Application of NAT10 and related biomolecules in the diagnosis and treatment of deep vein thrombosis

Abstract

This invention belongs to the fields of biomedicine and molecular biology, focusing on the application of NAT10 and related biomolecules in the diagnosis and treatment of deep vein thrombosis (DVT). Specifically, this invention has found that NAT10 is a key initiator of endothelial cell ferroptosis, and its expression is significantly increased in DVT. The results indicate that inhibiting NAT10 reduces ac4C levels in DVT, thereby alleviating endothelial ferroptosis. However, decreased NAT10 expression leads to reduced HMOX1 stability and decreased Fe... 2+ The release and inhibition of lipid peroxidation make targeting the NAT10 / HMOX1 axis a potential treatment for DVT, and therefore this invention has good potential practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of NAT10 and related biomolecules in the diagnosis and treatment of deep vein thrombosis. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Deep vein thrombosis (DVT) is a common peripheral vascular disease with an estimated annual incidence of over 1 in 1,000 people. The incidence of DVT is rising year by year, seriously impacting patients' health and quality of life. The most common causes of DVT are endothelial injury, blood stasis, and a hypercoagulable state. The underlying mechanisms of endothelial injury are crucial in the development of DVT. Despite significant efforts to explore the relevant underlying molecular mechanisms, feasible therapeutic targets for mitigating endothelial injury remain lacking in clinical practice.

[0004] Mounting evidence suggests a link between high iron levels and thrombosis, indicating that iron homeostasis plays a crucial role in thrombosis formation. Iron accumulation significantly accelerates thrombosis following vascular injury and increases vascular oxidative stress. Recent studies have shown that ferroptosis, a form of regulated cell death characterized by iron overload and lipid peroxidation, is closely associated with vascular injury. Furthermore, excessive free ferrous iron promotes ferroptosis by exacerbating intracellular oxidative stress. RNA modification may play a significant role in deep vein thrombosis (DVT), but its etiology remains unclear, requiring further investigation to identify novel targets for prevention and treatment.

[0005] n4-Acetylcytidine (ac4C) modification, a newly discovered mRNA modification, is considered a ubiquitous epigenetic marker of mRNA and plays a crucial role in regulating mRNA stability and translation efficiency. Acetyltransferase 10 (NAT10) is the only known ac4C-modifying enzyme in mammals, catalyzing the acetylation of cytidine residues in mRNA. Furthermore, NAT10 has been found in the nucleolus and regulates telomerase activity, ribosomal RNA transcription, and cytokinesis. Recent studies have revealed that NAT10-mediated ac4C acetylation is involved in various physiological and pathological processes, including aging, apoptosis, autophagy, and ferroptosis. However, the changes and roles of NAT10 in deep vein thrombosis (DVT) remain unclear.

[0006] Heme oxygenase 1 (HMOX1) is a ferroptosis activator that breaks down heme into carbon monoxide, biliverdin, and iron ions (Fe2+). 2+HMOX1 generates reactive oxygen species (ROS) via the Fenton reaction, promoting lipid peroxidation and accumulation. While studies have reported cytoprotective functions, increasing evidence suggests that HMOX1 can exhibit cytotoxic effects when its intracellular expression levels exceed a certain threshold. More importantly, HMOX1 overexpression promotes Fe... 2+ Overload leads to increased iron content, ROS generation, and exacerbated lipid peroxidation, resulting in endothelial cell ferroptosis. However, current research on the role of HMOX1 in the pathogenesis of deep vein thrombosis (DVT) is insufficient. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide the application of NAT10 and related biomolecules in the diagnosis and treatment of deep vein thrombosis (DVT). Specifically, the present invention has found that NAT10 is an important initiator of endothelial cell ferroptosis, and its expression is significantly increased in DVT. The results of this invention indicate that inhibiting NAT10 reduces ac4C levels in DVT, thereby alleviating endothelial ferroptosis. However, decreased NAT10 expression leads to reduced HMOX1 stability and decreased Fe... 2+ The release and inhibition of lipid peroxidation. Therefore, targeting the NAT10 / HMOX1 axis may be a potential treatment for DVT. Based on the above research findings, this invention is thus completed.

[0008] Specifically, the technical solution of the present invention is as follows:

[0009] In a first aspect, the invention provides the use of reagents for detecting the expression levels of NAT10 and related biomolecules in the preparation of deep vein thrombosis detection products.

[0010] Furthermore, the deep vein thrombosis detection product can be used for screening, (auxiliary) diagnosis, monitoring, or prognosis of deep vein thrombosis.

[0011] Specifically, the expression levels of NAT10 and its related biomolecules are positively correlated with the occurrence and development of deep vein thrombosis (DVT). In particular, NAT10 is significantly upregulated in venous tissue and is positively correlated with the severity of DVT. Therefore, it can be used for screening, (auxiliary) diagnosis, monitoring, or prognosis of DVT.

[0012] The related biomolecules of NAT10 include ac4C modification and HMOX1.

[0013] The substances used to detect the expression levels of NAT10 and its related biomolecules may include reagents for detecting the expression levels of genes encoding NAT10 and its related biomolecules based on real-time quantitative PCR, in situ hybridization, gene chips, and gene sequencing, and / or reagents for detecting the protein (enzyme) expression levels of NAT10 and its related biomolecules based on immunoassay methods.

[0014] In this invention, the product may be primers, probes, (gene or protein) chips, detection kits, detection devices, and detection equipment, etc.

[0015] A second aspect of the present invention provides a system for detecting deep vein thrombosis, the system comprising:

[0016] The acquisition module is configured to acquire the expression levels of NAT10 and its related biomolecules in the test samples of the subjects.

[0017] The analysis module is configured to analyze and judge the subject's disease status based on the expression levels of NAT10 and related biomolecules obtained by the acquisition module.

[0018] The related biomolecules of NAT10 include ac4C modification and HMOX1.

[0019] The sample to be tested can be a blood sample from the subject, or more specifically, a mononuclear cell sample from peripheral blood.

[0020] The detection of deep vein thrombosis can specifically be used for screening, (auxiliary) diagnosis, monitoring, or prognosis of deep vein thrombosis.

[0021] A third aspect of the invention provides the use of NAT10 and related biomolecules as targets in the preparation and / or screening of drugs for deep vein thrombosis.

[0022] Furthermore, based on the effects of candidate drugs on NAT10 and its related biomolecules before and after use, it can be determined whether candidate drugs can be used for the prevention and / or treatment of deep vein thrombosis.

[0023] Further methods for screening drugs to prevent or treat deep vein thrombosis include:

[0024] (I) Treat systems expressing and / or containing NAT10 and its related biomolecules with candidate substances; set up a control group that is not treated with candidate substances;

[0025] (II) After completing step (I), detect the expression level of NAT10 and its related biomolecules in the system; if the expression level of NAT10 and its related biomolecules in the system treated with the candidate substance is significantly downregulated compared with the control, the candidate substance can be used as a candidate drug for the prevention or treatment of deep vein thrombosis.

[0026] The related biomolecules of NAT10 include ac4C modification and HMOX1.

[0027] More specifically, step 2) is as follows: if the expression levels of NAT10 and / or acetylated HMOX1 are significantly downregulated in the system treated with the candidate substance, then the candidate substance can be used as a candidate drug for the prevention or treatment of deep vein thrombosis.

[0028] The system may be a solution system, cell system, tissue system, organ system, or animal system, without any specific limitation.

[0029] A fourth aspect of the invention provides the use of a substance that inhibits NAT10 expression or reduces its activity in any one or more of the following:

[0030] (a) Inhibiting HMOX1 expression or preparing products that inhibit HMOX1 expression;

[0031] (b) Inhibiting ferrodeath and iron overload or preparing products that inhibit ferrodeath and iron overload;

[0032] (c) Inhibiting the formation of lipid peroxides or preparing products that inhibit the formation of lipid peroxides;

[0033] (d) Products for the prevention and / or treatment of deep vein thrombosis.

[0034] Among them, substances that inhibit NAT10 expression or reduce its activity include, but are not limited to, RNA interference molecules or antisense oligonucleotides targeting NAT10, small molecule inhibitors (such as Remodelin), siRNA, shRNA, substances that carry out lentiviral infection or gene knockout, etc.

[0035] In (a)-(c), the product can act on endothelial cells, specifically venous endothelial cells (such as human umbilical vein endothelial cells).

[0036] The product can be a drug or an experimental reagent for non-pharmaceutical purposes, which can be used for basic research. For example, the product can be used to regulate ferroptosis in endothelial cells in vivo or in vitro, thereby preparing endothelial cell-related biological models and laying a material basis for the study of the mechanisms of diseases such as deep vein thrombosis.

[0037] According to the present invention, when the product is a drug, the drug further includes at least one inactive pharmaceutical ingredient.

[0038] The inactive pharmaceutical ingredient may be a carrier, excipient, or diluent commonly used in pharmaceuticals. Furthermore, the carriers, excipients, and diluents that may be included are well-known in the art, and those skilled in the art can determine that they meet clinical standards.

[0039] The medicament of the present invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via single or multiple doses. It will be understood by those skilled in the art that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0040] The drug can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, and chimpanzees.

[0041] A fifth aspect of the present invention provides a method for preventing and / or treating deep vein thrombosis, the method comprising administering to a subject the aforementioned substance that inhibits NAT10 expression or reduces its activity and / or the corresponding product.

[0042] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0043] The aforementioned technical approach reveals that NAT10 is a crucial regulator of ferroptosis in deep vein thrombosis (DVT). Specifically, NAT10 mediates ac4C modification to increase HMOX1 stability, leading to iron overload and lipid peroxidation, thus forming a positive feedback loop that exacerbates DVT. These results suggest that targeting NAT10 may be a promising therapeutic strategy for enhancing endothelial cell ferroptosis, and therefore possesses significant potential for practical application. Attached Figure Description

[0044] 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.

[0045] Figure 1In this embodiment of the invention, ferroptosis is involved in the formation of DVT; (A) mRNA microarray analysis of peripheral blood mononuclear cells, KEGG enrichment analysis of the top 10 pathways. (B) The top 10 KEGG pathways enriched in DVT mice (with thrombus) according to 4D Label Free. (C) Transmission electron microscopy showing mitochondrial morphology and cristae (red arrows) in vascular tissue. Scale bar = 1 micrometer. (DF) Ferrous ions (Fe) in DVT mice. 2+ Levels of malondialdehyde (MDA) and glutathione (GSH). (G, H) Rats treated with Ferrostatin-1 (Fer-1) were monitored by ELISA to detect plasma levels of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1). (I, J) Representative images of thrombi in each group were obtained by HE staining (magnification, ×100) and vascular ultrasound examination after Fer-1 treatment. Scale bar = 200 μm. ***P < 0.001.

[0046] Figure 2 In this embodiment of the invention, NAT10 knockdown significantly reduced ferrodesorption in DVT mice; (A) Dot blotting showed that the total ac4C level in the vascular tissue of DVT mice was high (n=5). (B) Real-time quantitative PCR was used to detect the relative expression level of NAT10 mRNA in DVT mice (n=10). (C) Volcano plots showed increased NAT10 expression in DVT mice. (D) Western blotting was used to detect NAT10 protein levels. (E, F) HE staining and vascular ultrasound were used to detect representative images of thrombi in NAT10 knockdown DVT mice. Scale bar = 200 μm. (GI) Fe in NAT10 knockdown DVT mice was detected. 2+ MDA and GSH levels. ***P<0.001.

[0047] Figure 3 In this embodiment of the invention, NAT10 deficiency inhibits ferroptosis in HUVECs; (A) Cell viability was detected using a CCK8 assay kit. (B) HUVECs were treated with RSL3 (4 μM) / FINO2 (40 μM) or remodeling (20 μM) for 12 h (magnification, x100). (C, E) Lipid peroxidation was detected by BODIPY™ 581 / 591C11 probe after NAT10 inhibition, followed by HUVECs treated with RSL3 (4 μM) or FINO2 (40 μM) for 12 h (magnification, ×600). Green images represent oxidized lipids, while red images represent non-oxidized lipids. Scale bar = 10 μm. (D, F) Fe2+ was observed in HUVECs after NAT10 inhibition by the FerroOrange probe, followed by 12 h of treatment with RSL3 or FINO2. 2+(Magnification, ×600). Red in the image represents Fe. 2+ Blue represents the cell nucleus. Scale bar = 10 μm. (G, H) MDA and GSH levels in HUVECs. *P<0.05,**P<0.01,***P<0.001.

[0048] Figure 4 In this embodiment of the invention, NAT10 mediates HMOX1 mRNA acetylation in vitro; (A) acRIP-seq analysis showed highly enriched ac4C motifs. (B) Venn diagrams showed mRNA prediction using acRIP-seq, 4D Label Free, and FerrDb V2 Database. (C) gRT-PCR was used to detect mRNA levels in vascular tissues of DVT mice (n=10) and controls (n=10). (D) gRT-PCR was used to detect the mRNA levels of NAT10 and HMOX1 in si-NAT10s. (E) Western blot was used to detect the HMOX1 protein level after NAT10 inhibition. (F) acRIP-qPCR was used to detect the ac4C modification level of HMOX1 mRNA. (G) NAT10RIP-qPCR was used to analyze HMOX1 mRNA in HUVECs. (H) The stability of HMOX1 was detected after treatment with actinomycin D. (IL) gRT-PCR and Western blotting were used to detect the mRNA and protein levels of NAT10 and HMOX1 in HUVECs and C166 cells in each treatment group. (M, N) MDA and GSH levels in HUVECs and C166 cells. *P<0.05,**P<0.01,***P<0.001.

[0049] Figure 5 In this embodiment of the invention, NAT10 downregulation alleviates ferroptosis by reducing HMOX1 expression. (A, B) Representative thrombus images of each group were detected by color Doppler ultrasound of blood vessels after treatment with HE staining (magnification, x100) and ZnPP (HMOX1 inhibitor). Scale bar = 200 μm. (CE) Fe in DVT mice 2+ (F) HUVECs cell viability was detected using the CCK8 assay kit. (G) Fe levels in HUVECs after 12 h of treatment with NAT10 and CoPP were inhibited. 2+ Horizontal, red in the figure represents Fe 2+Blue represents the cell nucleus (magnification × 600). Scale bar = 10 μm. (H, I) Protein levels of HMOX1 and GPX4 in si-NAT10 were detected by Western blotting. (J, K) MDA and GSH levels were determined in si-NAT10. **P < 0.01, ***P < 0.001.

[0050] Figure 6 In this embodiment of the invention, knocking out NAT10 inhibits HMOX1 and reduces the formation of deep vein thrombosis (DVT) in vivo. (A, B) NAT10 f / f Cdh5-Cre + Representative images of thrombi in (NAT10 knockout) DVT mice were obtained by HE staining (magnification, x100) and vascular color ultrasound examination. Scale bar = 200 μm. (CE) Fe in different treatment groups 2+ (F) Gluco-RT-PCR was used to detect the expression of HMOX1 in different treatment groups. (G) Western blot was used to detect the expression of HMOX1 and GPX4 in different treatment groups. (HK) Plasma ET-1, eNOS, TNF-α, and TGF-β1 were detected by ELISA. Detailed Implementation

[0051] 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.

[0052] 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.

[0053] The present invention will now 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 reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0054] 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. The following examples illustrate test methods with specific conditions, which are generally performed under conventional conditions.

[0055] Example

[0056] 1. Methods and Materials

[0057] 1.1 Microarray Analysis

[0058] Genome-wide analysis of mRNA expression in peripheral blood mononuclear cells from 6 patients with deep vein thrombosis (DVT) and 6 controls was performed using Human mRNA (4*180K, design ID: 084410). Sample labeling, microarray hybridization, and washing were performed by OE Biotech.

[0059] 1.2 Proteomics Analysis

[0060] Vascular tissues were collected from DVT mice (thrombotic group, n=6), DVT mice (non-thrombotic group, n=6), and control group (n=6) for proteomics analysis. Proteins were extracted by centrifugation and then subjected to 4D LFQ proteomics analysis, including liquid chromatography-MS / MS analysis and data analysis.

[0061] 1.3 Cell Culture and Transfection

[0062] Human umbilical vein endothelial cells (HUVECs) and C166 cells were obtained from Procell Life Technology Co., Ltd. (Wuhan, China). Cells were transfected with siRNA or a negative control (GenePharma, Shanghai, China) using Lipofectamine RNAIMAX (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions.

[0063] 1.4 Determination of Iron Content

[0064] The Fe content in vascular tissue was analyzed using a ferrous ion content assay kit (BC5415, Solarbio, Beijing, China). 2+ The relative content of.

[0065] 1.5 Determination of malondialdehyde (MDA) levels

[0066] The treated cells and veins were lysed by sonication, homogenized in an ice bath, and then centrifuged at 8,000 x g for 10 min at 4 °C. Relative MDA levels were detected using an MDA assay kit (BC0025, Solarbio, Beijing, China).

[0067] 1.6 Glutathione (GSH) Measurement

[0068] The treated cells and veins were lysed by sonication, homogenized in an ice bath, and then centrifuged at 8,000 x g for 10 min at 4 °C. Relative glutathione levels were determined using a reduced glutathione assay kit (BC1175, Solarbio, Beijing, China).

[0069] 1.7 Determination of lipid peroxidation (LPO)

[0070] Pretreated cells were incubated with BODIPY™ 581 / 591C11 probes (Invitrogen, Carlsbad, USA) at 37°C for 30 min. Cells were then seeded in confocal culture dishes and treated with ferroptosis activator (with or without NAT10 inhibitor) for 12 h. After washing three times with PBS, cell nuclei were stained with Hoechst (Beyotime, Shanghai, China). LPO fluorescence was detected using a scanning confocal microscope (LSM 880, Carl Zeiss AG, Oberkochen, Germany).

[0071] 1.8 Immunofluorescence Fe 2+ analyze

[0072] Detection of Fe by Immunofluorescence Staining 2+ Cells were seeded horizontally onto confocal culture dishes and treated with RSL3 or FINO2 for 12 hours, with or without the addition of NAT10 inhibitor. Cells were washed three times with PBS and stained with Hoechst. Finally, images were taken using a scanning confocal microscope.

[0073] 1.9 Cell viability assay

[0074] Cells were seeded in 96-well plates at a density of 8 × 10⁸ cells per well. 3 Cells. After each treatment, the culture medium was changed to 100 μL DMEM and 10 μL CCK8 solution (Beyotime, Shanghai, China) per well according to experimental requirements. After incubation at 37°C for 1 h, the absorbance was measured at 450 nm.

[0075] 1.10 Real-time quantitative PCR (qRT-PCR)

[0076] Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, USA) according to specifications. Approximately 1 μg of RNA was reverse transcribed into cDNA, and qRT-PCR analysis was performed using SYBR Green (Invitrogen, Carlsbad, USA). The results were then analyzed according to 2... -ΔΔCt The formula calculates the relative mRNA level.

[0077] 1.11 RNA Immunoprecipitation (RIP)

[0078] After ligating magnetic beads with NAT10 or IgG antibodies, the antigen was captured using a RIP kit, and then RNA was extracted and verified using qRT-PCR.

[0079] HUVECs were transfected using acetylated RNA immunoprecipitation sequencing (acRIP-seq) and sequenced via Yunxu Technology Co., Ltd. (Shanghai, China). RNA was first extracted from the samples (using TRIzol), and quality control was performed to determine its concentration and purity. Total RNA was immunoprecipitated according to the manufacturer's instructions. The samples were then sequenced using the NovaSeq platform (Illumina).

[0080] 1.12 ac4C-RIP

[0081] Testing and use The ac4C RIP kit (GS-ET-005, Cloudseq Biotech, Shanghai, China) was used according to the manufacturer's instructions. In short, total RNA (200 μg) was randomly digested into nucleotide chains of 100–200 bp, and a mixture of 5 μg ac4C antibody and magnetic beads was incubated with the disrupted RNA. HMOX1 mRNA enrichment was analyzed by qRT-PCR.

[0082] 1.13 Western Blotting

[0083] Samples were lysed in RIPA buffer with protease and phosphatase inhibitors (Solarbio, Beijing, China), treated with SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were then incubated with diluted primary antibodies and horseradish peroxidase-labeled secondary antibodies. Antibodies against NAT10 (1:1000, ab194297, Abcam, Cambridge, USA), HMOX1 (1:1000, ab68477, Abcam, Cambridge, USA), GPX4 (1:1000, ab125066, Abcam, Cambridge, USA), and GAPDH (1:1000, ab181603, Abcam, Cambridge, USA) were used in this study.

[0084] 1.14 RNA ac4C dot blot method

[0085] Total RNA was extracted from venous vessels and denatured at 95°C for 3 minutes. 5 μg of RNA was loaded onto a nylon membrane and cross-linked at 37°C for 30 minutes. The membrane was blocked and incubated overnight at 4°C with anti-ac4c antibody (ab252215, 1:500, Abcam, Cambridge, USA). After incubation with horseradish peroxidase-labeled anti-rabbit IgG secondary antibody (1:4000, ZGB-Bio, Beijing, China), the membrane was imaged using a Tanon 4800 multichemiluminescence imaging system (Tanon, Shanghai, China).

[0086] 1.15 RNA stability analysis

[0087] HUVECs were transfected with NC or si-NAT10s (sequences shown in Table 1). Cells were then treated with actinomycin D (AbMole, Houston, USA) at a final concentration of 5 μg / ml. Cells were collected at 0, 2, 4, and 6 h, and RNA was extracted for qRT-PCR.

[0088] Table 1 siRNA and NC sequences

[0089]

[0090] 1.16 Construction and generation of endothelial cell-specific NAT10 gene knockout mice

[0091] Endothelial cell-specific NAT10 gene knockout mice were purchased from Cyagen Biosciences (Suzhou, China). flox / flox Cdh5-Cre ERT2 Mice and Cdh5-Cre ERT2 Mice were mated and intraperitoneally injected with tamoxifen (60 mg / kg) daily for one week to establish endothelial cell-specific NAT10 conditional knockout mice (NAT10...). flox / flox Cdh5-Cre ERT2 PCR was used to identify the NAT10 gene knockout mouse genotype. Western blot was used to identify NAT10. flox / flox Cdh5-Cre ERT2 (NAT10 f / f Cdh5-Cre + Knockout efficiency in mice.

[0092] 1.17 DVT mouse model and treatment

[0093] Wild-type mice (C57BL / 6J, 8-week-old males) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). Animal experiments were approved by the Ethics Committee of Shandong University of Traditional Chinese Medicine. A DVT mouse model was constructed using the inferior vena cava (IVC) stenosis method.

[0094] Ferroplasmosis inhibitor experiment: Mice were randomly divided into 3 groups (15 mice / group): (1) sham operation group; (2) DVT group; (3) DVT + Ferrostatin-1 (Fer-1, ferroplasmosis inhibitor, aladdin) group. Fer-1 (5 mg / kg) was injected intraperitoneally for 3 consecutive days before and after inferior vena cava stenosis surgery.

[0095] HMOX1 inhibition was performed on mice, which were randomly divided into two groups (15 mice per group): (1) DVT group; (2) DVT + zinc protoporphyrin IX (ZnPP, HMOX1 inhibitor, aladdin, China) group. ZnPP (10 mg / kg) was administered intraperitoneally 3 days before IVC stenosis and continued for 3 days after surgery.

[0096] NAT10 inhibition treatment experiment: Mice were randomly divided into 3 groups (15 mice / group): (1) sham operation group; (2) DVT group; (3) DVT + Remodelin (NAT10 inhibitor, aladdin, China) group. NAT10 inhibitor (10 mg / kg) was injected via the tail vein for 3 consecutive days before inferior vena cava stenosis and for 3 consecutive days after surgery.

[0097] In the HMOX1 overexpression experiment, mice were randomly divided into 3 groups (15 mice / group): (1) DVT group (NAT10 f / f Cdh5-Cre + (2) DVT nat10 knockout group (NAT10) f / f Cdh5-Cre + (3) DVT nat10 gene knockout + cobalt protoporphyrin IX (CoPP, HMOX1 activator, Aladdin, China) group. CoPP (15mg / kg) was injected intraperitoneally for 3 consecutive days to establish an inferior vena cava stenosis model, and then injected intraperitoneally for 3 consecutive days after surgery.

[0098] Fresh specimen sections 2 mm below the ligation site of the inferior vena cava were analyzed for hematoxylin and eosin (H&E).

[0099] 1.8 Transmission Electron Microscopy (TEM) Analysis

[0100] Morphological characteristics of ferroptosis in vascular tissue, TEM images taken by Hitachi HT-7800 transmission electron microscope (Hitachi, Ibaraki Prefecture, Japan).

[0101] 1.19 Mouse Doppler ultrasound

[0102] Mice underwent vascular Doppler ultrasound examination under isoflurane-oxygen co-anesthesia. Thrombosis images were acquired using a small animal ultrasound imaging system (VINNO6 LAB, VINNO, Suzhou, China). Sections were embedded in paraffin and stained with hematoxylin and eosin (HE) according to standard procedures.

[0103] 1.20 ELISA

[0104] The protein expression of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) was detected using a mouse ELISA kit.

[0105] 1.21 Statistical Analysis

[0106] Results are expressed as mean SEM and compared using two-tailed Student's t-test or one-way ANOVA. Statistical analysis was performed using GraphPad Prism 8.0 software. Unless otherwise stated, all experiments had at least three independent representative trials. A p-value < 0.05 was considered statistically significant.

[0107] 2 Experimental Results

[0108] 2.1 Inhibiting ferroptosis can improve the formation of DVT.

[0109] KEGG analysis was used to analyze the correlation between differentially expressed genes upregulated by CHIP (P<0.05, FC>1.5) and 4D Label Free (P<0.05, FC>2) to elucidate the physiological function and pathogenesis of DVT. Among the top 10 significantly enriched pathways, we found significant differential enrichment in ferroptosis-related pathways (…). Figure 1 A, B). Similarly, the results showed that mitochondria in the vascular endothelial cells of DVT mice were shrunken and cristae were reduced ( Figure 1 C). Fe in DVT mice 2+ MDA levels were significantly elevated (Figure ID, E). Furthermore, GSH expression was decreased in DVT mice. Figure 1 F). To further confirm the dominant role of ferroptosis in deep vein thrombosis, we treated mice with an intraperitoneal injection of a ferroptosis inhibitor (Fer-1) before establishing a deep vein thrombosis model. As expected, Fer-1 treatment significantly reduced thrombus size and alleviated vascular endothelial damage (F). Figure 1 These results suggest that ferroptosis plays an important role in the formation of DVT.

[0110] 2.2 NAT10 exacerbates endothelial ferroptosis and ac4C RNA modification in DVT

[0111] Total ac4C acetylation of RNA, measured quantitatively by dot blot analysis, was significantly increased in the vascular endothelial tissue of DVT. Figure 2 A). Similarly, in the 4D Label Free experiment, NAT10, acting as a "writer" for ac4C synthesis, was significantly highly expressed in the vascular endothelial tissue of DVT mice. Figure 2 B). Further validation with a larger sample size revealed a significant increase in NAT10 in DVT mice. Figure 2 (C, D). To test this hypothesis, we pretreated mice with a NAT10 inhibitor, which reduced thrombus formation (Figure S2A, B). To investigate the role of NAT10 in endothelial cell ferroptosis, we used NAT10... f / + Mice and Cdh5-Cre ERT2 Through mouse hybridization, endothelial cell-specific NAT10 knockout mice were established. As expected, NAT10 silencing significantly reduced thrombus formation. Figure 2 E, F). Simultaneously, inhibition of NAT10 alleviates ferroptosis in endothelial cells of DVT mice (E, F). Figure 2 In summary, these results indicate that inhibiting NAT10 helps alleviate ferroptosis formation in DVT.

[0112] 2.3 Downregulating NAT10 can increase the ability to inhibit ferroptosis in vitro.

[0113] To further emphasize the role of NAT10 in vascular endothelial cell ferroptosis, we conducted in vitro loss-of-function experiments. We induced ferroptosis in HUVECs cells using two different ferroptosis inducers (RSL3 and FINO2). Inhibition of NAT10 significantly alleviated the RSL3 / FINO2-induced decrease in cell viability. Figure 3 A). Furthermore, after inhibiting NAT10, the ferroptosis morphology was significantly restored ( Figure 3 B). At the cellular level, we investigated changes associated with ferroptosis in depth. Downregulation of NAT10 led to RSL3 / FINO2-induced reductions in LPO and Fe. 2+ The level decreased significantly ( Figure 3 CF). Simultaneously, we observed that the absence of NAT10 had a direct impact on other key markers of ferroptosis. Specifically, the absence of NAT10 reduced the expression of MDA ( ). Figure 3 G), increased the expression of GSH induced by RSL3 / FINO2 in vitro ( Figure 3 In summary, these data indicate that NAT10 deficiency significantly inhibits ferroptosis in vitro.

[0114] 2.4NAT10 stabilizes HMOX1 by inducing ac4C modification of HMOX1.

[0115] To investigate the potential mechanisms by which NAT10 regulates vascular endothelial cell function, we performed RIP-seq on negative controls (NC) and si-NAT10 in HUVECs. Sequence analysis of ac4C modification showed that the typical CXXCXXCXX motif was highly enriched within the ac4C site. Figure 4 A). Consistent with previous reports, we found that in NC and si-NAT10, the ac4C peak was mainly located in the protein-coding region (CDS) of the mRNA transcript. Since NAT10 promotes ac4C modification and RNA stability, we selected downregulated genes from 4D Label Free (FC>2, P<0.05), FerrDb V2 Database (Driver), and acRIP-seq (FC>2, P<0.05) for Venn plotting. The results showed that AGPS, LGMN, FAR1, and HMOX1 were screened from the overlapping regions. Figure 4 B). Of these four genes, HMOX1 was significantly upregulated in DVT mice. Figure 4 C). Knocking down NAT10 significantly reduced HMOX1 mRNA and protein levels. Figure 4 D, E). Similarly, acRIP-qPCR results showed that the level of ac4c acetylated HMOX1 mRNA was continuously reduced in si-NAT10 HUVECs. Figure 4 F). We observed via RIP-qPCR that NAT10 can bind to HMOX1 mRNA (F). Figure 4 G). More importantly, inhibition of NAT10 promotes actinomycin D-induced degradation of HMOX1 mRNA (G). Figure 4 Therefore, we hypothesize that NAT10 regulates ferroptosis by acetylation of HMOX1 mRNA.

[0116] Investigating the function of NAT10-mediated HMOX1 inhibition, knocking down NAT10 inhibits the decrease in HMOX1 expression during ferroptosis. Figure 4 Furthermore, inhibition of NAT10 significantly reduced MDA expression during ferroptosis and promoted an increase in GSH (IL). Figure 4 (M, N). Overall, NAT10 promotes the stability of HMOX1 mRNA through ac4C modification, thereby regulating endothelial cell function.

[0117] 2.5 Inhibition of NAT10 can alleviate endothelial dysfunction by inhibiting HMOX1.

[0118] It has been reported that upregulation of HMOX1 is the cause of increased ferroptosis in endothelial cells of diabetic patients

[26] . To confirm whether HMOX1 alters the formation of DVT, we injected mice with ZnPP to neutralize the function of HMOX1. HE staining and Doppler showed that the thrombus volume in DVT mice treated with ZnPP decreased synchronously ( Figure 5 A, B), while ZnPP treatment reduced Fe2t and MDA levels in DVT mice ( Figure 5 C, D), ZnPP treatment increased GSH expression in DVT mice (C, D), Figure 5 E).

[0119] Furthermore, to further emphasize the role of HMOX1, we used CoPP (an HMOX1 agonist) to increase HMOX1 expression. The results showed that overexpression of HMOX1 significantly inhibited the cell viability of HUVECs. Figure 5 F). Meanwhile, overexpression of HMOX1 significantly increased Fe in vitro. 2+ level( Figure 5 G). It is worth noting that overexpression of HMOX1 leads to the accumulation of Fe. 2+ This further exacerbates ferroptosis in endothelial cells. Inhibition of NAT10 reduces MDA levels and increases the expression of GSH and GPX4, thereby reducing ferroptosis in endothelial cells. Figure 5 In summary, these data indicate that inhibiting NAT10 reduces HMOX1 expression, alleviates ferroptosis, and thus mitigates endothelial dysfunction.

[0120] 2.6NAT10 mitigates DVT formation by regulating HMOX14.

[0121] To verify the role of NAT10 in regulating HMOX1 and altering DVT formation, NAT10 knockout mice were injected with CoPP to activate HMOX1. Based on HE staining and Doppler ultrasound results, we found that thrombus reduction was observed in NAT10 knockout DVT mice. Figure 6 A, B). NAT10 silencing significantly reduced Fe 2+ And the expression of MDA, while the activation of HMOX1 actually increased ferroptosis ( Figure 6 C, D). Simultaneously, NAT10 silencing increases GSH and GPX4 expression, while HMOX1 activation has the opposite effect. Figure 6 E, G). Notably, NAT10 silencing leads to decreased HMOX1 expression (E, G). Figure 6 F, G). Knockdown of NAT10 significantly reduced the levels of ET1 and TNF-α in DVT mice and increased the levels of eNOS and TGF-β1. Figure 6These data indicate that inhibiting NAT10 can reduce HMOX1 expression, inhibit ferroptosis, and thus alleviate endothelial dysfunction and thrombus formation in DVT mice.

[0122] In summary, the current study found that NAT10 expression was significantly upregulated in the venous tissue of DVT mice compared to sham-operated mice, and this upregulation was positively correlated with disease severity. Notably, inhibiting NAT10 reduced thrombus formation in DVT mice. To emphasize the significance of NAT10, we constructed NAT10 knockout mice in endothelial cells to further explore its role. Using this mouse model, we found that NAT10 silencing may be a feasible therapeutic intervention for the prevention and treatment of DVT. Furthermore, silencing NAT10 reduced DVT formation by decreasing endothelial cell ferroptosis. Our study is the first to demonstrate that NAT10-mediated ac4C modification regulates HMOX1 mRNA stability and leads to aberrant upregulation of HMOX1 in DVT. Therefore, we hypothesize that NAT10 promotes ferroptosis by upregulating HMOX1, leading to iron overload and inhibiting lipid peroxide production, supporting our research data. These findings provide a new explanation for how NAT10-mediated ac4C modification functions in DVT. This study also found that HMOX1 promotes the release of free iron, leading to ferroptosis and ultimately promoting thrombus formation. Furthermore, we discovered that HMOX1 expression is highly upregulated in DVT, and inhibiting HMOX1 can reduce thrombus occurrence. Therefore, further inhibiting HMOX1 expression by knocking down NAT10 can not only suppress iron but also reduce lipid peroxidation, thereby further improving endothelial cell ferroptosis. Therefore, targeting NAT10 may be a promising therapeutic strategy for enhancing endothelial cell ferroptosis.

[0123] Matters not covered in this invention are common knowledge.

[0124] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting NAT10 expression levels in the preparation of deep vein thrombosis detection products.

2. The application as described in claim 1, characterized in that, The deep vein thrombosis detection product is used for screening, auxiliary diagnosis, diagnosis, monitoring, or prognosis of deep vein thrombosis.

3. The application as described in claim 1, characterized in that, The reagents for detecting NAT10 expression levels include reagents for detecting the expression level of the NAT10-encoding gene based on real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing, and / or reagents for detecting the protein expression level of NAT10 based on immunoassay methods. The products include primers, probes, chips, detection kits, detection devices, and detection equipment. The chips include gene chips and protein chips.

4. A system for detecting deep vein thrombosis, characterized in that, The system includes: The acquisition module is configured to acquire the expression level of NAT10 in the test sample of the subject. The analysis module is configured to analyze and judge the disease status of the subject based on the expression level of NAT10 obtained by the acquisition module.

5. The system as described in claim 4, characterized in that, The sample to be tested is a blood sample from the subject; The blood sample is a mononuclear cell sample from peripheral blood.

6. The system as described in claim 4, characterized in that, The detection of deep vein thrombosis specifically refers to the screening, auxiliary diagnosis, diagnosis, monitoring, or prognosis of deep vein thrombosis.

7. The use of Remodelin in the preparation of products for the prevention and / or treatment of deep vein thrombosis.

8. The application as described in claim 7, characterized in that, The product acts on endothelial cells; The endothelial cells are venous endothelial cells; The product is a drug or a laboratory reagent for non-medical purposes.

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