT - / - Lox-1 - / - In the mouse aorta, overexpression of Trim31 significantly downregulated the expression of inflammatory factors Tnf-α, Il-6, and Il-1β (Fig. 8I); however, this inhibitory effect disappeared in mice with reconstructed Lox-1-K12R (Fig. 8J), further highlighting the importance of K12-specific ubiquitination for TRIM31 function.

[0090] To further verify the necessity of LOX-1 for TRIM31 activity, we conducted experiments on Apoe. - / - With Apoe - / - Lox-1 - / - Mice were treated with AAV9-Lyz2 overexpression of Trim31. Blood lipid levels were similar across groups. Results showed that in Apoe - / - In mice, Trim31 overexpression significantly reduced lesion area and necrotic core proportion, while increasing collagen deposition, enhancing smooth muscle coverage, thickening the fibrous cap, and reducing the vulnerable plaque index; however, in Apoe... - / - Lox-1 - / - In mice, Trim31 overexpression had almost no effect on plaque morphology, demonstrating that LOX-1 is essential for the anti-atherosclerotic effect of TRIM31. These changes are consistent with alterations in the expression of aortic inflammatory factors.

[0091] In in vitro experiments, we used Lox-1 - / - Trim31 was overexpressed in mouse-derived PMs. Consistent with in vivo results, Trim31 overexpression did not alter Lox-1 levels. - / -Lipid accumulation or DiI-oxLDL uptake in macrophages did not affect the expression of inflammatory genes or the secretion of cytokines. Western blot results further showed that, in the absence of LOX-1, Trim31 could not regulate ox-LDL-induced phosphorylation levels of the NF-κB and MAPK pathways.

[0092] In summary, these results demonstrate that ubiquitination of LOX-1 at lysine 12 is both necessary and a crucial structural basis for the inhibitory effect of TRIM31 on atherosclerosis. The TRIM31-LOX-1 axis constitutes a core molecular pathway regulating the progression and stability of atherosclerosis by modulating foam cell formation, inflammatory responses, and plaque vulnerability, providing a clear theoretical basis for targeted intervention strategies based on this axis.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting TRIM31 in the manufacture of any one or more of the following: (a1) an atherosclerosis diagnosis or auxiliary diagnosis product; (a2) an atherosclerosis prognosis evaluation or auxiliary prognosis evaluation product.

2. A system for atherosclerosis diagnosis and / or prognosis assessment, characterized in that, The system at least comprises: an acquisition module configured to acquire the expression level of TRIM31 of a subject; an evaluation module configured to evaluate the disease condition of the subject according to the expression level of TRIM31 obtained by the acquisition module.

3. The system of claim 2, wherein, The TRIM31 is a TRIM31 coding gene and / or a TRIM31 protein; The TRIM31 is derived from macrophages.

4. Use of TRIM31 as a target in screening atherosclerosis prevention and / or treatment drugs.

5. The use according to claim 4, wherein the compound is ###0002### The method for screening atherosclerosis prevention and / or treatment drugs comprises: 1) treating a system expressing and / or containing TRIM31 with a candidate substance; setting a parallel control without treating with the candidate substance; 2) after completing step 1), detecting the expression level of TRIM31 in the system; if the expression amount of TRIM31 in the system treated with the candidate substance is significantly increased compared with the parallel control, the candidate substance can be used as a candidate glioma drug.

6. Use of an expression promoter of TRIM31 in any one or more of the following: (a) manufacturing a product for inhibiting lipid accumulation and / or foam cell formation; (b) manufacturing a product for inhibiting inflammatory response; (c) manufacturing a product for promoting LOX-1 degradation; (d) manufacturing a product for preventing and / or treating atherosclerosis and related diseases.

7. Use according to claim 6, wherein The expression promoter of TRIM31 comprises a substance capable of promoting the expression and / or activity of TRIM31 gene and / or protein, and further comprises at least one of a recombinant expression vector containing a TRIM31 gene or a gene fragment having 90% or more homology with the TRIM31 gene, a polypeptide, a protein, a nucleic acid and a nucleic acid aptamer capable of up-regulating the expression of TRIM31 gene and / or TRIM31 protein, a polysaccharide, a natural active substance, and a biological agent; The recombinant expression vector comprises any one or more of a vector-based eukaryotic expression plasmid, an adenovirus, an adeno-associated virus, a lentivirus, a retrovirus, an LNP liposome, a microinjection technology, a gene editing system element, and a homologous recombination vector that specifically targets TRIM31 gene and / or TRIM31 protein.

8. The use according to claim 6, characterized in that, In the application (b), the inhibition of inflammatory response specifically manifests as inhibition of the expression of inflammatory factors and reduction of the phosphorylation level of key proteins in the inflammatory signaling pathway, the inflammatory factors include Tnf-α, Il-6 and Il-1β, and the key proteins in the inflammatory signaling pathway include p65, ERK, JNK and p38.

9. Use of an expression inhibitor of TRIM31 in constructing an atherosclerosis model.

10. The use according to claim 9, wherein the compound is ###0002### The atherosclerosis model is a cell model, a tissue model, an organ (or organoid) model, or a non-human animal model; the non-human animal model is a non-human mammal, and further a mouse. The expression inhibitor of TRIM31 includes a substance capable of inhibiting the expression and / or activity of the TRIM31 gene and / or protein, and specifically can include shRNA, small interfering RNA, dsRNA, microRNA, antisense nucleic acid, or a construct capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microRNA, antisense nucleic acid; and an antibody against the TRIM31 protein, and a compound-based inhibitor. The expression inhibitor of TRIM31 includes a substance capable of inhibiting the expression and / or activity of the TRIM31 gene and / or protein, and specifically can include shRNA, small interfering RNA, dsRNA, microRNA, antisense nucleic acid, or a construct capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microRNA, antisense nucleic acid; and an antibody against the TRIM31 protein, and a compound-based inhibitor.

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