Application of selenoprotein M in the preparation of drugs for the treatment of abdominal aortic aneurysms

Abstract

This invention discloses the application of selenoprotein M in the preparation of drugs for treating abdominal aortic aneurysms. This invention also provides the application of selenoprotein M in the preparation of drugs for reducing macrophage inflammation. This invention can effectively delay the progression of abdominal aortic aneurysms and reduce macrophage inflammation, and has broad prospects for medical applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of selenoprotein M in the preparation of drugs for the treatment of abdominal aortic aneurysms. Background Technology

[0002] Abdominal aortic aneurysm (AAA) refers to a localized, aneurysmal dilation of the abdominal aorta, diagnosed when its diameter exceeds 1.5 times the normal value (or increases by more than 50%). Currently, surgical treatment and management of risk factors are the main treatment options for AAA. However, surgical treatment is often accompanied by serious complications such as kidney injury, spinal cord injury, and arterial intimal leakage, and there is no significant benefit for AAA patients with aneurysm diameter <55 mm (male) or <50 mm (female). While interventions targeting risk factors such as smoking cessation, blood pressure control, and lipid-lowering can slow the progression of AAA, their therapeutic effects are limited. Therefore, there is an urgent clinical need to find new drugs for the treatment of AAA.

[0003] Inflammatory cell infiltration and a strong inflammatory response play a crucial role in the initiation and progression of aortic aneurysm (AAA). Clinical studies have found a high correlation between aortic wall inflammation in AAA patients and the dilation and rupture of AAA. Single-cell sequencing technology has confirmed that AAA is a chronic inflammatory disease in which activated macrophages play a vital role. Macrophages infiltrating the adventitia primarily mediate inflammation by secreting chemokines and cytokines, leading to aortic wall cell apoptosis and vascular smooth muscle cell phenotypic transformation, resulting in aortic dilation and rupture. However, the specific molecular mechanisms of macrophage infiltration and inflammation in the progression of AAA are not yet fully elucidated.

[0004] Selenium is an essential trace element that the human body cannot synthesize on its own. It has been reported to possess anti-inflammatory and antioxidant properties, inhibiting processes such as macrophage polarization, vascular smooth muscle cell apoptosis, and extracellular matrix degradation. Increasing research indicates a close correlation between selenium deficiency and the development of aneurysms (AAA). According to relevant literature, AAA patients have reduced selenium levels in arterial tissue and plasma, and plasma selenium levels are significantly negatively correlated with the diameter of AAA aneurysms. Mice fed a selenium-deficient diet have a higher probability of developing AAA and larger aneurysms; the mechanism involves increased hydrogen peroxide content and matrix metalloproteinase (MMP)-2 expression, promoting the anabolic conversion of vascular smooth muscle cells. Various forms of selenium are ultimately converted to selenocysteine ​​via H2Se in vivo, which is then used to synthesize selenoproteins for their function. Studies have found that four patients with selenoprotein deficiency due to mutations in selenocysteine ​​insertion sequence binding protein 2 (SECISBP2) exhibited early-onset, progressive, aneurysmal dilation of the aorta. Currently, there are 25 types of selenoproteins in mammals, including the GPX family, TXNRD family, DIO family, MSRB1, and SEPHS2.

[0005] Selenoprotein M (Selenom) is a member of the selenoprotein family, containing a selenocysteine ​​(Sec) residue. It participates in various physiological and pathological processes, including redox regulation, inflammation, calcium homeostasis, glucose metabolism, and lipid metabolism. Evidence suggests that Selenom is associated with several diseases, such as obesity, cardiomyocyte damage, and Alzheimer's disease. For example, Selenom deficiency can induce adipose tissue inflammation and obesity by regulating the LATS2-YAP / TAZ-ROS signaling pathway. However, the role of Selenom in AAA (autoimmune inflammatory response) has not yet been investigated. Summary of the Invention

[0006] One objective of this invention is to provide a drug that can effectively treat abdominal aortic aneurysms, addressing the technical problems mentioned above.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides the use of selenoprotein M in the preparation of a medicament for the treatment of abdominal aortic aneurysm.

[0009] Secondly, the present invention provides the use of selenoprotein M in the preparation of a drug for reducing macrophage inflammation.

[0010] Thirdly, the present invention provides the application of a recombinant vector overexpressing selenoprotein M in the preparation of a drug for treating abdominal aortic aneurysm.

[0011] Preferably, the recombinant vector is a recombinant adeno-associated virus vector.

[0012] Preferably, the serotype of the recombinant adeno-associated virus vector is any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9, with AAV9 being the most preferred.

[0013] Preferably, the recombinant vector contains a nucleotide sequence as shown in SEQ ID NO.1.

[0014] Preferably, the nucleotide sequence encoding the selenoprotein M is shown in SEQ ID NO.2.

[0015] Compared with existing technologies, this invention can effectively delay the progression of abdominal aortic aneurysm and reduce macrophage inflammation, and has broad prospects for medical applications. Attached Figure Description

[0016] Figure 1 Selenin M expression levels are reduced in abdominal aortic aneurysms / dissections. (A) Western blot analysis of Selenom protein levels in human aortic dissection (AAD) tissue; (B) Western blot analysis of Selenom protein levels in human aortic dissection tissue; (C) RT-qPCR analysis of Selenom gene levels in human aortic dissection tissue; (D) Western blot analysis of Selenom protein levels in AngII-induced mouse abdominal aortic aneurysms; (E) Western blot analysis of Selenom protein levels in AngII-induced mouse abdominal aortic aneurysms; (F) RT-qPCR analysis of Selenom gene levels in AngII-induced mouse abdominal aortic aneurysms; (G) Western blot analysis of Selenom protein levels in PPE-induced mouse abdominal aortic aneurysms; (H) Western blot analysis of Selenom protein levels in PPE-induced mouse abdominal aortic aneurysms; (I) RT-qPCR analysis of Selenom gene levels in PPE-induced mouse abdominal aortic aneurysms. Data are expressed as mean ± SD. For comparisons between the two groups, Student's t-test of two-tailed analysis was used.

[0017] Figure 2Overexpression of Selenom delayed PPE-induced AAA. (A) Maximum tumor diameter in AAA model mice overexpressing Selenom (OE-Selenom); (B) Statistical analysis of the maximum tumor diameter in AAA model mice; (C) Ultrasound imaging detection of the maximum tumor diameter in AAA model mice; (D) HE and EVG staining (Vangissen staining for elastic fibers) detection of morphological changes and elastic fiber breakage in different layers of the abdominal aorta; (E) Elastic fiber degradation score; (F) Western blot detection of Selenom protein levels in arterial tissue of AAA mice overexpressing Selenom; (G) Statistical analysis of Selenom protein levels in arterial tissue of AAA mice overexpressing Selenom using Western blot. Data are expressed as mean ± SD. Student's t-test with two-tailed analysis was used for comparisons between two groups.

[0018] Figure 3 Selenom reduces macrophage inflammation. (A) Heatmap of differentially expressed selenoproteins in macrophages; (B) Immunofluorescence showing the localization and decreased expression of Selenom in AAA macrophages; (C) GO analysis of differentially expressed genes in Selenom-positive and Selenom-negative macrophages; (D) Western blot detection of Selenom overexpression in macrophages; (E) Statistical analysis of Selenom overexpression in macrophages using Western blot; (F) Western blot detection of Selenom overexpression reducing macrophage inflammation; (G) Statistical analysis of Selenom overexpression decreasing INOS expression using Western blot; (H) Statistical analysis of Selenom overexpression decreasing TNF-α expression using Western blot; (I) Western blot detection of Selenom knockdown exacerbating macrophage inflammation; (J) Statistical analysis of Selenom knockdown increasing INOS expression using Western blot; (K) Statistical analysis of Selenom knockdown increasing TNF-α expression using Western blot. Data are expressed as mean ± SD. For comparisons between the two groups, Student's t-test of two-tailed analysis was used.

[0019] Figure 4 Schematic diagram of the construction of a recombinant adeno-associated virus vector overexpressing Selenom. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0021] Unless otherwise specified, all instruments and reagents used in the examples are conventional instruments or reagents in the art and are commercially available products. Unless otherwise specified, all specific experimental operations involved in the text are understandable or known to those skilled in the art based on their common knowledge or conventional technical means, and will not be described in detail here.

[0022] I. Experimental Methods:

[0023] 1. Western blot:

[0024] Cells and abdominal aortic tissue were washed with phosphate-buffered saline (PBS; Gibco) at 4°C, followed by protein extraction using RIPA lysis buffer (FD009, Fdbio Science) combined with 1% protease inhibitor (FD1001, Fdbio Science) and 1% benzyl sulfonyl fluoride (PMSF; FD0100, Fdbio Science). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (ISEQ00010, Merck Millipore). The PVDF membrane was blocked with 5% skim milk for 1–2 hours at room temperature and then incubated overnight with primary antibody at 4°C. It was subsequently washed three times with tris(hydroxymethyl)aminomethane-buffered saline (TBST) containing Tween-20 and incubated with secondary antibody (FDR007, Fdbio Science) for 1 hour at room temperature. Protein bands were visualized using an ECL kit (FD8000, Fdbio Science) and analyzed using ImageJ software. The antibodies used include: anti-α-Tubulin (66031-1-Ig, Proteintech), anti-GAPDH (60004-1-Ig, Proteintech), and anti-Selenom (sc-514952, Santa Cruz; 29929, Signalway Antibody).

[0025] 2. RT-qPCR:

[0026] Total RNA was extracted from abdominal aortic tissue and cells using TRIzol reagent (AG21101, AG). The total RNA was then converted to cDNA for real-time quantitative PCR (qPCR) using a Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix (11141ES60, Yeasen). qPCR was performed on a Light Cycler 480 II instrument (Roche Diagnostics, Basel, Switzerland) using Hieff® qPCR SYBR Green Master Mix (11201ES08, Yeasen). β-actin (ACTB) mRNA expression was detected as an internal reference, and relative gene expression was normalized using the 2-ΔΔCt method.

[0027] The primers used were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are shown in Table 1 below.

[0028] Table 1. Primer sequences

[0029]

[0030] 3. Collection of human tissue samples:

[0031] Human tissue samples were obtained from the abdominal aorta of patients who underwent open surgical repair. Adjacent non-aneurysmal aortic sections were also obtained from these patients as a control group. The excised aortic tissue was rapidly cryopreserved in liquid nitrogen and stored at -80°C for subsequent processing.

[0032] 4. Establish a mouse model of abdominal aortic aneurysm (AAA):

[0033] (1) Establishment of an angiotensin II (AngII)-induced AAA model. Mice were anesthetized with 2% isoflurane. An osmotic micropump (2004W, RWD LifeScience Co. Ltd.) equipped with AngII (1000 ng / kg / min; A9525, Sigma) or saline was implanted in the subcutaneous tissue of the back of the neck. Four weeks later, the abdominal aorta was collected for further analysis.

[0034] (2) Establishment of a porcine pancreatic elastase (PPE)-induced AAA model. The mice were completely anesthetized prior to surgery. The abdominal aortic segment from the renal artery to the iliac artery was separated from the surrounding retroperitoneal tissue. A gelatin sponge soaked in PPE solution (E1250, Sigma) or physiological saline was placed around the abdominal aorta for 30 minutes. Two weeks later, the abdominal aorta was collected for further analysis.

[0035] 5. Construction and use of recombinant adeno-associated virus (AAV) overexpressing Selenom:

[0036] AAV virus packaging and detection services were provided by Guangzhou Dahong Biotechnology Co., Ltd. The serotype was AAV9, promoter F4 / 80, and gene name EGFP-F2A-Selenom (CDS+3UTR). One month prior to AAA modeling, the viral titer was injected via tail vein to a level of 5 × 10⁻⁶. 11 The viral fluid was injected into mice. Its expression efficiency was verified using Western blot and other experimental methods. A schematic diagram of the construction of the recombinant adeno-associated virus vector overexpressing Selenom is shown below. Figure 4 As shown.

[0037] Selenom encoded nucleotide sequence:

[0038] >NC_000077.7:3464684-3467351 Mus musculus strain C57BL / 6J chromosome11, GRCm39

[0039]

[0040] EGFP-F2A-Selenom (CDS+3UTR) sequence:

[0041]

[0042] 6. Single-cell RNA sequencing (scRNA-seq) data processing:

[0043] Sequencing data were obtained from the Gene Expression Comprehensive Database (access number: GSE233625). Cross-sample adjustment, processing, and quality control were performed using the Seurat package (version 5.0.1) in R software (version 4.3.2). After removing low-quality cells, the gene expression matrix was normalized using the Normalize Data function, and the Find VariableFeatures function was used to identify 2000 features with high inter-cell variance. Dimensionality reduction and cell clustering were performed using the Run PCA, Find Neighbors, and Find Clusters functions, followed by dimensionality reduction using the Run Uniform Manifold Approximation and Projection (UMAP) and Runtsne functions. Differentially expressed genes (DEGs) were screened within each cell cluster using the FindAllmarkers function. Cell markers were searched using the CellMarker 2.0 database (http: / / 117.50.127.228 / CellMarker / ) to manually label cell types.

[0044] Log-Two-Fold Change > 1.0 and P < 0.05 were used as screening criteria to obtain DEGs. Functional richness analysis of differentially expressed genes was performed using visualization and comprehensive discovery (DAVID), Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analysis tools. p Functions with values ​​less than 0.05 are considered to have significant differences.

[0045] 7. Lentiviral transduction and overexpression:

[0046] Full-length or 3'-UTR-containing mouse Selenom cDNA was amplified and inserted into a pCDH lentiviral vector via Hanbio Biotechnology Co., Ltd. (Guangzhou, China). Empty pCDH lentiviral vectors or vectors encoding Selenom were transfected into HEK293T cells along with packaging plasmids using Lipofectamine 3000 reagent (L3000015, ThermoFisher Scientific) according to the manufacturer's instructions to produce lentiviral particles. The supernatant was collected and added to macrophages.

[0047] 8. Silent Selenom ShRNA in macrophages:

[0048] To prepare lentiviral particles, the lentiviral vector pLVX encoding SelenomShRNA, provided by Hanyi Biosciences Co., Ltd. (Guangzhou, China), was transfected into HEK293T cells along with the packaging vector.

[0049] The sequence of Selenom ShRNA is: 5'-GCCGAAATTACCAGGAACTAG-3'.

[0050] 9. Immunofluorescence:

[0051] Tissue sections or cultured cells were first fixed in 4% paraformaldehyde for 30 minutes, then washed and treated with 0.1% Triton X-100 for 15 minutes, followed by blocking with 5% BSA for 1 hour at room temperature. Next, they were incubated with primary antibodies, and then with fluorescent secondary antibodies (Alexa Fluor 488 and / or 594; Abcam) for 1 hour at room temperature. Following this, nuclear staining was performed using 4',6-diamidinyl-2-phenylindole (DAPI; C0065, Solarbio), and the final images were captured using a confocal laser scanning microscope (Leica). The primary antibodies used were CD68 (1:50; E307V; CST) and Selenom (1:50; sc-514952, Santa Cruz).

[0052] II. Experimental Results:

[0053] 1. Selenom protein M expression levels are reduced in abdominal aortic aneurysms / dissections.

[0054] Human tissue samples were obtained from the abdominal aorta of four patients who underwent open surgery to repair abdominal aortic aneurysms. Adjacent non-aneurysmal aortic sections were obtained from the same patients as a control group.

[0055] The patient's clinical information is shown in Table 2 below.

[0056] Table 2. Patient Clinical Information

[0057]

[0058] Western blot and RT-qPCR were used to detect the expression of Selenom in human aortic dissection tissue. The results showed that the expression of Selenom in human aortic aortic dissection tissue was decreased compared with that in non-aneurysmal aorta. Figure 1 AC in the middle.

[0059] Angiotensin II (AngII) was used to construct an AAA model mouse. Western blot and RT-qPCR results showed that Selenom expression was decreased in mouse AAA artery tissue. Figure 1 (DF in the text).

[0060] AAA model mice were constructed using elastase (PPE). Western blot and RT-qPCR results showed that Selenom expression was decreased in mouse AAA artery tissue. Figure 1 (GI in the text).

[0061] 2. Overexpression of Selenom delayed PPE-induced AAA in mice.

[0062] To determine whether Selenom prevents AAA formation, an AAV serotype 9 (AAV9) overexpressing Selenom (including the CDS+3UTR sequence) was constructed and injected into mice via tail vein. One month later, a mouse abdominal aortic aneurysm model was established using PPE.

[0063] The results showed that gross observation and ultrasound imaging indicated that overexpression of Selenom could reduce the diameter of AAA tumors. Figure 2 AC in the middle.

[0064] HE and EVG staining showed that after Selenom overexpression, the diameter of AAA tumors decreased and the degree of elastic fiber breakage was reduced. Figure 2 DE in the middle.

[0065] Furthermore, Western blot analysis showed that Selenom expression was increased in the arterial tissue of AAA mice overexpressing Selenom. Figure 2 (FG in the middle).

[0066] 3. Selenom reduces macrophage inflammation

[0067] Analysis of differentially expressed selenoprotein genes in macrophages revealed that the decrease in Selenom was the most significant. Figure 3 (A in the middle).

[0068] Furthermore, decreased Selenom expression was confirmed in macrophages from mouse abdominal aortic aneurysm samples, and immunofluorescence assays also revealed significant colocalization of Selenom in macrophages. Figure 3 (B in the middle).

[0069] To elucidate the potential role of macrophage-derived Selenom in abdominal aortic aneurysms (AAA), enrichment analysis was performed on the data based on single-cell RNA sequencing (scRNA-seq). GO and KEGG analyses showed that immune responses and immune-related pathways (NF-κB, TGF-β signaling pathways) were significantly enriched in Selenom-negative macrophages. Figure 3 (C in the middle).

[0070] Macrophages play a crucial role in the pathogenesis of aortic inflammatory disease (AAA). They primarily aggregate in the adventitia, acting as essential producers and reactive agents of inflammatory mediators, coordinating the complex inflammatory dynamics within the aortic lumen. In in vitro experiments, stably transfected macrophages expressing silenced and overexpressing Selenom were constructed using lentivirus and treated with LPS. Western blot analysis showed that overexpression of Selenom reduced the expression of inflammatory factors (INOS and TNF-α). Figure 3 (DH in the middle).

[0071] Conversely, macrophages lacking Selenom exhibited increased expression of inflammatory cytokines (INOS and TNF-α). Figure 3 (IK in the middle).

[0072] The above results indicate that macrophage-derived Selenom deficiency exacerbates LPS-induced inflammatory cell infiltration and the accompanying inflammatory response, while increased Selenom expression has the opposite effect.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The use of selenoprotein M in the preparation of a drug for treating abdominal aortic aneurysm, wherein the nucleotide sequence encoding said selenoprotein M is shown in SEQ ID NO.

2.

2. The application of a recombinant vector overexpressing selenoprotein M in the preparation of a drug for treating abdominal aortic aneurysm, wherein the nucleotide sequence encoding the selenoprotein M is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, The recombinant vector is a recombinant adeno-associated virus vector.

4. The application according to claim 3, characterized in that, The serotype of the recombinant adeno-associated virus vector is any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

5. The application according to claim 2, characterized in that, The recombinant vector contains a nucleotide sequence as shown in SEQ ID NO.1.

Images