Use of fusobacterium nucleatum and its channel proteins as targets in preparation of products for detecting, inhibiting or delaying abdominal aortic aneurysm

Abstract

The application discloses Fusobacterium nucleatum and a path protein thereof as a target in preparation of products for detecting, inhibiting or delaying abdominal aortic aneurysm, and belongs to the technical field of medicines.The application first proves that Fusobacterium nucleatum is specifically planted in AAA aneurysm tissue, establishes the oral pathogenic bacteria as a key promoting factor for occurrence and development of AAA, directly acts on vascular smooth muscle cells through specific virulence proteins, and drives a core molecular mechanism of pathological phenotype conversion.Based on this, the application of a substance for detecting Fusobacterium nucleatum in preparation of products for detecting progression of abdominal aortic aneurysm can provide a key microbiological marker for early warning, risk stratification and disease course monitoring of AAA, and also provides a crucial diagnostic basis and intervention window for subsequent development of a new therapy (such as an antibacterial, antitoxin or signal path inhibitor) for the target, has a clear value for solving the current dilemma of lack of drug strategies in clinical management of AAA.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Fusobacterium nucleatum and its pathway proteins as targets in the preparation of products for detecting, inhibiting or delaying abdominal aortic aneurysms. Background Technology

[0002] Abdominal aortic aneurysm (AAA) is a common and life-threatening vascular disease in the elderly, with its main risk being the extremely high mortality rate due to aneurysm rupture. Current clinical treatment relies on surgery, and there is a lack of effective drugs, stemming from the unclear molecular mechanisms involved. Phenotypic transformation and apoptosis of vascular smooth muscle cells (VSMCs) are core events in AAA pathology, but the upstream signals and environmental factors driving this transformation have not been fully elucidated.

[0003] In recent years, the association between the microbiota within pathological tissues (such as bacteria within atherosclerotic plaques) and the development of chronic diseases has been increasingly revealed. Notably, patients with AAA often also have periodontitis, suggesting that the systemic migration of oral pathogens may be involved in the disease process. Among them, *Fusobacterium nucleatum*, as the dominant pathogen of periodontitis, has been shown to colonize various extraintestinal lesions at long distances and drive inflammation and lesions through interactions with host cells via its specific virulence proteins. However, whether this bacterium is widely present in AAA tissues and whether it also plays a role in AAA formation through pathogenic proteins remains unconfirmed. This scientific blind spot limits a deeper understanding of the etiology of AAA from the novel perspective of "bacterium-host" interactions and hinders the discovery of relevant intervention targets. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of Fusobacterium nucleatum and its pathway proteins as targets in the preparation of products for detecting, inhibiting, or delaying abdominal aortic aneurysms.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows.

[0006] In a first aspect, the present invention provides the use of Fusobacterium nucleatum as a biomarker in the preparation of products for detecting the progression of abdominal aortic aneurysms.

[0007] In some embodiments of the present invention, the product for detecting the progression of abdominal aortic aneurysm is capable of detecting the content of Fusobacterium nucleatum in abdominal aortic aneurysm tissue.

[0008] In some embodiments of the present invention, the product for detecting the progression of abdominal aortic aneurysm is a kit.

[0009] A second aspect of the present invention provides the use of a substance that blocks the adhesion of Fusobacterium nucleatum to vascular smooth muscle cells in the preparation of products that inhibit or delay the occurrence and / or development of abdominal aortic aneurysms.

[0010] In some embodiments of the present invention, the substance that blocks the adhesion of Fusobacterium nucleatum to vascular smooth muscle cells includes a substance that interferes with receptor proteins in vascular smooth muscle cells, the receptor proteins being capable of specifically binding to Fusobacterium nucleatum virulence proteins.

[0011] In some embodiments of the present invention, the virulence protein of *Fusobacterium nucleatum* is a dipeptide-binding protein, and the receptor protein is Fibronectin 1.

[0012] In some embodiments of the present invention, the substance that interferes with receptor proteins in vascular smooth muscle cells is an RNA interference molecule or antisense oligonucleotide, small molecule inhibitor, siRNA, shRNA targeting the receptor protein, or a substance that performs lentiviral infection or gene knockout, as well as a specific antibody against itself or its upstream or downstream molecules.

[0013] In some embodiments of the present invention, the substance that interferes with receptor proteins in vascular smooth muscle cells is siRNA, the nucleic acid sequence of which is shown in SEQ ID NO.1.

[0014] A third aspect of the present invention provides a composition comprising a substance that blocks the adhesion of Fusobacterium nucleatum to vascular smooth muscle cells, said substance being siRNA with a nucleic acid sequence as shown in SEQ ID NO.1.

[0015] A fourth aspect of the invention provides the use of the composition described in the third aspect in the preparation of products that inhibit or delay the occurrence and / or development of abdominal aortic aneurysms.

[0016] The beneficial effects of this invention are as follows: This invention is the first to demonstrate the specific colonization of *Fusobacterium nucleatum* within AAA tumor tissue, establishing this oral pathogen as a key contributing factor to the occurrence and development of AAA, and providing a novel perspective on the "oral flora-vascular lesion" axis for understanding the etiology of AAA. It further reveals the core molecular mechanism by which this bacterium directly acts on vascular smooth muscle cells through its specific virulence proteins, driving their pathological phenotypic transformation, thereby discovering a series of novel potential therapeutic targets. Based on this, the application of substances detecting *Fusobacterium nucleatum* in the preparation of products for detecting the progression of abdominal aortic aneurysms can provide crucial microbiological biomarkers for early warning, risk stratification, and disease monitoring of AAA. It also provides vital diagnostic evidence and intervention windows for the subsequent development of novel therapies targeting this site (such as antibacterial, antiviral, or signaling pathway inhibitors), and has clear value in addressing the current shortage of drug strategies in the clinical management of AAA. Attached Figure Description

[0017] 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.

[0018] Figure 1 In Embodiment 1 of the present invention F. nucleatum Bioinformatics and clinicopathological evidence enriched in AAA specimens. Here, A represents the 16S rRNA dataset PRJNA1128157. F. nucleatum Annotation results and related Zotu expression heatmaps for each AAA sample. B represents DNA from AAA samples. F. nucleatum Specific primer agarose gel electrophoresis experiment, +ve is F. nucleated The bacterial culture is the positive control, and -ve is the negative control for DNA extraction reagent. C represents the immunofluorescence and FISH experiments of the control group. D represents the immunofluorescence and fluorescence in situ hybridization (FISH) experiments of AAA tissue, with green fluorescent labeling of the VSMC marker protein α-SMA and red probe labeling. F. nucleatum The white arrow marks the AAA organization. F. nucleatum Enrichment.

[0019] Figure 2 In Embodiment 2 of the present invention F. nucleatum Animal experiments to promote AAA formation were validated. A is a schematic diagram of the animal model experiment. B is a gross representation of the aorta in five groups of mice. C is the incidence of abdominal aortic aneurysm. D is the maximum diameter of the abdominal aorta. E is a schematic diagram of the survival curve.

[0020] Figure 3 In Embodiment 2 of the present invention F. nucleatum Animal experimental results promoting AAA formation. A shows ultrasound detection of abdominal aortic aneurysm in mice. B shows micro-computed tomography / micro-CT (Micro-CT) detection of periodontitis severity. C shows immunofluorescence combined with FISH staining of aortic tissue; green fluorescent marker α-SMA in mice is used, and red fluorescent marker is used. F. nucleatum Blue fluorescent DAPI labels the cell nuclei. D represents periodontal tissue bound to FISH staining, with red fluorescent labeling. F. nucleatum The cell nucleus was labeled with blue fluorescent DAPI. P <0.05.

[0021] Figure 4 In Embodiment 3 of the present invention F. nucleatum The impact on VSMC adhesion and invasion. Where A represents... F. nucleatedLaser confocal microscopy image of invasive and adherent VSMCs. Red fluorescence indicates phalloidin-labeled cytoskeleton, and green fluorescence indicates carboxyfluorescein diacetate succinimidyl ester (CFSE) staining. F. nucleatum The yellow arrow indicates F. nucleatum The bacteria are currently in the process of invading the VSMC; some of the bacteria have already entered the VSMC, indicated by the red arrow. F. nucleatum Complete intrusion into VSMC, indicated by the blue arrow. F. nucleatum Adhesive to the surface of the VSMC membrane. B represents... F. nucleatum Scanning electron microscope image of the adhering and invading VSMC. C represents... F. nucleatum Flow cytometry image of VSMCs that can adhere to and invade them. P <0.01, **** P <0.0001.

[0022] Figure 5 In Embodiment 3 of the present invention F. nucleatum The impact on VSMC phenotypic transformation. Where A represents the VSMC morphology at 0 h, 6 h, 12 h, 24 h, and 48 h. B represents the migration ability of VSMC at 12 h.

[0023] Figure 6 In Embodiment 3 of the present invention F. nucleatum Immunofluorescence assay results and related protein expression of VSMC phenotypic transformation. In A, the immunofluorescence assay shows red fluorescence labeling of the shrinking VSMC marker protein SM22α and green fluorescence labeling of the synthetic VSMC marker protein OPN. In B, the results show... F. nucleatum Protein expression of α-SMA, SM22α, OPN, CNN1, and GAPDH after stimulation.

[0024] Figure 7 In Embodiment 3 of the present invention F. nucleatum Immunofluorescence assay results and related protein expression of VSMC phenotypic transformation. A shows the immunofluorescence assay of mouse aortic VSMC phenotype, with green fluorescence labeling of the contractile VSMC marker proteins α-SMA, CNN1, and SM22α, red fluorescence labeling of the synthetic VSMC marker protein OPN, and blue fluorescence labeling of the cell nucleus with DAPI. B shows the relative protein expression level of the contractile VSMC marker protein α-SMA. C shows the relative protein expression level of the contractile VSMC marker protein SM22α. D shows the relative protein expression level of the contractile VSMC marker protein CNN1. E shows the relative protein expression level of the synthetic VSMC marker protein OPN.

[0025] Figure 8This is a schematic diagram of the histidine tag (His) pull-down experiment and high performance liquid chromatography-mass spectrometry analysis.

[0026] Figure 9 This is an example of an in vitro experiment to detect the effect of the key toxic protein DBP on the phenotypic transformation of VSMCs in Example 3 of the present invention. Wherein, A represents the morphology of VSMCs at 0h, 6h, 12h, 24h, and 48h. B represents the migration ability of VSMCs at 6h.

[0027] Figure 10 This is a diagram from Example 3 of the present invention, showing the in vitro detection of the expression levels of contractility and synthetic markers of the key toxic protein DBP after stimulation. A shows the Western blot (WB) graphs of the contractility and synthetic markers after DBP stimulation. B shows the relative protein expression levels of α-SMA. C shows the relative protein expression levels of SM22α. D shows the relative protein expression levels of CNN1. E shows the relative protein expression levels of OPN.

[0028] Figure 11 This is an immunofluorescence assay performed in Example 3 of the present invention to detect the key toxic protein DBP after stimulation of VSMCs. The assay uses green fluorescence to label the characteristic proteins α-SMA, CNN1, and SM22α of contractile VSMCs, red fluorescence to label the characteristic protein OPN of synthetic VSMCs, and blue fluorescence to label the cell nucleus with DAPI.

[0029] Figure 12 This diagram illustrates the effect of knocking down the key toxic protein DBP on VSMC phenotypic transformation in Example 3 of this invention. A shows a simulated molecular docking diagram of DBP and FN1. B shows an immunofluorescence colocalization map of DBP and FN1, with red representing FN1, yellow representing DBP, and blue representing the cell nucleus. C shows an immunoprecipitation assay (coIP) of DBP and FN1 binding. D shows a surface plasmon resonance (SPR) assay of DBP and FN1.

[0030] Figure 13 This image shows the characterization of VSMCs after knocking down the key toxic protein DBP in Example 3 of this invention. A is a scanning electron microscope image of VSMCs. B is a flow cytometry diagram of bacterial adhesion after FN1 knockdown. C is an immunofluorescence diagram of cell phenotypic transformation indicators after FN1 knockdown.

[0031] Figure 14 The results of VSMCD apoptosis and migration experiments after knocking down the key toxic protein DBP in Example 3 of this invention are shown. In this figure, A represents the apoptosis detection kit after FN1 knockdown, where the horizontal axis represents the fluorescein isothiocyanate area signal (FITC-A), and the vertical axis represents the phycoerythrin area signal (PE-A). B is a schematic diagram of the cell migration experiment after FN1 knockdown.

[0032] Figure 15 This is a Western blot (WB) image of proteins related to phenotypic transformation after FN1 knockdown in Example 3 of this invention. Detailed Implementation

[0033] It remains unclear whether *Fusobacterium nucleatum*, a common pathogen of periodontitis, is prevalent in AAA tissues, and whether this bacterium plays a role in the formation and development of AAA by acting on VSMCs through its pathogenic proteins and influencing their phenotypic transformation. This knowledge gap hinders a deeper understanding of the pathogenesis of AAA from the perspective of microbe-host interactions and the search for new intervention targets. To address this, this invention proposes the application of *Fusobacterium nucleatum* and its pathway proteins as targets in the preparation of products for the detection, inhibition, or delay of abdominal aortic aneurysms.

[0034] This invention, through clinical sample validation, animal model construction, in vitro intervention experiments, and bioinformatics analysis, confirms... F. nucleated It is significantly enriched in the lesion tissue of AAA patients, and can drive the occurrence and development of AAA by adhering to and invading VSMCs, inducing VSMC phenotypic transformation and apoptosis, and activating downstream inflammatory and fibrotic signaling pathways; intervention on VSMC membrane receptors can alleviate F. nucleated The impact on VSMC phenotypic transformation. This invention is based on... F. nucleatum Targeting the aortic aneurysm and its regulatory pathways, this study provides a complete theoretical basis, experimental evidence, and technical solutions for the preparation and screening of candidate drugs or formulations for treating abdominal aortic aneurysm, filling the gap in clinical drug treatment of AAA and having significant clinical significance and application value for early intervention and targeted therapy of AAA.

[0035] A first typical embodiment of the present invention provides the application of Fusobacterium nucleatum as a biomarker in the preparation of products for detecting the progression of abdominal aortic aneurysms.

[0036] The product for detecting the progression of abdominal aortic aneurysms can detect the progression of abdominal aortic aneurysms by detecting the content level of Fusobacterium nucleatum in the aneurysm tissue. Studies of this invention have shown that Fusobacterium nucleatum is significantly overexpressed in patients with abdominal aortic aneurysms, and therefore it can serve as a biomarker for the detection of abdominal aortic aneurysms.

[0037] This invention clarifies the close association between *Fusobacterium nucleatum* and the progression of acute cerebral inflammatory disease (AAA), and confirms that the specific binding of its key toxic protein DBP to the VSMC receptor FN1 is the core mechanism mediating AAA progression. Therefore, it proposes using substances that detect *Fusobacterium nucleatum* as the core detection tool to prepare products for detecting AAA progression. This is not simply using *Fusobacterium nucleatum* as a detection target, but rather closely integrating it with the complete mechanism previously validated in this invention. On one hand, it constructs a precise detection system of "molecular detection + mechanism support." Detecting the presence and content of *Fusobacterium nucleatum* not only reflects the progression status of AAA, but also clarifies whether AAA progression is mediated by *Fusobacterium nucleatum*, providing clear guidance for subsequent targeted interventions (such as interfering with DBP and FN1 binding), achieving precise linkage between "detection and intervention." On the other hand, the substance for detecting Fusobacterium nucleatum can be flexibly adapted to various detection methods (such as nucleic acid detection, protein detection, and immunoassay), and can be prepared into various products such as reagent kits, test strips, and in vitro diagnostic reagents. Compared with existing imaging detection, it has the advantages of convenient operation, lower cost, and the ability to achieve early screening and dynamic monitoring. It is suitable for various scenarios such as routine clinical testing and community screening, and solves the problems of complex operation, high cost, and difficulty in popularization of existing detection methods.

[0038] In this invention, the product for detecting the progression of abdominal aortic aneurysms (AAAs) can detect the content of *Fusobacterium nucleatum* in the AAA tissue. By directly detecting the content of this bacterium in AAA lesion tissue, the colonization level of *Fusobacterium nucleatum* in the lesion site can be accurately reflected, thereby clarifying its driving role in AAA progression. This fills the technical gap in existing imaging examinations, which can only observe the macroscopic morphology of the aneurysm and cannot clarify the content of the core pathogenic factors in the lesion site. At the same time, this content detection can accurately distinguish between *Fusobacterium nucleatum*-mediated and non-mediated AAA progression, providing a direct basis for clinical screening of patients suitable for the targeted intervention program of this invention (interference with DBP and FN1 binding). It can also dynamically monitor changes in bacterial content during the intervention process, intuitively judge the intervention effect, realize a closed loop of "detection-intervention-monitoring", and further improve the level of precision prevention and treatment of AAA.

[0039] In this invention, the abdominal aortic aneurysm tissue is derived from humans or other mammals. These mammals include mice, rats, guinea pigs, rabbits, dogs, pigs, and chimpanzees.

[0040] In this invention, the product for detecting the progression of abdominal aortic aneurysms includes, in addition to the kit, in vitro diagnostic reagents adapted for tissue sample testing, test strips, chips (such as protein chips and nucleic acid chips), sequencing library construction reagents, and supporting detection auxiliary products (such as tissue sample processing reagents, nucleic acid extraction reagents, protein extraction reagents, and fluorescence detection reagents). These products can all achieve accurate detection of the content of Fusobacterium nucleatum in abdominal aortic aneurysm tissue and can be flexibly selected according to clinical testing scenarios and accuracy requirements.

[0041] A second typical embodiment of the present invention provides the use of a substance that blocks the adhesion of *Fusobacterium nucleatum* to vascular smooth muscle cells in the preparation of products that inhibit or delay the occurrence and / or development of abdominal aortic aneurysms. The present invention discovers that *Fusobacterium nucleatum* drives abdominal aortic aneurysms by promoting phenotypic transformation of vascular smooth muscle cells and directly translates this discovery into a novel precision treatment strategy. It proposes a therapeutic pathway for developing specific blocking agents targeting the key initiating step of "bacterial-cell" adhesion, providing an original solution to the current clinical situation lacking effective drugs, and laying the foundation for the development of innovative drugs to prevent and treat abdominal aortic aneurysms by intervening in the "oral-vascular axis".

[0042] In this invention, the substance that blocks the adhesion of *Fusobacterium nucleatum* to vascular smooth muscle cells includes a substance that interferes with receptor proteins in vascular smooth muscle cells, wherein the receptor proteins can specifically bind to *Fusobacterium nucleatum* virulence proteins. This invention precisely targets the treatment strategy from "blocking communication between bacteria and cells" to a specific molecular interaction interface (virulence protein-receptor protein), clarifying the direct target of drug design (the receptor protein of the host cell). This makes it possible to develop highly specific inhibitors (such as antibodies and small molecules), enabling precise intervention and potentially avoiding the side effects of broad-spectrum antibacterial agents.

[0043] In this invention, the virulence protein of *Fusobacterium nucleatum* is dipeptide-binding protein (DBP), and the receptor protein is Fibronectin 1 (FN1). This invention provides a unique and well-defined target for developing highly selective inhibitors (such as blocking antibodies, competitive peptides, or small molecules) against this specific protein-protein interaction (PPI).

[0044] In this invention, the substance that interferes with receptor proteins in vascular smooth muscle cells is an RNA interference molecule or antisense oligonucleotide, small molecule inhibitor, siRNA, shRNA, or a substance that performs lentiviral infection or gene knockout, as well as a specific antibody against itself or its upstream or downstream molecules.

[0045] In this invention, the substance that interferes with receptor proteins in vascular smooth muscle cells is siRNA, and its nucleic acid sequence is shown in SEQ ID NO.1.

[0046] A third typical embodiment of the present invention provides a composition comprising a substance that blocks the adhesion of nucleated spindles to vascular smooth muscle cells, said substance being siRNA with a nucleic acid sequence as shown in SEQ ID NO.1.

[0047] In this invention, the composition further includes pharmaceutical excipients and / or pharmaceutical carriers.

[0048] In one or more embodiments, the pharmaceutical excipients include, but are not limited to, adhesives, stabilizers, solubilizers, buffers, preservatives, etc.

[0049] In one or more embodiments, the pharmaceutical carrier includes, but is not limited to, serum protein, lecithin, aluminum stearate, aluminum oxide, etc.

[0050] A fourth typical embodiment of the present invention provides the use of the composition described herein in the preparation of products that inhibit or delay the occurrence and / or development of abdominal aortic aneurysms.

[0051] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0052] Example 1: F. nucleatum Enrichment validation in AAA patient tissues Based on data and literature searches, this embodiment retrieved data PRJNA1128157(AAA), PRJNA748211, and PRJNA224116(Control) from the SRA database, and these datasets were 16S amplicon data. Raw sequencing data from blood vessel samples were selected from these three datasets. After quality control using FASTP software, the data were processed separately using amplicon analysis software workflow (the variable regions of the sequencing data differ depending on the sequencing batch) to obtain the bacterial composition and abundance of each dataset.

[0053] like Figure 1 As shown in Figure A, there are 11 Zotu annotations in the AAA samples. F. nucleatum Although the abundance varied among the samples, overall... F. nucleatum It was detected in all AAA samples; however, it was not noted in the Control samples. F. nucleatum (The influence of factors such as sequencing depth and variable region differences cannot be excluded).

[0054] Currently, aneurysm samples have been collected from patients who underwent open surgery for abdominal aortic aneurysms at Qilu Hospital of Shandong University (AAA, n=10) and healthy abdominal aortic specimens from organ donors (Control, n=4). DNA was extracted from partial tissue samples for further analysis. F. nucleated Specific primer agarose gel electrophoresis experiment; immunofluorescence experiment on paraffin sections and binding F. nucleated Specific probe detection F. nucleatum Whether it is enriched in AAA samples.

[0055] Both experimental results showed that AAA tissue was found F. nucleatum Enrichment ( Figure 1(Figures B and D) No traces were found in the Control tissue. F. nucleatum Enrichment ( Figure 1 (Figure C); the two detection results are consistent with the above bioinformatics analysis results.

[0056] Example 2: F. nucleatum Animal experiments to promote AAA formation Frozen F. nucleatum (ATCC 25586) Provided by the Oral Microbiome Laboratory of Shandong University. Animal experimental groups are as follows: control group, F. nucleatum Group, Ang II model group, Ang II+ F. nucleatum The patients were divided into two groups: the Ang II group and the Ang II+ ligature group. They were fed a high-fat diet for 28 days, and the abdominal aortic diameter was measured by vascular ultrasound weekly. The incidence and survival rate of AAA were recorded.

[0057] Animal experiment grouping: Control group: Saline solution was pumped in; Ang II was not required. F. nucleatum Related processing.

[0058] F. nucleatum Group: Ten-week-old male ApoE- / - mice were selected and fed with appropriate feed and sterile water. After one week, metronidazole powder was added to their diet at a dose of 20 mg / kg / day for one week to eliminate native anaerobic bacteria in the oral cavity and digestive tract of the rats. Mice were anesthetized by intraperitoneal injection of 0.08% sodium pentobarbital (40 mg / kg) to induce deep anesthesia, and then the maxillary second molar was ligated with silk suture at 4-0. Bacterial agents were applied topically to the ligation site in the oral cavity of the mice twice a week at a dose of 1×10⁻⁶. 9 CFU / each / time, modeling time 28 days.

[0059] Ang II model group: 10-week-old male ApoE - / - Mice were anesthetized by intraperitoneal injection of 0.08% sodium pentobarbital (40 mg / kg). The skin on the back was aseptically cut along the lower edge of the scapula, and an implantable capsule osmotic pump was embedded subcutaneously. The skin was then sutured. Mice were fed a high-fat diet (containing 0.25% cholesterol and 15% fat) daily, with Ang II (1000 ng / kg / min) continuously infused at a constant rate for 28 days.

[0060] Ang II+ F. nucleatum Group: Ang II model group and F. nucleatum Group processing.

[0061] Ang II+ligature group: Ang II model group and maxillary second molar 4-0 were ligated with silk sutures simultaneously, but no ointment was applied. F. nucleatum .

[0062] The results are as follows Figure 2 and Figure 3 As shown. Figure 2 and Figure 3 for F. nucleatum Results of animal experiments promoting abdominal aortic aneurysm formation. Gross observations showed ( Figure 2 Figure B), control group and F. nucleatum The aorta in the first group was normal in morphology and without aneurysmal dilatation; significant aortic dilatation and aneurysmal structures were observed in the Ang II group and the Ang II+ Ligature group; Ang II+ F. nucleated The aortic dilation was most significant in this group, with larger aneurysms and thinner vessel walls. (AAA incidence statistics) Figure 2 Figure C shows that the control group and F. nucleatum The incidence rate was 0% in the Ang II group; the incidence rate was approximately 50% in the Ang II and Ang II+ Ligature groups; Ang II+ F. nucleatum The incidence rate in the group increased significantly to about 70%, indicating that F. nucleatum It can significantly increase the incidence of AAA in conjunction with Ang II. Maximum diameter of the abdominal aorta ( ) Figure 2 The figure (D) shows that the control group and F. nucleatum Group diameter is within normal range; Ang II+ F. nucleatum The expansion was most pronounced in the Ang II group, exceeding that of the Ang II+Ligature group, suggesting... F. nucleatum It can synergistically increase tumor diameter with Ang II. Survival curve results (Figure 2, E plot) show that the control group and... F. nucleated All groups survived the entire process, including the Ang II group, the Ang II+Ligature group, and the Ang II+ F. nucleatum Mice in all groups died, and Ang II+ mice... F. nucleatum The survival rate of the Ang II group was significantly lower than that of the Ang II group, suggesting that... F. nucleatum Significantly reduced the survival rate of AAA mice. Dynamic ultrasound monitoring ( Figure 3 Figure A shows that as the modeling time increases, Ang II+ F. nucleatum The abdominal aortic dilatation appeared earlier, progressed faster, and had a more prominent aneurysm in the group, indicating that F. nucleatum It can accelerate the progression of AAA. Micro-CT and pathological staining ( Figure 3 Figure B shows that, F. nucleatum Group and Ang II+ F. nucleatumThe maxillary bone resorption and periodontal inflammation in the group were significantly higher than those in the control group, confirming the successful establishment of the periodontitis model. Aortic immunofluorescence-FISH staining (…) Figure 3 (Figure C) shows that Ang II+ F. nucleatum Group F. nucleatum The bacteria extensively colonized the aortic wall and highly co-localized with α-SMA-positive vascular smooth muscle cells; the bacterial signals in other groups were weak or absent; FISH staining of periodontal tissue ( Figure 3 (D diagram) shows F. nucleatum It can successfully colonize periodontal tissue. The above results confirm that... F. nucleatum It can enter the circulation through periodontal implantation and target vascular smooth muscle cells, synergistically promoting AAA occurrence, accelerating tumor progression, and reducing animal survival rate in conjunction with Ang II, thus clarifying... F. nucleatum It plays a key role in promoting the development and progression of abdominal aortic aneurysms. F. nucleated It can significantly exacerbate Ang II-induced abdominal aortic dilatation, increase the AAA tumorigenesis rate, and aggravate vascular elastic fiber degradation, collagen deposition disorder, and VSMC apoptosis, confirming... F. nucleatum It can promote the occurrence and development of AAA.

[0063] Example 3: F. nucleatum In vitro mechanism of VSMC phenotypic transformation Human aortic VSMCs were cultured and administered Ang II at concentrations of 1000 ng / mL or MOI=50 in groups. F. nucleated Ang II merger F. nucleatum After co-culturing cells for 24 hours, the expression of contractile proteins (α-SMA, SM22α) and synthetic proteins (OPN) was detected by immunofluorescence, Western blot, and RT-PCR. The levels of inflammatory factors, MMPs, and signaling pathway proteins were also measured. Results showed: F. nucleatum It can significantly inhibit the expression of VSMC contractile markers, upregulate synthetic markers, induce the release of inflammatory factors and MMPs activation. The results were screened and validated using pull-down, LC-MS, and immunoprecipitation experiments. F. nucleatum Key virulence factors bind specifically to VSMC membrane receptors.

[0064] 1) Detection F. nucleatum Impact on VSMC adhesion and invasion: Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining marker F. nucleatumAfterwards, the cells were co-cultured with VSMCs for 2 hours; the cytoskeleton was stained with phalloidin and the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), and observed under a laser confocal microscope. F. nucleatum Invasion and adhesion to VSMC; discovery F. nucleated It can adhere to the surface of VSMC and penetrate into the VSMC. Figure 4 (Figure A); Observation using a scanning electron microscope revealed... F. nucleatum It can invade and adhere to VSMC ( Figure 4 (Figure B). CFSE staining markers were used. F. nucleatum, Different concentrations were used at 3h, 6h, 12h, and 24h respectively. F. nucleatum Stimulating VSMCs and detecting them using flow cytometry F. nucleated The situation regarding the intrusion into VSMC revealed that, with... F. nucleatum With increasing concentration and prolonged stimulation time, the number of attacked VSMCs also increases. Figure 4 (Figure C in the middle)

[0065] 2) F. nucleatum Impact on VSMC phenotypic transformation: use F. nucleatum VSMCs were infected with Ang II as a positive control. VSMC cell morphology was assessed at 0 h, 6 h, 12 h, 24 h, and 48 h. Cell scratch assay was performed at 12 h to assess VSMC cell migration ability. Experimental results are as follows: Figure 5 As shown in Figures A and B, this confirms... F. nucleatum This can facilitate VSMC migration and change the VSMC morphology from "spindle-shaped" to "synthetic". Figure 5 As shown in Figure A, compared to group NC, F. nucleatum Infection alone can induce the transformation of VSMCs from the typical long spindle-shaped contractile pattern to a flattened, disordered synthetic pattern, with an effect comparable to that of the positive control Ang II; Ang II combined with F. nucleatum After processing, the degree of VSMC phenotypic transformation was further aggravated. For example... Figure 5 As shown in Figure B, the scratch test results show that... F. nucleatum Infection alone significantly enhances the migration ability of VSMCs and has a synergistic effect with Ang II. F. nucleatum The migration ability of the group with VSMC was significantly higher than that of the group treated alone. These results indicate that... F. nucleatumIt can participate in the occurrence and development of abdominal aortic aneurysms by inducing VSMC phenotypic transformation and enhancing their migration ability, thus providing a target for... F. nucleatum The intervention scheme for -VSMC interaction provides experimental evidence.

[0066] To further verify F. nucleatum To investigate the regulatory role of VSMC phenotypic transformation, this embodiment used immunofluorescence assays and Western blotting to detect relevant indicators of VSMC phenotypic transformation. The results are as follows: Figure 6 As shown. Immunofluorescence results show ( Figure 6 (Figure A) In the NC group, VSMCs highly expressed the contractile biomarker SM22α (red fluorescence) and lowly expressed the synthetic biomarker OPN (green fluorescence), maintaining the normal contractile phenotype; F. nucleatum Following infection alone, SM22α expression in VSMCs was significantly downregulated, while OPN expression was significantly upregulated, indicating phenotypic transformation in VSMCs. The Ang II-treated group alone exhibited similar phenotypic transformation characteristics, validating the effectiveness of the positive control. Ang II combined with... F. nucleatum After treatment, SM22α expression further decreased, OPN expression further increased, and the degree of phenotypic transformation was significantly aggravated. Western blot results further quantitatively verified this. Figure 6 (Figure B), compared with group NC, F. nucleatum Group, Ang II group, Ang II+ F. nucleatum In this group, the expression levels of contractile markers α-SMA, SM22α, and CNN1 proteins were significantly downregulated, while the expression levels of synthetic marker OPN proteins were significantly upregulated; among them, AngII+ F. nucleatum The expression levels of the contractile biomarkers in the control group were significantly lower than those in the control group alone, while the expression level of OPN was significantly higher than that in the control group alone. These results indicate that... F. nucleatum It can promote phenotypic transformation of VSMCs and exacerbate the severity of Ang II-induced phenotypic transformation of VSMCs.

[0067] Immunofluorescence was used to detect the expression of proteins related to phenotype transformation in mouse aortic VSMCs, and the results are as follows: Figure 7 As shown. Compared to the AAA model group, AAA+ F. nucleatum The aortic tissue of mice showed signs of contractile VSMC marker proteins (SM22α, CNN1, α-SMA). Figure 7 The expression of the synthetic VSMC marker protein (OPN) was significantly reduced in Figures B, C, and D. Figure 7 E-plot (in the middle part of the graph) expression significantly increased. The above results indicate that... F. nucleatum In vivo, it can significantly promote the phenotypic transformation of the aortic VSMCs in mice with abdominal aortic aneurysms from contractile to synthetic types, thereby accelerating the occurrence and development of abdominal aortic aneurysms.

[0068] 3) Screening F. nucleatum Key toxic protein: Identification F. nucleatum Identifying virulence factors (effective proteins) that interact with VSMCs is a crucial step in elucidating the pathogenesis of this pathogenic bacterium. Five virulence factors that may interact with VSMC membrane proteins were identified using the His pull-down assay and high-performance liquid chromatography-mass spectrometry. F. nucleatum Candidate proteins were identified (Table 1). These five proteins were screened using the Uniprot database, and the results showed that dipeptide-binding protein (DBP) is a dipeptide-binding protein mainly located in the periplasmic space of bacterial cells and is part of the ATP-binding cassette (ABC) transporter complex. Therefore, DBP was selected as a candidate protein for subsequent experiments.

[0069] Table 1. F. nucleatum candidate proteins

[0070] 4) In vitro experiments to detect the effect of the key toxic protein DBP on VSMC phenotypic transformation: use F. nucleatum DBP and Ang II were used as positive controls. VSMC cell morphology was detected at 0 h, 6 h, 12 h, 24 h, and 48 h, respectively. The results are as follows: Figure 9 As shown in Figure A; a 6-hour cell scratch assay was performed to detect migration ability, and the results are as follows. Figure 9 As shown in Figure B.

[0071] Figure 9 Figure A shows the dynamic observation results of VSMC cell morphology under different treatment conditions. Compared with NC, the VSMC morphology of the DBP group and Fn group changed from the typical long spindle-shaped contractile type to a flat, irregular synthetic type. The degree of VSMC phenotypic transformation was aggravated after AngII treatment, and the degree of phenotypic transformation of the AngII+DBP group and AngII+Fn group was significantly higher than that of the AngII group alone, confirming that DBP can independently induce VSMC morphological changes and synergistically aggravate phenotypic transformation with AngII.

[0072] Figure 9 Figure B shows the results of the VSMC scratch assay. At 0 h, the scratch widths were consistent across all groups. After 6 h, the scratches in the NC group showed no obvious closure, while the scratches in the DBP and Fn groups showed significantly increased closure, indicating that DBP can independently enhance the migration ability of VSMCs. The scratches in the AngII group showed significantly higher closure than those in the NC group, and the migration ability in the AngII+DBP and AngII+Fn groups was further enhanced, confirming that DBP can promote VSMC migration and amplify this effect in synergy with AngII.

[0073] To further verify the regulatory role of DBP protein in VSMC phenotypic transformation, this embodiment used a combination of Western blotting and immunofluorescence techniques to detect phenotypic transformation-related indicators. The results are as follows: Figure 10 and Figure 11 As shown. Western Blot detection results ( Figure 10 Figure A shows that, compared with NC, the systolic marker α-SMA (in the DBP group VSMC) was significantly lower. Figure 10 Figure B), SM22α ( Figure 10 (Figure C) and CNN1 ( Figure 10 The protein expression level of the synthetic biomarker OPN (Figure D) was significantly reduced. Figure 10 The protein expression level in the middle (Figure E) was significantly increased; F. nucleatum The single-treatment groups showed a similar phenotypic transformation trend. The Ang II treatment group, as a positive control, also induced VSMC phenotypic transformation. Furthermore, the expression of contractile markers further decreased and OPN expression further increased in the Ang II combined with DBP treatment group and the Ang II combined with Fn treatment group. Immunofluorescence staining results ( Figure 11 Further confirmation revealed that VSMCs in the NC group highly expressed α-SMA, CNN1, and SM22α, and lowly expressed OPN; the fluorescence intensity of the contractile markers was significantly reduced in the DBP and Fn groups, while the fluorescence intensity of the synthetic marker OPN was significantly increased; the phenotypic transformation characteristics were obvious in the Ang II treatment groups, and the phenotypic transformation was even more significant in the Ang II combined with DBP treatment group and the Ang II combined with Fn treatment group. These results indicate that DBP can independently promote the phenotypic transformation of VSMCs from contractile to synthetic forms, and can synergistically act with Ang II to further aggravate the degree of VSMC phenotypic transformation, providing a basis for the use of DBP as a phenotypic marker. F. nucleatum Key toxic proteins that promote the development and progression of abdominal aortic aneurysms provide direct molecular and cellular biological evidence.

[0074] 5) Screening for key receptor proteins in VSMCs: Identifying receptor proteins that interact between DBP and VSMCs is an important step in further elucidating the intracellular mechanisms of VSMCs. Using a biotin pull-down assay and high-performance liquid chromatography-mass spectrometry (HPLC-MS), five receptor proteins that may interact with VSMC membrane proteins were identified. F. nucleatum Candidate proteins were identified (Table 2). These five proteins were screened using the Uniprot database and binding assays. The results showed that Fibronectin 1 (FN1) is a core multifunctional glycoprotein of the extracellular matrix. It binds to molecules such as integrins, collagen, fibrin, and heparin through multiple domains, regulating almost all cell-matrix interactions.

[0075] Table 2. Candidate proteins for VSMC

[0076] 6) The effect of knockdown of the key toxic protein DBP on VSMC phenotypic transformation: To further clarify F. nucleatum The molecular mechanism by which the key toxic protein DBP regulates VSMC phenotypic transformation was investigated in this embodiment. This study used molecular docking, immunofluorescence co-localization, coIP, and SPR techniques to jointly verify the direct interaction between DBP and the VSMC surface receptor fibronectin 1 (FN1). Molecular docking simulation results ( Figure 12 Figure A shows that DBP and FN1 have a stable high-affinity binding site; immunofluorescence co-localization ( Figure 12 Figure B in the middle section confirms that the two are co-distributed within the VSMC; the coIP experiment ( Figure 12 (Figure C) further demonstrates that DBP and FN1 specifically bind in vivo; SPR experiment ( Figure 12 Figure D in the diagram confirms from a biophysical perspective that DBP can directly and with high affinity bind to the FN1 protein. Functional experiments show that... F. nucleatum Infection can induce the morphological transformation of VSMCs from contractile to synthetic types. Figure 13 Figure A in the middle), and significantly enhanced bacterial adhesion ( Figure 13 Figure B), promoting apoptosis ( Figure 14 Figure A) and abnormal migration (Figure B in Figure 14) significantly downregulated the expression of contractile markers α-SMA, CNN1, and SM22α, and upregulated the expression of synthetic marker OPN (Figure C in Figure 13). Figure 15 After knocking down FN1 via siRNA, F. nucleatum The adhesion ability with VSMCs was significantly reduced, and the morphology, migration and apoptosis levels of VSMCs were significantly restored. The expression trend of phenotypic transformation-related proteins was also significantly reversed.

[0077] The siRNA sequence is: GCCGGUUGUUAUGACAAUTTAUUGUCAUAACAACCGGGCTT (SEQ ID NO.1).

[0078] The above results collectively confirm that FN1 is F. nucleatum The key toxic protein DBP binds to a critical receptor on the surface of VSMCs. DBP induces phenotypic transformation of VSMCs, enhances migration, and disrupts vascular wall homeostasis by binding to FN1. Targeting and knocking down FN1 can effectively block this pathological process, providing a novel key molecular target and intervention strategy for the prevention and treatment of abdominal aortic aneurysms.

[0079] 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. The use of a substance that blocks the adhesion of Fusobacterium nucleatum to vascular smooth muscle cells in the preparation of products that inhibit or delay the development of abdominal aortic aneurysms; said substance is siRNA with a nucleic acid sequence as shown in SEQ ID NO.

1.

2. A composition, characterized in that, The substance includes a substance that blocks the adhesion of Fusobacterium nucleatum to vascular smooth muscle cells, said substance being siRNA with a nucleic acid sequence as shown in SEQ ID NO.

1.

3. The use of the composition of claim 2 in the preparation of a product that inhibits or delays the development of abdominal aortic aneurysm.

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