Application of Ackermania muciniphila as biomarker in preparation of product for diagnosing and / or treating atherosclerosis

Abstract

The invention provides application of Ackermania muciniphila as a biomarker in preparation of a product for diagnosing and / or treating atherosclerosis, and belongs to the technical field of biological medicines. The invention provides an application of Akkermansia muciniphila (Akkermansia muciniphila) as a biomarker in the preparation of a kit for diagnosing and / or detecting atherosclerosis (AS). The Akkermansia muciniphila can be used for detecting atherosclerosis (AS). A high fat diet induced ApoE gene knockout mouse copies an AS mouse model, metagenomics analysis of a control group and a model group shows that the abundance difference of Ackermania muciniphila is significant, and the AUC value under an ROC curve is 1, which indicates that Ackermania muciniphila is a potential biomarker strain of AS. The Ackermania muciniphila can also significantly reduce the AS plaque area of the aortic sinus of the AS model mouse, and the Ackermania muciniphila can play an anti-AS role through a two-component system, ABC transport and other metabolic pathways.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to Akkermansia myxophilus (… Akkermansia muciniphila, A. muciniphila The application of biomarkers in the preparation of products for the diagnosis and / or treatment of atherosclerosis (AS). Background Technology

[0002] Atherosclerosis (AS) is a key pathological basis for cardiovascular and cerebrovascular events. Based on the degree of vascular lesions and ischemia in affected organs, it is classified into aortic atherosclerosis, coronary atherosclerosis, cerebral artery atherosclerosis, renal artery atherosclerosis, mesenteric artery atherosclerosis, and lower extremity artery atherosclerosis. Coronary atherosclerosis, in particular, can lead to angina pectoris, myocardial infarction, arrhythmias, and even sudden death. Therefore, early detection and diagnosis of Atherosclerosis are crucial for the intervention and treatment of this disease.

[0003] Numerous reports have been published on biomarkers for AS, including lipids, specific genes, proteins, RNA, and alkaloids. These biomarkers are often used for early diagnosis.

[0004] Currently, an increasing number of studies indicate that changes in the gut microbiota are closely related to the formation and development of atherosclerosis (AS). In recent years, metagenomics technology has been widely used in research on the correlation between the pathogenesis of AS and the gut microbiota. Previous metagenomic studies on the gut contents of patients with atherosclerotic cardiovascular disease (ACVD) have shown that, compared to healthy individuals, ACVD patients have a relative decrease in Lactobacillus and Prevotella, and an increase in the abundance of Escherichia coli and Streptococcus. Through a search of human-related microorganisms in the Human Microbiome Project database, core gut microbiota genera of AS patients were identified (…). Lachnoclostridium ) and Clostridium ( Clostridium The expression of [specific marker] was significantly elevated compared to normal individuals, suggesting that gut microbiota may be an effective target for the diagnosis or treatment of AS. However, to date, there are no reports of microbial biomarkers serving as targets for the diagnosis and treatment of AS. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of Akkermansia myxophilus as a biomarker in the preparation of diagnostic and / or detection kits for AS.

[0006] This invention provides the application of Akkermansia myxophilus as a biomarker in the preparation of kits for the diagnosis and / or detection of AS.

[0007] This invention provides the application of reagents for detecting Akkermansia myxophilus in the preparation of diagnostic and / or detection kits for Acetobacter amyloliquefaciens.

[0008] Preferably, the reagent includes at least one of the following: intestinal contents DNA extraction reagent, library construction reagent, and sequencing reagent.

[0009] This invention provides the use of Akkermansia myxophilus in the preparation of medicaments for the prevention and / or treatment of AS.

[0010] Preferably, Akkermansia myxophilus, in combination with other probiotics, is used in the preparation of medicaments for the prevention and / or treatment of AS.

[0011] Preferably, the other probiotics include at least one of the following: Bacteroides ( Bacteroides sp) strain CAG927.

[0012] This invention provides the use of a reagent for increasing the abundance of Akkermansia myxophilus in the preparation of medicaments for the prevention and / or treatment of AS.

[0013] Preferably, the reagent comprises Akkermansia myxophilus strain ATCC BAA-835.

[0014] Preferably, the dosage form of the drug includes an oral dosage form.

[0015] Preferably, the oral dosage form includes at least one of the following: tablets, powders, and capsules.

[0016] This invention provides the application of *Ackermania myxophilus* as a biomarker in the preparation of kits for the diagnosis and / or detection of ankylosing spondylitis (AS). Using ApoE gene knockout mice induced by a high-fat diet as an AS animal model, metagenomic analysis was employed to screen for microbial species showing significant differences between the control and model groups. The results showed that *Ackermania myxophilus* was more abundant in the control group, while its abundance was significantly reduced in the model group, demonstrating a significant difference between the two groups. The area under the ROC curve (AUC value) was 1, indicating that *Ackermania myxophilus* is a potential biomarker for AS. Further validation experiments confirmed that *Ackermania myxophilus* can significantly reduce the area of ​​AS plaques in the aortic sinus of AS model mice. *Ackermania myxophilus* may exert its anti-AS effect through metabolic pathways such as a two-component system and ABC transport. Therefore, this invention provides *Ackermania myxophilus* as a biomarker for the diagnosis and / or detection of AS. Attached Figure Description

[0017] Figure 1 HE staining results of mouse aortic sinus; magnification 100×, C is normal control group, M is model group; Figure 2The results are composed of the original data; Host reads: the proportion of the number of reads belonging to the host; Low quality: the proportion of low-quality reads filtered out out of the total number of raw reads; Clean reads: the proportion of clean reads obtained after filtering out of the total number of raw reads. Figure 3 This is a graph showing the error rate distribution of base sequencing; the horizontal axis represents the base position of the reads, and the vertical axis represents the single base error rate. Figure 4 This is a graph showing the distribution of ATGC content; the horizontal axis represents the base position of Reads, and the vertical axis represents the proportion of single bases. Figure 5 This is a map showing the distribution of gene catalog lengths in mouse intestinal contents metagenomics; the horizontal axis represents gene length, and the vertical axis represents the number of genes. Figure 6 The results show the differential microbial composition of mouse intestinal contents (phylum level); the horizontal axis represents the group, the vertical axis represents the relative abundance of the top 30 phyla, C is the normal control group, and M is the model group; Figure 7 The results show the differential microbial composition of mouse intestinal contents (genus level); the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 30 genera; C is the normal control group, and M is the model group. Figure 8 The results show the differential microbial composition of mouse intestinal contents (species level); the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 30 species; C is the normal control group, and M is the model group. Figure 9 This is a heatmap of species abundance clustering in mouse intestinal contents (phylum level); the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 50 phyla; C is the normal control group, and M is the model group. Figure 10 The results of clustering heatmap analysis of species abundance in mouse intestinal contents (genus level); the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 50 genera; C is the normal control group, and M is the model group. Figure 11 The results of clustering heatmap analysis of species abundance in mouse intestinal contents (species level); the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 50 species; C is the normal control group, and M is the model group. Figure 12 The results of Alpha diversity analysis of mouse intestinal contents are shown; the horizontal axis represents the group, and the vertical axis represents the Shannon, Simpson, and invsimpson indices. Figure 13The results are from principal coordinates analysis (PCoA) of mouse intestinal contents; the horizontal axis represents the first principal component and its contribution to the sample differences, and the vertical axis represents the second principal component and its contribution to the sample differences; C represents the normal control group, and M represents the model group. Figure 14 The results of Lefse analysis (phylum level) of differential species among mouse intestinal contents groups are shown; the horizontal axis represents LDA scores, and the vertical axis represents the dominant phylum in each group; purple represents the dominant phylum in group C, and gray represents the dominant phylum in group M. Figure 15 The results of Lefse analysis (genus level) of differential species among mouse intestinal contents groups are shown; the horizontal axis represents LDA score, and the vertical axis represents the dominant bacterial genera in each group; purple represents the dominant bacterial genera in group C, and gray represents the dominant bacterial genera in group M. Figure 16 The results of LAFSE analysis (species level) of differential species among mouse intestinal contents groups are shown; the horizontal axis represents LDA scores, and the vertical axis represents the dominant bacterial species in each group; purple represents the dominant bacterial species in group C, and gray represents the dominant bacterial species in group M. Figure 17 ROC curve; Figure 18 The results of HE staining in mice are shown; magnification is 100×; M is the model group, and AKK is the Akkermansia myxophilus group. Figure 19 This is a map showing the distribution of gene catalog lengths in mouse intestinal contents metagenomics; the horizontal axis represents gene length, and the vertical axis represents the number of genes. Figure 20 The results show the differential microbial composition of mouse intestinal contents (phylum level); the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 30 phyla; M is the model group, and AKK is the Akkermansia myxophilus group. Figure 21 The results of differential microbial composition in mouse intestinal contents (genus level) are shown; the horizontal axis represents the group and the vertical axis represents the relative abundance of the top 30 genera; M is the model group and AKK is the Akkermansia myxophilus group. Figure 22 The results of differential microbial composition in mouse intestinal contents (species level) are shown; the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 30 species; M is the model group, and AKK is the Akkermansia myxophilus group. Figure 23 This is a heatmap of species abundance clustering in mouse intestinal contents (phylum level); the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 50 phyla; M is the model group, and AKK is the Akkermansia myxophilus group. Figure 24This is a heatmap of species abundance clustering in mouse intestinal contents (genus level); the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 50 genera; M is the model group, and AKK is the Akkermansia myxophilus group. Figure 25 This is a heatmap of species abundance clustering in mouse intestinal contents (species level); the horizontal axis represents the group, and the vertical axis represents the relative abundance of the top 50 species; M is the model group, and AKK is the Akkermansia myxophilus group. Figure 26 The results of Alpha diversity analysis of mouse intestinal contents are shown; the horizontal axis represents the group, and the vertical axis represents the Shannon, Simpson, and invsimpson indices. Figure 27 The results of PCoA analysis of mouse intestinal contents are shown; the horizontal axis represents the first principal component and its contribution to the sample differences, and the vertical axis represents the second principal component and its contribution to the sample differences; M is the model group, and AKK is the Akkermansia myxophilus group. Figure 28 The results of Lefse analysis (genus level) of differential species among mouse intestinal contents groups; the horizontal axis represents LDA score, and the vertical axis represents the dominant bacterial genera in each group; purple represents the dominant bacterial genera in group M, and blue represents the dominant bacterial genera in group AKK. Figure 29 The results of Lefse analysis (species level) of differential species among mouse intestinal contents groups are shown; the horizontal axis represents LDA scores, and the vertical axis represents the dominant bacterial species in each group; purple represents the dominant bacterial species in group M, and blue represents the dominant bacterial species in group AKK. Figure 30 Results of KEGG analysis of mouse intestinal contents (LEVEL B); Figure 31 The results of KEGG analysis of mouse intestinal contents (LEVEL C). Detailed Implementation

[0018] This invention provides the application of Akkermansia myxophilus as a biomarker in the preparation of diagnostic and / or detection kits for Acetaminophen (AS).

[0019] In this invention, the AS preferably includes at least one of the following: aortic atherosclerosis, coronary atherosclerosis, cerebral artery atherosclerosis, renal artery atherosclerosis, mesenteric artery atherosclerosis, lower extremity artery atherosclerosis, etc., and more preferably coronary atherosclerosis.

[0020] In this invention, ApoE gene knockout mice induced by a high-fat diet are used as an AS animal model for screening biomarkers. The high-fat diet preferably contains at least 20% fat by mass, more preferably 21%–25%, and most preferably 21.5%. The fat preferably includes animal fats and / or vegetable oils. The animal fats preferably include at least one of the following: lard, mutton fat, beef tallow, etc. The vegetable oils preferably include at least one of the following: rapeseed oil, corn oil, sunflower oil, and peanut oil, etc. In this embodiment of the invention, the AS animal model is preferably fed a basal diet containing 21% lard and 0.15% cholesterol by mass.

[0021] In this invention, the abundance of *Ackermania myxophilus* showed a significant difference between the AS animal model group and the healthy control group. Lefse analysis of differentially expressed species between the groups showed that *Ackermania myxophilus* was significantly enriched in the healthy control group, and the area under the ROC curve (AUC value) was 1, indicating that... A. muciniphila This is a potential biomarker species for Ackermansia myxophilus (AS). Based on the reliability, stability, and accuracy of bioinformatics analysis data, the described *Ackermania myxophilus* exhibits good detection sensitivity and specificity. Further validation experiments confirmed that... A. muciniphila It can significantly reduce the area of ​​aortic AS sinus plaques in AS model mice. A. muciniphila It may exert its anti-AS effect through metabolic pathways such as two-component systems and ABC transport.

[0022] In this invention, to elucidate the intrinsic mechanism of AS biomarkers, this invention explores AS biomarkers from the perspective of the "gut axis." Specifically, metagenomics technology was used to analyze the intestinal contents of mice, and inter-group difference analysis was performed on the intestinal contents of control and model mice. Species abundance clustering heatmap results showed a significant difference in the abundance of gut microbiota between the model and control groups. Lefse analysis revealed that *Ackermania myxophilus* was significantly enriched at the species level in the control group, while its abundance was significantly downregulated in the model group, with an ROC value of 1, indicating that *Ackermania myxophilus* has high diagnostic accuracy as a biomarker for AS. This invention uses *Ackermania myxophilus* not only to adjust the abundance and diversity of the intestinal microbial community in AS model mice, but also to regulate multiple metabolic pathways, suggesting that *Ackermania myxophilus* may continue to promote anti-AS development through the "gut axis."

[0023] This invention provides the use of reagents for detecting Akkermansia myxophilus in the preparation of kits for diagnosing and / or detecting AS.

[0024] In this invention, the detection method is preferably metagenomic library sequencing or Akkermansia myxophilus 16S sequencing. The reagents preferably include at least one of the following: intestinal content DNA extraction reagent, library construction reagent, and sequencing reagent. In this embodiment, the intestinal content DNA extraction reagent is preferably the Magnetic Soil And Stool DNA Kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.). The library construction reagent is preferably the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (purchased from NEB). This invention does not impose any special limitations on the type of sequencing reagent; sequencing reagents well-known in the art can be used. In this embodiment, the sequencing method preferably uses the NovaSeq 6000 sequencing platform with a PE150 read length.

[0025] In this invention, the preferred method for diagnosing AS using the kit involves extracting DNA from the sample to be tested using an intestinal contents DNA extraction reagent, constructing a library using the DNA as material and the library construction reagent, sequencing the constructed library using a sequencing reagent to obtain sequencing results, removing low-quality reads from the sequencing results, performing genome assembly, gene prediction analysis and functional annotation analysis, and cluster heatmap analysis of Akkermansia myxophilus species abundance. When the abundance of Akkermansia myxophilus in the sample to be tested is significantly lower than that in the healthy control group, it is determined that the sample to be tested may have AS, and further verification using clinical testing methods is required.

[0026] This invention provides the use of Akkermansia myxophilus in the preparation of medicaments for the prevention and / or treatment of AS.

[0027] In this invention, *Akkermansia myxophilus* is preferably used in combination with other probiotics in the preparation of medicaments for the prevention and / or treatment of AS. The other probiotics preferably include at least one of the following: *Bacteroides* (…). BacteroidesThe drug is strain CAG927 (Multi-omics reveals the mechanism of rumen microbiome and its metabolome together with host metabolome participating in the regulation of milk production traits in dairy buffaloes. 2024 Mar 8:15:1301292. doi:10.3389 / fmicb.2024.1301292.). The dosage form of the drug includes an oral dosage form. The oral dosage form preferably includes at least one of the following: tablets, powder, and capsules. The viable concentration of *Akkermansia myxophilus* in the drug is not less than 1 × 10⁻⁶. 8 CFU / g, more preferably 5×10 8 CFU / g ~10×10 8 CFU / g.

[0028] This invention provides the use of a reagent for increasing the abundance of Akkermansia myxophilus in the preparation of medicaments for the prevention and / or treatment of AS.

[0029] In this invention, the reagent preferably includes Akkermansia myxophilus strain ATCC BAA-835.

[0030] The following examples illustrate the application of Akkermansia myxophilus as a biomarker in the preparation of products for diagnosing and / or treating AS, but these examples should not be construed as limiting the scope of protection of this invention.

[0031] Example 1 1. Preparation and grouping of experimental animals and models Specific pathogen-free (SPF) ApoE - / - Male mice and male C57BL / 6J mice of the same strain (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were fed at the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine. - / - Mice served as the model group (Model group, M group, 12 weeks old) and were fed a high-fat diet for 12 weeks; C57BL / 6J mice served as the control group (Control group, C group, 12 weeks old) and were fed a normal mouse diet for 12 weeks. The high-fat diet formula for mice consisted of the normal basal diet for mice + 21wt% lard + 0.15wt% cholesterol.

[0032] 2. HE staining of mouse aortic sinus Mice in both the model group and control group were anesthetized with 2.5% sodium pentobarbital and perfused with 4% paraformaldehyde. The aorta along with the heart was harvested and preserved in 10% formalin for paraffin sectioning and HE staining. HE-stained images were acquired using a fully automated digital slide scanner.

[0033] HE staining results showed that group C mice had smaller AS plaques in the aortic sinus; compared with group C, group M mice had significantly larger AS plaque areas in the aortic sinus. Figure 1 The above results indicate that the AS mouse model was successfully constructed.

[0034] 3. Metagenomic analysis of mouse intestinal contents 3.1 Sample Processing Intestinal contents samples from mice in groups M and C were slowly thawed at 4°C. A suitable amount of sample was then added to the Magnetic Soil And Stool DNA Kit (TIANGEN) to extract DNA. DNA concentration was measured using the Qubit® dsDNA Assay Kit on a Qubit® 2.0 Fluromete (rLife Technologies, CA, USA). Sequencing libraries were generated according to the manufacturer's recommendations using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB). Finally, sequencing was performed using the NovaSeq 6000 sequencing platform with a PE150 read length.

[0035] 3.2 Bioinformatics Analysis First, the high-throughput sequencing results are identified by sequence base recognition and converted into raw sequencing reads, also known as raw reads. These reads include the name, base sequence, and corresponding sequencing quality information for each read. Second, before data analysis, it is necessary to ensure that the reads are of sufficiently high quality to guarantee the accuracy of subsequent analysis. Therefore, by eliminating low-quality reads, high-quality Clean Data is obtained. Figure 2 Meanwhile, the sequencing error rate is related to the base sequencing quality. QC30 represents a base identification error probability of 0.001 and a base identification accuracy of 99.9%. A higher base quality value indicates more reliable base identification and higher accuracy. Figure 3 Base type distribution testing is used to detect the presence of AT and GC segregation. Since the sequence being tested consists of randomly fragmented DNA fragments, theoretically, due to random fragmentation and the principle of complementary base pairing, the contents of G and C, and A and T, should be equal in each sequencing cycle, indicating high stability throughout the testing process. Figure 4Based on genome assembly, gene prediction analysis is performed, followed by gene set construction and functional annotation analysis of the predicted genes. Python is used to count the length of the gene catalogue, and the results are as follows. Figure 5 As shown, genes with a length of 200-600 bp account for a large proportion, with the 500 bp gene being the most numerous. As the gene length increases, the number of genes decreases accordingly.

[0036] 4. Differential analysis of intestinal contents among mouse groups 4.1. Bar Chart Analysis The results are as follows Figure 6~Figure 8 As shown, the bar chart displays the species composition at the taxonomic level, which clearly shows the species composition of the samples between groups and the proportion of different species in the samples between groups.

[0037] according to Figure 6 It can be seen that at the phylum level, compared with group C, group M has an increase in Firmicutes, Actinobacteria, and Proteobacteria, and a decrease in Bacteroidetes and Verrucomicrobia, with an upward adjustment in the Firmicutes / Bacteroidetes ratio; according to Figure 7 It can be seen that, at the genus level, compared to group C, group M has a higher proportion of *Femtobacter* genus ( Faecalibaculum Bifidobacterium spp. Bifidobacterium ), Eubacteria ( Eubacterium ) genus Rocherton Roseburia The number of Chlamydia species increased. Chlamydia ), Candidatus_ Amulumruptor genus *Alternaria* ( Alistipes ), Duborella ( Dubosiella Bacteroides ( Bacteroides ), Prevotella spp. Prevotella Reduce; according to Figure 8 It can be seen that, at the species level, compared to group C, the *Rodentium* species in group M (…) Faecalibaculum_rodentium Bifidobacterium species ( Bifidobacterium_ pseudolongum ), Eubacterium _sp_14-2、 Roseburia _sp_1XD42-69 increased, Chlamydia ( Chlamydia_ abortus ) Candidatus_Amulumruptor _caecigallinarius, Duborella strain ( Dubosiella_ newyorkensis )reduce.

[0038] 4.2 Clustering heatmap analysis of species abundance Cluster heatmaps are a graphical representation of data matrices that uses color gradients to represent the magnitude of values ​​and clusters species based on abundance similarity.

[0039] The results are as follows Figure 9~Figure 11 As shown. Differences between the top 50 species at the phylum, genus, and species levels in groups C and M.

[0040] 4.3 Alpha Diversity Analysis Alpha diversity analysis can reflect the abundance and diversity of microbial communities.

[0041] The results are as follows Figure 12 As shown, the M group showed a relatively higher level of bacterial diversity compared to the C group, indicating that the M group had a higher bacterial diversity than the C group.

[0042] 4.4. PCoA Analysis Further analysis using PCoA, a classic unconstrained ordination analysis method, expands the sample distance matrix in a low-dimensional space by projection, preserving the distance relationships of the original samples to the greatest extent possible.

[0043] The results are as follows Figure 13 As shown, the differences between the two groups are significant.

[0044] 4.5 Lefse analysis of species differences between groups LEFSE analysis of species with significant differences between groups is used to screen species with significant differences between groups. The rank-sum test is used to detect species with significant differences between different groups, and dimensionality reduction and the influence of species with significant differences are achieved by LDA (linear discriminant analysis) to obtain LDA value. The significance of species with significant differences is determined based on the LDA value.

[0045] The results are as follows Figure 14~Figure 16 As shown. It can be seen that at the phylum level, Chlamydia, Bacteroidetes, and Verrucous are the dominant phyla in group C, while Firmicutes are the dominant phylum in group M; at the genus level, the genera significantly enriched in group C include: g_Chlamydia, g_Prevotella, g_Bacteroidales_unclassified, g_Akkermansia, g_ Angelakisella, g_Barnesiella, g_Corynebacterium, g_Porphyromonas, g_Odoribacter, g_Flavobacterium, g_Dysgonomonas, g_Alloprevotella The genera significantly enriched in group M include: g_ Faecalibaculum, f_Erysipelotrichaceae_unclassified, g_Staphylococcus, g_ Erysipelatoclostridium, g_Geobacillus, g_Limosilactobacillus, g_Streptomyces At the species level, the top ten species significantly enriched in group C include: s_Chlamydia_abortus, s_Bacteroidales_ bacterium, s_Akkermansia_muciniphila, s_Bacteroides _sp_CAG927、 s_Bacteroidales_ bacterium _52_46、 s_Muribaculum_intestinale, s_Prevotella _sp_CAG873、 s_Alistipes _sp_CHKCI003、s_Prevotella _sp_MGM2、 s_Prevotella _sp_CAG1031; The bacterial species significantly enriched in group M include: s_Faecalibaculum_rodentium, s_Erysipelatoclostridium_ramosum, s_ Geobacillus _sp_BCO2、 s_Erysipelotrichaceae_bacterium _2_2_44A、 s_ Limosilactobacillus_fermentum, s_Staphylococcus_aureus, s_Streptomyces _sp_SPB78、 s_Enterococcus_mundtii, s_Gardnerella_vaginalis According to the above information, Akkermansia myxophilus (… Akkermansia_muciniphila AKK was significantly enriched at the C group level, while its abundance was downregulated in the model group.

[0046] Previous studies have shown that Akkermansia muciniphila can alleviate atherosclerosis by reducing inflammation, decreasing metabolic endotoxin levels and the expression of intestinal tight junction proteins, and reducing intestinal permeability (Li J, Lin S, Vanhoutte PM, et al. Akkermansia muciniphila protects against atherosclerosis by preventing metabolic endotoxemia-induced inflammation in Apoe− / − mice[J]. Circulation, 2016, 133(24): 2434-2446.). Other studies have confirmed that Akkermansia muciniphila intervention in ApoE induced by a high-fat diet can reduce inflammation. - / - In mice, the serum levels of TG, LDL-C, IL-1I, L-6, IL-8, CRP, TNF-α, and MCP-1 were significantly lower than those in the model group (Li Liya, Liu Yumeng, Zhang Yong, et al. Effects of Akkermansia myxophilus on lipid metabolism and inflammatory factors in high-fat diet-induced obese ApoE gene knockout mice [J]. Chinese Journal of Geriatric Multiorgan Diseases, 2022, 21(10):780-785.).

[0047] ROC curves of Akkermansia myxophilus in groups C and M ( Figure 17 The graph shows the AUC (Ackeratophilic Acid) ratio, where the horizontal axis represents the false positive rate and the vertical axis represents the true positive rate. The AUC ranges from 0 to 1; a higher AUC value indicates a higher accuracy in target prediction. An AUC value of 1 indicates this. Therefore, *Ackermania pseudomallei* may be a potential biomarker for ankylosing spondylitis (AS).

[0048] Example 2 1. Preparation and grouping of experimental animals and models Specific pathogen-free (SPF) ApoE - / -Male mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were fed at the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine.

[0049] Randomly divided into model group (Model group, M group, 12 weeks old) and Akkermansia myxophilus group ( A. muciniphila The mice (AKK group, 12 weeks old) were fed a high-fat diet for 12 weeks. The high-fat diet formula for mice was: regular basal diet for mice + 21 wt% lard + 0.15 wt% cholesterol.

[0050] The Akkermansia myxophilus bacterial suspension was prepared and provided by Ningbo Taisto Biotechnology Co., Ltd. The bacterial suspension was prepared using PBS as a solvent, and the bacterial cell concentration was 1×10⁻⁶. 9 CFU / mL. Mice in the AKK group were administered 0.2 ml / day by gavage. The animal experiment intervention lasted for 12 weeks.

[0051] 2. Mouse aortic sinus & HE staining Mice were anesthetized with 2.5% sodium pentobarbital, and perfused sequentially with 4% paraformaldehyde and physiological saline. The heart and aorta were removed and preserved in 10% formalin for paraffin sectioning and HE staining. HE-stained images were acquired using a fully automated digital slide scanner.

[0052] HE staining results showed that the aortic sinus AS plaque area was larger in group M mice; compared with group M, the aortic sinus AS plaque area was significantly reduced in group AKK mice. Figure 18 The results showed that Akkermansia myxophilus had a significant ameliorative effect on AS.

[0053] 3. Metagenomic analysis of mouse intestinal contents First, the high-throughput sequencing results are identified by sequence base recognition and converted into raw sequencing sequences, called RawReads. These reads include the name of each sequence, its base sequence, and corresponding sequencing quality information. Second, before data analysis, it's crucial to ensure the reads are of sufficiently high quality to guarantee the accuracy of subsequent analyses. Therefore, low-quality reads are excluded to obtain high-quality Clean Data. Sequencing error rate is related to base sequencing quality. Base type distribution checks are used to detect the presence of AT and GC segregation. Since the sequenced DNA fragments are randomly fragmented, due to random fragmentation and the principle of complementary base pairing, theoretically, the content of G and C, and A and T should be equal in each sequencing cycle, and the GC content should fluctuate around 50%. QC30 represents a base recognition error probability of 0.001 and a base recognition accuracy of 99.9%. Based on genome assembly, gene prediction analysis is performed, followed by gene set construction and functional annotation analysis. Python is used to count the length of the gene catalog, and the results are as follows. Figure 19 As shown. Figure 19 Results and Figure 5 The results showed a consistent trend in gene length and number.

[0054] 4. Differential analysis of intestinal contents among mouse groups 4.1 Bar Chart Analysis The results are as follows Figures 20-22 As shown, the bar chart displays the species composition at the taxonomic level, which visually shows the species composition of the samples between groups and the proportion of different species in the samples between groups.

[0055] according to Figure 20 It can be seen that at the phylum level, compared with the M group, the abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Deferribacteres was upregulated in the AKK group, while the abundance of Chlamydiae and Actinobacteria was downregulated in the AKK group. Figure 21 It can be seen that, at the genus level, compared to group M, group AKK... Oscillibacter, Lachnospiraceae; unclassified, Firmicutes; unclassified, Anaerotruncus Abundance increased, AKK group Alistipes, Schaedlerella Abundance decreased; Figure 22 It can be seen that, at this level, compared to group M, group AKK... Oscillibacter _sp_1-3、 Butyricicoccus _sp_1XD8-22 abundance increased, AKK group Schaedlerella_arabinosiphila, Eubacterium_plexicaudatum, Roseburia The abundance of _sp_1XD42-69 was downgraded.

[0056] 4.2 Clustering heatmap analysis of species abundance Cluster heatmaps are a graphical representation of data matrices that uses color gradients to represent the magnitude of values ​​and clusters species based on abundance similarity. The results are as follows: Figures 23-25 As shown, the differences between the M group and the AKK group in the top 50 species at the phylum, genus, and species levels are evident.

[0057] 4.3 Alpha Diversity Analysis Alpha diversity analysis can reflect the abundance and diversity of microbial communities.

[0058] The results are as follows Figure 26 As shown, the AKK group showed a relatively higher level of activity compared to the M group, indicating that the Akkermansia myxophilus group had higher bacterial diversity than the M group.

[0059] 4.4 PCoA Analysis Further analysis using PCoA, a classic unconstrained ordination method, involves projecting the sample distance matrix into a low-dimensional space while preserving the original sample distance relationships to the greatest extent possible. The results are as follows... Figure 27 As shown, the differences between the two groups are significant.

[0060] 4.5 Lefse analysis of species differences between groups LEFSE analysis of species with significant differences between groups is used to screen species with significant differences between groups. The rank-sum test is used to detect species with significant differences between different groups, and dimensionality reduction and the influence of species with significant differences are achieved by LDA (linear discriminant analysis) to obtain LDA value. The significance of species with significant differences is determined based on the LDA value.

[0061] The results are as follows Figures 28-29 As shown. At the genus level, Ligilactobacillus It is the dominant genus in group M. Candidatus_Amulumruptor, Anaerotruncus, Flavonifractor, Butyricicoccus, Firmicutes. unclassified, Firmicutes, Pseudoflavonifractor, Acutalibacter, Ruminococcus, Eubacteriales, Eubacteriales. unclassified, Faecalibacterium It is the dominant genus in the AKK group. At the species level, Chlamydia_trachomatis, Ligilactobacillus_murinus, Blautia_ hansenii It is the dominant bacterial species in group M. Candidatus_Amulumruptor_caecigallinarius, Lachnospiraceae_bacterium _A4、 Butyricicoccus _sp_1XD8_22、 Acutalibacter _sp_1XD8_33、 Eubacterium _sp_CAG180、 Eubacterium _sp_CAG786、 Anaerotruncus _sp_G32012、 Eubacterium _sp_CAG115、 Roseburia _sp_CAG303、uncultured_ Flavonifractor _sp、Flavonifractor_plautii , Oscillibacter _sp_PC13 is the dominant strain in the AKK group.

[0062] 4.6 KEGG pathway The relevant metabolic pathways between the M and AKK groups in KEGG LEVEL B level are as follows: Figure 30 As shown, these include amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, glycan biosynthesis and metabolism, replication and repair, lipid metabolism, cell motility, cell community-prokaryotes, cell community-prokaryotes, membrane transport, energy metabolism, and signal transduction. The relevant metabolic pathways between the M and AKK groups in KEGG LEVEL C level are as follows: Figure 31 As shown, it includes two-component systems, ABC transporters, ribosomes, purine metabolism, amino sugar and nucleotide sugar metabolism, quorum sensing, and starch and sucrose metabolism.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Akkermansia myxophilus ( Akkermansia muciniphila The application of biomarkers in the preparation of kits for the diagnosis and / or detection of atherosclerosis.

2. Application of reagents for detecting Akkermansia myxophilus in the preparation of kits for the diagnosis and / or detection of atherosclerosis.

3. The application according to claim 2, characterized in that, The reagents include at least one of the following: intestinal contents DNA extraction reagents, library construction reagents, and sequencing reagents.

4. The use of Akkermansia myxophilus in the preparation of drugs for the prevention and / or treatment of atherosclerosis.

5. The application according to claim 4, characterized in that, Application of Akkermansia myxophilus in combination with other probiotics in the preparation of drugs for the prevention and / or treatment of atherosclerosis.

6. The application according to claim 5, characterized in that, The other probiotics include at least one of the following: Bacteroides ( Bacteroides sp) strain CAG927.

7. The use of an agent for increasing the abundance of Akkermansia myxophilus in the preparation of drugs for the prevention and / or treatment of atherosclerosis.

8. The application according to claim 7, characterized in that, The reagent includes Akkermansia myxophilus strain ATCCBAA-835.

9. The application according to claim 4 or 7, characterized in that, The dosage form of the drug includes oral dosage forms.

10. The application according to claim 9, characterized in that, The oral dosage form includes at least one of the following: tablets, powders, and capsules.

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