Application of Phocaecola vulgaris and metabolite tetrahydrofolic acid of Phocaecola vulgaris in treatment of abdominal aortic aneurysm

Abstract

The invention discloses a Phocaecola vulgaris bacterium and an application of a metabolite tetrahydrofolic acid of the Phocaecola vulgaris bacterium in treatment of abdominal aortic aneurysm. According to the application disclosed by the invention, the fact that the abundance of the Phocaecola vulgaris and the metabolite is in negative correlation with the progress of the aneurysm is proved for the first time, and an animal model verifies that the expansion of the angiotensin-induced abdominal aortic aneurysm of a mouse can be obviously inhibited by oral administration of the Phocaecola vulgaris or intraperitoneal injection of tetrahydrofolic acid, so that the survival rate is increased, and an innovative strategy is provided for the treatment of the abdominal aortic aneurysm.

Description

Technical Field

[0001] The invention relates to application of Phocaeicola vulgatus and its metabolite tetrahydrofolate in treating abdominal aortic aneurysm, belonging to the technical field of biomedicine. Background Art

[0002] Abdominal aortic aneurysm (AAA) is a fatal vascular disease characterized by abnormal dilation of the aortic wall, with a rupture mortality rate as high as 80%. Currently, the main treatment options are open surgery or endovascular repair, but these procedures carry high risks and are not suitable for patients with early-stage or small aneurysms. Regarding pharmacological treatment, no clear and effective medications exist to inhibit aneurysm progression or reduce the risk of rupture. Recent studies have shown that the development of AAA is closely associated with vessel wall inflammation, elevated matrix metalloproteinase (MMP) activity, oxidative stress, and vascular smooth muscle cell apoptosis.

[0003] The role of the gut microbiota and its metabolites in cardiovascular disease is gaining increasing attention. For example, Roseburia intestinalis and its metabolite butyrate promote the development of abdominal aortic aneurysms by upregulating neutrophil extracellular trap-induced inflammation; short-chain fatty acids (SCFAs) inhibit atherosclerosis by modulating immune cell function; and certain microbial metabolites (such as trimethylamine oxide) may exacerbate vascular inflammation. Recent studies have shown that the gut microbiota composition of patients with abdominal aortic aneurysms differs significantly from that of healthy controls, suggesting that microbial imbalance may influence disease progression through metabolite-mediated immune regulation. Tetrahydrofolate, the active form of folate, participates in DNA synthesis and repair, the methylation cycle, and the regulation of oxidative stress, but its role in abdominal aortic aneurysms remains unclear.

[0004] Currently, no specific bacterial strains or their metabolites have been used in the clinical treatment of abdominal aortic aneurysms. Therefore, targeting the gut microbiota and its metabolites may provide a new therapeutic strategy for abdominal aortic aneurysms. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the existing technology and provide an effective therapeutic drug for abdominal aortic aneurysm by using Phocaeicola vulgatus and its metabolite tetrahydrofolate in the treatment of abdominal aortic aneurysm.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides use of Phocaeicola vulgatus and / or its metabolite tetrahydrofolate in the preparation of a medicament for treating abdominal aortic aneurysm.

[0008] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is Phocaeicola vulgatus, and / or tetrahydrofolic acid, and / or a salt form of tetrahydrofolic acid (such as calcium tetrahydrofolate).

[0009] In a second aspect, the present invention provides a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is Phocaeicola vulgatus and / or tetrahydrofolic acid or a salt form of tetrahydrofolic acid.

[0010] In a third aspect, the present invention provides use of a product for detecting Phocaeicola vulgatus and / or its metabolite tetrahydrofolate in the preparation of a product for diagnosing, staging or evaluating the prognosis of abdominal aortic aneurysms.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] (1) The present invention is the first to discover that the abundance of Phocaeicola vulgatus and tetrahydrofolate is negatively correlated with the progression of abdominal aortic aneurysms. Clinical data analysis showed that the abundance of this bacterium in the feces and the plasma tetrahydrofolate level of AAA patients were significantly lower than those in healthy people, and were negatively correlated with the diameter of the aneurysm. Animal experiments confirmed that oral administration of Phocaeicola vulgatus or intraperitoneal injection of tetrahydrofolate can significantly inhibit the expansion of angiotensin-induced abdominal aortic aneurysms in mice and improve survival rates.

[0013] (2) Tetrahydrofolate alone can reduce ferroptosis of vascular wall smooth muscle cells in angiotensin-induced abdominal aortic aneurysm mouse model. Therefore, the present invention provides a potential small molecule compound for the treatment of abdominal aortic aneurysm, which has good clinical translation and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The relationship between the abundance of Phocaeicola vulgatus and tetrahydrofolate and the progression of abdominal aortic aneurysm is shown; A is the comparison of Phocaeicola vulgatus abundance in feces between AAA patients (n=40) and healthy people (n=38); B is the comparison of plasma tetrahydrofolate levels in feces between AAA patients (n=40) and healthy people (n=38); C is the Spearman correlation analysis of Phocaeicola vulgatus abundance and tetrahydrofolate; D is the Spearman correlation analysis of Phocaeicola vulgatus abundance and the maximum diameter of abdominal aortic aneurysm; E is the Spearman correlation analysis of plasma tetrahydrofolate content and the maximum diameter of abdominal aortic aneurysm.

[0015] Figure 2 The results show that oral gavage with Phocaeicola vulgatus inhibits the progression of abdominal aortic aneurysm in a mouse model; A is the maximum diameter of the abdominal aorta in the abdominal aortic aneurysm mouse model 4 weeks after oral gavage with Phocaeicola vulgatus (n=12); B is the tumor formation rate in the abdominal aortic aneurysm mouse model 4 weeks after oral gavage with Phocaeicola vulgatus (n=12); C is the Kaplan-Meier analysis of the survival of the two groups of mice (n=12).

[0016] Figure 3 The tetrahydrofolate content in the supernatant of Phocaeicola vulgatus incubated with PBS or folic acid for 48 h is shown (n=3), indicating that Phocaeicola vulgatus can produce tetrahydrofolate through substrate metabolism.

[0017] Figure 4 The results show that intraperitoneal injection of tetrahydrofolic acid inhibits the progression of abdominal aortic aneurysm mouse model; A is the maximum diameter of the abdominal aorta in the abdominal aortic aneurysm mouse model 4 weeks after intraperitoneal injection of tetrahydrofolic acid (n=12); B is the tumor formation rate in the abdominal aortic aneurysm mouse model 4 weeks after intraperitoneal injection of tetrahydrofolic acid (n=12); C is the Kaplan-Meier analysis of the survival of the two groups of mice (n=12). DETAILED DESCRIPTION

[0018] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0019] In the following examples, the experimental methods without specific conditions were carried out according to conventional methods and conditions, or selected according to the product specifications; the materials and reagents used were all conventional commercially available products unless otherwise specified.

[0020] The present study collected stool and plasma samples from 40 AAA patients and 38 healthy controls. Metagenomic sequencing and untargeted metabolomics analysis revealed that the abundance of Phocaeicola vulgatus was significantly lower in AAA patients than in healthy controls. High-throughput untargeted metabolomics revealed decreased levels of 126 metabolites in the plasma of patients with abdominal aortic aneurysm compared with controls. Metorigin data was used to predict the possible bacterial origin of these metabolites. Tetrahydrofolate was found to be closely associated with Phocaeicola vulgatus and to be significantly decreased in the plasma of patients with abdominal aortic aneurysm.

[0021] Based on this, the present invention aims to provide the therapeutic effects of Phocaeicola vulgatus and its metabolite tetrahydrofolate on abdominal aortic aneurysm.

[0022] Example

[0023] 1. Materials

[0024] 1.1 Clinical tissue specimens

[0025] The fecal and blood samples used in this study were obtained from patients with confirmed abdominal aortic aneurysm diagnosed at Zhongshan Hospital, Fudan University. This study has been approved by the Ethics Committee of Zhongshan Hospital, Fudan University, and all patients signed informed consent.

[0026] 1.2 Animals

[0027] All animal experiments used in this paper followed the procedures approved by the Institutional Animal Care and Use Committee (IACUC) of Zhongshan Hospital, Fudan University. - / - Mice, 8 weeks old, were purchased from Weitonglihua Company (Shanghai, China).

[0028] 1.3 Phocaeicola vulgatus strain

[0029] The Phocaeicola vulgatus bacteria used in the present invention were purchased from the American Type Culture Collection (ATCC) with the accession number being ATCC 8482.

[0030] 2. Experimental Methods

[0031] 2.1 Metagenomic Sequencing

[0032] Using sterile fecal collection tubes, researchers collected fresh samples and immediately placed them in a -80°C ultra-low temperature freezer to avoid repeated freezing and thawing. The magnetic bead-based soil and fecal genomic DNA extraction kit from Tiangen Biotechnology (Beijing) Co., Ltd. was used, and agarose gel electrophoresis and The quality of the DNA was checked by using a 2.0 fluorometer. The qualified DNA was sonicated into 350 bp fragments for Illumina library construction. Ultra TMDNA library preparation kit. The library was sequenced on the Illumina Novaseq 6000 platform, generating 150 bp paired-end reads. All bioinformatics analyses were performed by Shanghai Meiji Biotechnology Co., Ltd. The raw data were quality controlled using fastp, and potential host contamination was removed using BWA. Metagenomic assembly was performed using MEGAHIT, and contigs larger than 800 bp were retained. Gene prediction and abundance analysis were performed using Prodigal and CD-HIT software, generating a non-redundant gene catalog with 95% identity and 90% coverage. Clean reads were aligned to the non-redundant gene catalog using Bowtie2, and gene abundance (TPM) was calculated based on the number of reads and gene length. Representative sequences were selected from the non-redundant gene catalog and compared to the NCBI NR database using BLASTP (version 2.2.28+) with an e-value cutoff of 1e-5 for taxonomic annotation. For alignment against the Kyoto Encyclopedia of Genes and Genomes database (KEGG), BLASTP (version 2.2.28+) was used with an e-value cutoff of 1e-5.

[0033] 2.2 Non-targeted metabolome sequencing

[0034] Fasting venous blood was collected from patients with abdominal aortic aneurysm (n = 40) and healthy controls (n = 38). After EDTA anticoagulation, plasma was separated by centrifugation at 1500 × g for 10 minutes at 4°C and stored in aliquots at −80°C. Metabolites were extracted using a modified methanol-acetonitrile two-phase extraction method: 100 μL of plasma was added to 400 μL of pre-chilled methanol-acetonitrile (1:1, v / v). The mixture was vortexed and sonicated on ice for 10 minutes (300 W power, 4°C). Protein was precipitated at −20°C for 1 hour. The supernatant was centrifuged at 14,000 × g for 15 minutes, concentrated by nitrogen purging, and reconstituted in 50 μL of 80% methanol. The supernatant was filtered through a 0.22 μm filter before injection. A mixed quality control (QC) sample was inserted into each batch to assess reproducibility (RSD < 15%).

[0035] An Agilent 1290 UHPLC system coupled to an Agilent 6545Q-TOF mass spectrometer was used for simultaneous acquisition in positive and negative ion modes. Chromatographic conditions included a ZORBAX Eclipse Plus C18 column (2.1×100 mm, 1.8 μm), column temperature at 40°C, flow rate at 0.3 mL / min; mobile phase A (0.1% formic acid in water)-B (0.1% formic acid in acetonitrile), gradient elution (5% to 95% B phase, 0-15 min; hold at 95% for 15-18 min). Mass spectrometry parameters included an electrospray ionization (ESI) source, drying gas temperature at 350°C, capillary voltage at 3500 V, scan range m / z 50-1000, and MS / MS collision energy of 10-40 eV.

[0036] The raw data were peak extracted, aligned, and normalized using Progenesis QI software, and metabolites were annotated using the HMDB and KEGG databases. Orthogonal partial least squares discriminant analysis (OPLS-DA, VIP>1.0) and t-test (p<0.05) were used to screen for differential metabolites associated with abdominal aortic aneurysm, and a random forest model was further constructed to evaluate and screen metabolite indicators with diagnostic potential (AUC>0.9). Innovations include biphasic extraction combined with polar gradient elution technology (increasing small molecule metabolite coverage by>30%) and multimodal ion fragment matching strategy (annotation accuracy>85%). Significance was determined using an unpaired Student's t-test, and p<0.05 was considered statistically significant.

[0037] 2.3 ELISA detection of tetrahydrofolate

[0038] A commercial THF kit (tetrahydrofolate detection kit (ELISA method)) was used to perform the detection according to the method recommended in the kit instructions.

[0039] 2.4 Construction of mouse model

[0040] 8-week-old ApoE - / - Male mice (C57BL / 6 background) were anesthetized with isoflurane and implanted subcutaneously with an Alzet osmotic pump (model 2004) in the back. Ang II (1000 ng / kg / min, dissolved in 0.9% saline) was continuously infused for 28 days. A control group was implanted with a saline pump. Survival of the mice was recorded daily, and 28-day survival was calculated (Kaplan-Meier analysis).

[0041] On days 7, 14, 21, and 28 of infusion, the maximum diameter of the abdominal aorta (longitudinal and transverse scans) was measured using a high-frequency ultrasound system (VisualSonics Vevo 3100, probe frequency 30 MHz). The dilation rate of the abdominal aorta was calculated based on the adjacent normal vessel segment (1 mm from the renal artery bifurcation). Mice were euthanized 28 days after modeling. After euthanasia, the abdominal aorta was quickly dissected and exposed. The surrounding connective tissue was gently stripped to avoid traction injury. After stripping, the abdominal aorta was immediately immersed in 4% paraformaldehyde (PFA) and fixed for 24 hours. After fixation, the vascular specimens were placed under a stereomicroscope (Olympus SZX16), and the following parameters were measured using a calibrated microscale (accuracy 0.01 mm): normal vessel diameter (no dilated segment); maximum tumor diameter (the adventitial value at the maximum diameter of the entire vessel). The hemangioma and adjacent normal segments were embedded in paraffin and sliced to a thickness of 5 μm for the following: HE staining: to evaluate the integrity of the vascular wall, inflammatory cell infiltration and thrombosis. Elastic fiber staining (EVG): to quantify the proportion of elastic fiber rupture in the tunica media (ImageJ software analysis, the rupture area ratio > 30% is considered pathologically positive). The AAA model was considered successful if any of the following conditions was met: 1. Morphological criteria: hemangioma diameter / normal vessel diameter ≥ 1.5 (i.e., expansion rate ≥ 50%). 2. Pathological criteria: EVG staining showed continuous rupture of elastic fibers accompanied by degeneration of the tunica media, and HE staining confirmed full-thickness structural destruction of the vascular wall. 3. Clinical endpoint: death due to aneurysm rupture during the experiment (autopsy confirmed peritoneal hemorrhage and vascular rupture site).

[0042] 2.5 In vivo experiments in mice

[0043] In the oral gavage experiment, mice were gavaged with 1×10 9 The mice were treated with 1000 colony-forming units (CFU) of Phocaeicola vulgatus or 50 mg / kg tetrahydrofolate in 200 μl of PBS until the end of the study. The control group mice were given the same volume of PBS.

[0044] 2.6 Phocaeicola vulgatus culture

[0045] Phocaeicola vulgatus strains were cultured anaerobically using Difco Reinforced Clostridial Medium (BD Biosciences, Catalog No. 218081) in an anaerobic incubator (Coy Laboratory Products) maintained at 37°C in an atmosphere of 10% CO₂, 10% H₂, and 80% N₂. All manipulations involving Phocaeicola vulgatus were performed in an anaerobic chamber, and the culture medium and reagents were pre-deoxygenated for at least 48 hours.

[0046] 3. Data Analysis

[0047] All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using SPSS 20.0 software. When comparing two groups of data, a normal distribution test was performed first, followed by the Student t test. If normal distribution or homogeneity of variance was not satisfied, the Mann-Whitney U test was used to compare the two groups. When the experimental design involved comparisons of more than two groups, a normal distribution test was performed first, followed by analysis of variance (ANOVA) to compare differences between groups, and the least significant difference (LSD) test was used for multiple comparisons between pairs. If normal distribution or homogeneity of variance was not satisfied, the Kruskal-Wallis test was used to compare differences between groups, and the Hodges-Lehmann test was used for multiple comparisons between pairs. A p value of less than 0.05 was considered statistically significant. p values are represented by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0048] 4. Implementation Plan and Results

[0049] 4.1 Phocaeicola vulgatus and tetrahydrofolate are associated with the development and progression of abdominal aortic aneurysms

[0050] To investigate the relationship between microorganisms and metabolites and the development of abdominal aortic aneurysms, metagenomic and non-targeted metabolome sequencing was performed on the feces and plasma of 40 patients with abdominal aortic aneurysms and 38 healthy controls. The results showed that the levels of Phocaeicola vulgatus and tetrahydrofolate in patients with abdominal aortic aneurysms were significantly lower than those in healthy controls ( Figure 1 AB). At the same time, correlation analysis between the two showed that the abundance of Phocaeicola vulgatus and tetrahydrofolate was positively correlated ( Figure 1 C). Since the maximum diameter of the aorta is the main diagnostic and prognostic indicator of abdominal aortic aneurysm, we further analyzed the correlation between the abundance of Phocaeicolavulgatus and tetrahydrofolate and the maximum diameter of the aorta, and found that both were significantly negatively correlated with the maximum diameter of abdominal aortic aneurysm ( Figure 1 These results indicate that the abundance of Phocaeicola vulgatus and tetrahydrofolate is significantly reduced in patients with abdominal aortic aneurysm, and that Phocaeicola vulgatus and tetrahydrofolate are significantly associated with the occurrence and development of abdominal aortic aneurysm.

[0051] Phocaeicola vulgatus and its derived metabolite tetrahydrofolate effectively inhibit the progression of abdominal aortic aneurysms

[0052] 1) In order to clarify the therapeutic effect of Phocaeicola vulgatus on abdominal aortic aneurysm, an angiotensin-induced abdominal aortic aneurysm mouse model was established in this example. 1×10 9 After four weeks of continuous gavage, the incidence and mortality of abdominal aortic aneurysms in the Phocaeicola vulgatus treatment group were significantly reduced compared with the angiotensin-induced group. At the same time, a significant decrease in the maximum diameter of the abdominal aorta was observed ( Figure 2 AC). To verify that tetrahydrofolate is a derivative metabolite of the angiotensin-induced group, an incubation experiment was conducted between the angiotensin-induced group and folic acid, the substrate of tetrahydrofolate. The results showed that after the addition of folic acid, the tetrahydrofolate content in the angiotensin-induced group fluid increased significantly ( Figure 3 ), indicating that tetrahydrofolate can be metabolized by angiotensin-induced groups. Next, the inhibitory effect of intraperitoneal injection of tetrahydrofolate on abdominal aortic aneurysms was explored using an angiotensin-induced abdominal aortic aneurysm mouse model. It was found that intraperitoneal injection of 50mg / kg tetrahydrofolate had the effect of inhibiting the occurrence and development of abdominal aortic aneurysms ( Figure 4 AC). This indicates that Phocaeicola vulgatus and its derived metabolite tetrahydrofolate can effectively inhibit the occurrence and development of abdominal aortic aneurysms.

[0053] The above description is only a preferred embodiment of the present invention and does not constitute any formal or substantial limitation to the present invention. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of Phocaeicola vulgatus and / or its metabolite tetrahydrofolate in the preparation of a medicament for treating abdominal aortic aneurysm.

2. The use according to claim 1, characterized in that The medicine comprises an active ingredient and a pharmaceutically acceptable carrier or excipient. The active ingredient is Phocaeicola vulgatus bacteria, and / or tetrahydrofolic acid, and / or a salt form of tetrahydrofolic acid.

3. A pharmaceutical composition, characterized in that The invention comprises an active ingredient and a pharmaceutically acceptable carrier or excipient; the active ingredient is Phocaeicola vulgatus, and / or tetrahydrofolic acid, and / or a salt form of tetrahydrofolic acid.

4. Use of a product for detecting Phocaeicola vulgatus and / or tetrahydrofolate in the preparation of a product for diagnosing, staging, or evaluating the prognosis of abdominal aortic aneurysm.

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