Application of viscosus in preparation of product for treating hyperlipidemia periodontitis

Abstract

The application discloses application of visceric odor bacteria in preparation of a product for treating hyperlipidemia periodontitis, and belongs to the technical field of biological medicine. The application aims to reveal the key role of visceric odor bacteria in the pathogenesis of hyperlipidemia periodontitis. It is found through experiments that visceric odor bacteria are enriched in the intestines of control mice, and the abundance of visceric odor bacteria is significantly reduced in hyperlipidemia periodontitis mice. Compared with the hyperlipidemia periodontitis mice, the mice treated by visceric odor bacteria show a significantly reduced CEJ-ABC distance, improved bone microstructure parameters, and less inflammatory infiltration, effectively reducing periodontal destruction and systemic metabolic disorders caused by hyperlipidemia periodontitis, and providing a new and integrated intervention means for hyperlipidemia periodontitis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of Visceral Odorbacterium in the preparation of products for treating hyperlipidemia and periodontitis. Background Technology

[0002] Hyperlipidemic periodontitis is a complex disease characterized by the coexistence of periodontitis and hyperlipidemia, marked by the bidirectional exacerbation of metabolic and inflammatory dysregulation. Globally, 45-50% of adults are affected by periodontitis, which is associated with major systemic pathological mechanisms such as diabetes and cardiovascular disease. Hyperlipidemia significantly exacerbates periodontal destruction; for example, elevated triglyceride levels can increase the risk of periodontitis by more than 700%. Conversely, the persistent inflammation and microbial dysregulation associated with periodontitis further disrupt systemic lipid metabolism. Traditional local treatments, failing to address the underlying metabolic-inflammatory dysregulation, are insufficient to address this pathological interaction, resulting in limited therapeutic efficacy.

[0003] The role of the gut microbiota as a key link between metabolic diseases and oral health is increasingly being confirmed. Hyperlipidemia and periodontitis can both cause gut microbiota dysbiosis, typically manifested as a decrease in the abundance of beneficial symbiotic bacteria and an increase in the abundance of pro-inflammatory bacteria, thereby exacerbating host metabolic disorders and inflammatory states. This dysbiotic gut microbiota affects host metabolic pathways, alters circulating metabolites, and may impair the integrity of the intestinal barrier, triggering systemic inflammation and thus remotely activating inflammatory pathways in oral tissues, promoting bone resorption.

[0004] However, the existing technologies mentioned above have the following problems and shortcomings in the treatment of hyperlipidemic periodontitis: 1. Although the concept of the "gut-mouth" axis is widely recognized in connecting systemic metabolic diseases and local oral health, in the complex comorbidity of hyperlipidemic periodontitis, it is not yet clear which key gut microbes are effective therapeutic targets, and how they regulate disease progression through specific molecular mechanisms. 2. Existing treatment strategies, including traditional local periodontal therapy, mainly focus on controlling local inflammation and plaque, but fail to fundamentally address the systemic metabolic disorders inherent in hyperlipidemic periodontitis, nor can they effectively intervene in the periodontal tissue damage exacerbated by this. 3. Currently, there is a lack of systematic and multi-level research on the mechanisms of action of gut microbes and their functional metabolites involved in the pathophysiological processes of hyperlipidemic periodontitis. Therefore, how to accurately identify key functional bacteria with therapeutic potential in hyperlipidemic periodontitis and systematically elucidate their multi-level mechanisms of action of "bacteria-metabolites-host response" is a major challenge that urgently needs to be addressed in the prevention and / or treatment of hyperlipidemic periodontitis. Summary of the Invention

[0005] The purpose of this invention is to provide the application of *Visceral Osmidrosis* in the preparation of products for treating hyperlipidemic periodontitis, thereby addressing the problems existing in the prior art. This invention aims to reveal the key role of *Visceral Osmidrosis* depletion in the pathogenesis of hyperlipidemic periodontitis, and to explore therapeutic strategies for reshaping the gut microbiome, improving systemic metabolic dysfunction, and alleviating periodontal inflammation and bone loss by supplementing *Visceral Osmidrosis*, thus providing a novel and integrative intervention for hyperlipidemic periodontitis.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] In a first aspect, the present invention provides the use of Visceral Odorbacterium in the preparation of products for treating hyperlipidemic periodontitis.

[0008] Preferably, the product is a drug.

[0009] Preferably, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0010] Preferably, the drug refers to a drug that can alleviate hyperlipidemic periodontitis.

[0011] Preferably, the dosage form of the drug is selected from one or more of the following: emulsion, capsule, pill, tablet, suspension, syrup, ointment, oral liquid, injection, and granule.

[0012] Secondly, the present invention also provides a product for treating hyperlipidemic periodontitis, the product comprising Visceral Odorbacterium.

[0013] Preferably, the product also includes a pharmaceutically acceptable carrier or excipient.

[0014] Preferably, the dosage form of the product is selected from one or more of the following: emulsion, capsule, pill, tablet, suspension, syrup, ointment, oral liquid, injection, and granule.

[0015] Thirdly, the present invention also provides the application of the above-mentioned product in the preparation of a medicament for treating hyperlipidemic periodontitis.

[0016] The present invention discloses the following technical effects:

[0017] 1. Improves gut microbiota dysbiosis in patients with hyperlipidemic periodontitis and mouse models: Clinical data show that the abundance of *Aeromonas viscerata* in the gut is significantly reduced in patients with hyperlipidemic periodontitis, and animal model experiments have also confirmed a close relationship between hyperlipidemic periodontitis and the depletion of *Aeromonas viscerata*. Supplementing with *Aeromonas viscerata* can effectively reshape the gut microbiome and improve the dysbiosis caused by hyperlipidemic periodontitis.

[0018] 2. Effectively reduces periodontal bone loss and inflammation: Oral administration of *Bacillus viscerans* significantly reduced alveolar bone resorption, improved bone microstructural parameters, and reduced inflammatory cell infiltration in mice with hyperlipidemic periodontitis. This effect was superior to heat-inactivated *Bacillus viscerans*, indicating that the dynamic metabolic activity of live bacteria and their ability to regulate the microbial ecosystem are key to their therapeutic effect.

[0019] 3. Alleviation of systemic metabolic dysfunction: Mice treated with *Ostridium visceratum* showed significant improvements in body weight, blood glucose, and lipid abnormalities (such as total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C)). Fat accumulation and hepatic steatosis were also significantly reduced, and no side effects on major organs were observed. This indicates that the therapeutic effect of *Ostridium visceratum* on hyperlipidemic periodontitis is systemic, capable of simultaneously addressing both local inflammation and systemic metabolic disorders.

[0020] In summary, the visceral ozone bacterium provided by this invention can fundamentally intervene in the pathological process of hyperlipidemic periodontitis by targeting the gut-mouth axis, achieving synergistic treatment of local periodontal lesions and systemic metabolic disorders, and providing an innovative and effective strategy for the prevention and treatment of hyperlipidemic periodontitis. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Example 1 shows the clinical study design and quantitative analysis of key bacterial species; where A is a schematic diagram of the clinical study design; B is a bar chart of gut bacteria (species level) with significant differences in abundance between the HPD patient group and the NC group.

[0023] Figure 2 The table shows the gut-oral microbiota and metabolomics characteristics of HPD patients in Example 1. A represents the partial least squares discriminant analysis score of gut microbiota composition between the two groups; B represents a heatmap of gut bacteria (species level) with significant abundance differences between the two groups; C represents a non-metric multidimensional scaling analysis score of oral microbiota composition between the two groups; and D represents a heatmap of oral bacteria (genus level) with significant abundance differences between the two groups.

[0024] Figure 3The images show the gut-oral microbiota and metabolomics characteristics of HPD patients in Example 1. A is a volcano plot (positive ion mode) of fecal metabolites from HPD patients, with red and blue dots representing significantly upregulated and downregulated metabolites in HPD patients (p<0.05, VIP>1), respectively. B is a volcano plot (negative ion mode) of fecal metabolites from HPD patients, with red and blue dots representing significantly upregulated and downregulated metabolites in HPD patients (p<0.05, VIP>1), respectively. C is a Spearman correlation heatmap of differentially expressed gut microbiota species and differentially expressed oral microbiota genera, with red indicating positive correlation and blue indicating negative correlation. D shows the differentially expressed gut microbiota species and the top 50 differentially expressed metabolites. Spearman correlation heatmap: A9, 2-amino-1-phenylethanol; A12, DL-phenylalanine; A22, Arginine; A37, 4-benzofuranethanamine, 2,3-dihydro-α-methyl-; A38, O-toluidine; A46, Benzyl alcohol, p-fluoro-α-[1-(methylamino)ethyl]-, erythro-(.+ / -.)-; A47, 3'-fluoromethcathinone; *p<0.05, **p<0.01, ***p<0.0001;

[0025] Figure 4 This diagram illustrates the establishment and phenotypic characteristics of the HPD mouse model in Example 2. A shows a schematic diagram of the animal experimental design; B shows the weight change curves of mice in each group; C shows a quantitative bar chart of serum biochemical indicators (blood glucose, TC, TG, HDL-C, LDL-C); D shows H&E staining of adipose tissue and Oil Red O staining of liver, scale bar = 50 μm; E shows a quantitative bar chart of adipose index and liver index; *p<0.05, **p<0.01, ***p<0.0001;

[0026] Figure 5 The following are the pathological and molecular marker analyses of the major organs in the HPD mouse model of Example 2; where A is the H&E staining map of the heart, spleen, kidney and ileum, scale bar = 50 μm; B is the quantitative bar chart of the heart, spleen and kidney indices; C is the Western blot analysis results of related proteins in alveolar bone tissue; *p<0.05, **p<0.01, ***p<0.0001;

[0027] Figure 6 The following are the pathological, molecular, and microbial characteristics of hyperlipidemia exacerbating periodontitis in Example 2; where A is the alveolar bone Micro-CT 3D reconstruction, 2D sagittal image, and hematoxylin and eosin staining of periodontal tissues, scale bar = 1 mm; B is the quantitative analysis of alveolar bone loss and bone microstructure parameters in the maxillary second molar region; C is the protein expression level analyzed by Western blot; D is the heatmap (genus level) of the relative abundance of the top 30 oral bacterial genera in PD and HPD mice; *p<0.05, **p<0.01, ***p<0.0001;

[0028] Figure 7 This section presents the pathological, molecular, and microbial characteristics of hyperlipidemia exacerbating periodontitis in Example 2. A shows the effect size analysis of linear discriminant analysis of the gut microbiota in NC and HPD mice; B shows the inter-group difference analysis of the relative abundance of *Ostomella viscerata* in NC and HPD mice; C shows the Spearman correlation heatmap of *Ostomella viscerata* and differentially expressed oral genus in NC and HPD mice; D shows the volcano plot of the correlation between *Ostomella viscerata* and differentially expressed intestinal metabolites in NC and HPD mice, with red dots representing positively correlated metabolites and blue dots representing negatively correlated metabolites. Data are expressed as mean ± standard deviation (SD); *p<0.05, **p<0.01, ***p<0.0001.

[0029] Figure 8 Example 3 illustrates the experimental design of fecal microbiota transplantation (FMT) and its effects on the systemic phenotype and microbiota of recipient mice. A shows a schematic diagram of the FMT experimental design; B shows a curve illustrating the change in the recipient mouse's body weight; C shows a quantitative bar chart of fasting blood glucose levels; D shows the results of Western blot analysis of related proteins in alveolar bone; E shows the results of quantitative immunofluorescence staining analysis of related proteins in alveolar bone; F shows the PCoA analysis of oral microbiota; G shows the Alpha diversity index analysis of oral microbiota; H shows the PCoA analysis of gut microbiota; I shows the Alpha diversity index analysis of gut microbiota; *p<0.05, **p<0.01, ***p<0.0001;

[0030] Figure 9The following table shows the effects of the FMT experiment in Example 3 on the pathology and microbiota of periodontal tissues in recipient mice. A shows Micro-CT 3D reconstruction, 2D sagittal images, and H&E staining (scale bar = 1 mm); B shows quantitative analysis of alveolar bone loss and bone microstructure parameters; C shows Western blot analysis of osteogenic-related proteins and bone resorption / inflammatory proteins in alveolar bone tissue; D shows representative immunofluorescence staining of alveolar bone (RUNX2, green; RANKL, NLRP3, red; DAPI, blue) (scale bar = 20 μm); E shows TRAP staining of alveolar bone sections (red indicates multinucleated osteoclasts) (scale bar = 100 μm); F shows hierarchical clustering analysis at the genus level of the oral microbiota; G shows LEfSe analysis of differentially expressed bacterial genera in the oral microbiota; H shows a heatmap of differentially expressed bacterial species in the gut microbiota. Data are expressed as mean ± SD; *p<0.05, **p<0.01, ***p<0.0001.

[0031] Figure 10 The images show the pathological analysis of the liver and other organs of the fecal microbiota transplant recipient mice in Example 3; where A is the H&E staining and Oil Red O staining image of the liver; B is the quantitative bar chart of the liver index; C is the H&E staining image of the heart, spleen, kidney and ileum; D is the quantitative bar chart of the heart, spleen and kidney index; scale bar = 50 μm;

[0032] Figure 11 This document presents the experimental design and molecular and metabolic phenotypic analysis of the *Ostomella visceralis* intervention model in Example 4. A shows a schematic diagram of the *Ostomella visceralis* intervention experiment design; B shows the results of Western blot analysis of related proteins in alveolar bone; C shows the quantitative analysis results of immunofluorescence staining of related proteins in alveolar bone; D shows the curves of body weight changes in mice of each group during the intervention period; E shows a quantitative bar chart of serum biochemical indicators; *p<0.05, **p<0.01, ***p<0.0001;

[0033] Figure 12Example 4 illustrates the therapeutic effect of visceral Osterix intervention on HPD and its regulation of intestinal metabolism. A shows representative Micro-CT 3D reconstruction, 2D sagittal plane images, and H&E staining of periodontal tissues of the mouse maxilla (scale bar = 1 mm); B shows quantitative analysis of alveolar bone loss and bone microstructure parameters; C shows NMDS analysis of oral microbiota based on Bray-Curtis distance; D shows Western blot analysis of osteogenic-related proteins and osteoclast / inflammation-related proteins in alveolar bone tissue; E shows immunofluorescence staining of RUNX2, Osterix (green), RANKL, NLRP3, and interleukin-1β (IL-1β) (red) in alveolar bone tissue, with cell nuclei counterstained with DAPI (blue) (scale bar = 20 μm); *p<0.05, **p<0.01, ***p<0.0001;

[0034] Figure 13This section illustrates the therapeutic effect of *Ostomyces viscerans* intervention on HPD and its regulation of intestinal metabolism in Example 4. A shows TRAP-stained osteoclasts (scale bar = 100 μm); B is a metabolite volcano plot (positive and negative ion mode); C is a correlation network between differentially expressed intestinal metabolites and microorganisms (red lines indicate positive correlation, green lines indicate negative correlation); D is a Spearman correlation heatmap of intestinal microbiota and metabolites: A1, 1-palmitoyl-sn-glycero-3-phosphocholine; A2, 1-stearoyl-sn-glycero-3-phosphocholine. (LPC(18:0))); A3, 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine; A4, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine; A5, 1-stearoyl-sn-glycero-3-phosphocholine 3-phosphocholine); A6, 1-(1z-hexadecenyl)-sn-glycero-3-phosphocholine; A7, 1-pentadecanoyl-sn-glycero-3-phosphocholine; A8, 1-heptadecanoyl-sn-glycero-3-phosphocholine; A9, 1-myristoyl-sn-glycero-3-phosphocholine; *p<0.05, **p<0.01, ***p<0.001;

[0035] Figure 14The image shows the effect of visceral odor bacillus intervention on the improvement of systemic metabolism and major organ morphology in mice in Example 4. A shows H&E staining images of different adipose tissues (iWAT, eWAT, pWAT, BAT) and H&E and Oil Red O staining images of the liver, scale bar = 50 μm; B shows quantitative analysis images of adipose tissue index, white / brown adipose tissue ratio, and liver index; C shows representative H&E staining images of the heart, spleen, kidney, and ileum, scale bar = 50 μm; D shows quantitative analysis images of organ indices of the heart, spleen, and kidney. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] The visceral odor bacillus used in this invention was purchased from the Beijing National Culture Collection Center (BNCC, Beijing, China), with accession number BNCC359789.

[0042] *Visceral Odorbacterium* BNCC359789 is a Gram-negative, non-spore-forming, obligate anaerobic bacillus. Morphologically, it is characterized by short rods. This strain grows well under anaerobic conditions (85% nitrogen, 10% hydrogen, and 5% carbon dioxide) on Columbia blood agar plates or modified minced meat medium at approximately 37°C and pH 7.0, forming round, smooth, translucent, or grayish-white colonies. Its metabolic characteristics include the ability to ferment various carbohydrates, producing short-chain fatty acids such as acetic acid and propionic acid. This strain has the potential to regulate intestinal flora balance, maintain intestinal homeostasis, and modulate the immune system in the host.

[0043] Strain purification and amplification: Single colonies of strain BNCC359789 were isolated and streaked onto fresh Columbia blood agar plates and incubated at 37°C for 4-5 days in an anaerobic incubator (85% N2, 10% H2, 5% CO2).

[0044] Liquid culture: The obtained colonies were inoculated into a modified minced meat medium for liquid amplification. During the logarithmic growth phase (optical density 600 nm (OD)... 600 (≈0.8-1.0, usually after culturing for 24-36 hours) bacteria are harvested.

[0045] Bacterial collection and washing: Bacterial cells were collected by centrifugation (3000 rpm, 10 minutes, 4°C) and washed with sterile PBS to remove culture medium residue.

[0046] Concentration adjustment: The bacterial pellet was resuspended in sterile PBS, and the bacterial concentration was adjusted to 1×10⁻⁶ using turbidimetric and plate count methods. 9 CFU / mL.

[0047] Preparation of heat-inactivated bacteria: To prepare heat-inactivated bacteria, a suspension of live bacteria was incubated at 70°C for 30 minutes, then rapidly cooled to 4°C, and successful inactivation was confirmed by culturing on CBA plates and observing no growth.

[0048] Example 1: Microbiome Characterization Analysis of Patients with Hyperlipidemia and Periodontitis and Healthy Volunteers

[0049] method:

[0050] This study recruited 20 patients diagnosed with hyperlipidemic periodontitis (HPD) according to the 2017 American College of Periodontology / European Federation of Periodontology guidelines and 20 healthy volunteers (normal lipid levels and no periodontal disease), aged 25 to 60 years. Exclusion criteria included use of antibiotics or probiotics within the past 3 months, pregnancy, severe systemic diseases, or immunodeficiency. Saliva samples were collected from participants for 16S rRNA sequencing, and stool samples were collected for metagenomic sequencing and non-targeted metabolomics analysis. A schematic diagram of the clinical study design is available below. Figure 1A.

[0051] Results and Analysis:

[0052] Partial least squares discriminant analysis (PLS-DA) revealed significant differences in gut microbiota composition between patients with hyperlipidemia and periodontitis and healthy volunteers. Figure 2 (A). At the species level, the relative abundance of *Ostomyces viviparus* in the gut of patients with hyperlipidemic periodontitis was significantly reduced, exhibiting the most significant depletion. Figure 1 B, Figure 2 (B). Meanwhile, oral 16S rRNA sequencing results showed significant differences in oral microbiota composition between patients with hyperlipidemic periodontitis and healthy volunteers. Figure 2 In patients with hyperlipidemic periodontitis, periodontal pathogens such as Treponema, Aggregatibacter, Porphyromonas, Prevotella, Neisseria, and Tannerella are enriched. Figure 2 In addition, fecal non-targeted metabolomics analysis identified 290 differentially metabolites (D). Figure 3 (AB). Visceral odor bacilli showed a significant negative correlation with various periodontal pathogens ( Figure 3 (CD). These clinical data indicate that hyperlipidemic periodontitis is characterized by depletion of gut visceral odor bacilli and enrichment of oral pathogens, suggesting a potential gut-oral axis connection.

[0053] Example 2: The effect of hyperlipidemia on periodontitis and its correlation with the depletion of visceral ozone bacteria in a mouse model of hyperlipidemic periodontitis.

[0054] method:

[0055] Eight-week-old specific-pathogen-free (SPF) male C57BL / 6J mice were randomly divided into four groups (n=6 per group): control group (NC), periodontitis group (PD), hyperlipidemia group (HLP), and hyperlipidemia-periodontitis group (HPD). Mice in the hyperlipidemia and hyperlipidemia-periodontitis groups were fed a high-fat diet (HFD; 60% kcal from fat) for 20 weeks, while the control and periodontitis groups were fed a standard diet. At week 19, under isoflurane anesthesia, the necks of the bilateral maxillary second molars were ligated using 5-0 sterile sutures to induce a periodontitis model in mice in the periodontitis and hyperlipidemia-periodontitis groups. The ligation was maintained for one week. See the schematic diagram of the animal experimental design. Figure 4 A.

[0056] At the end of the experiment, mouse body weight, fasting blood glucose, serum total cholesterol (TC), serum triglycerides (TG), and serum LDL-C levels were assessed. In vivo whole-body fat composition analysis and isolated maxillary bone microstructure analysis were performed using a micro-computed tomography (Micro-CT) system to quantify alveolar bone loss (cementoenamel junction to alveolar bone crest distance, CEJ-ABC) and bone microstructure parameters (bone mineral density, BMD), bone volume / total volume (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp)). Histological analysis was performed on the heart, liver, spleen, kidney, ileum, four types of adipose tissue (inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), perirenal white adipose tissue (pWAT), and brown adipose tissue (BAT)) and maxilla (hematoxylin and eosin (H&E) staining and Oil Red O staining).

[0057] Western blotting was used to analyze osteogenic proteins (osteoprotegerin (OPG), osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), Osterix, and osteocalcin (OCN)) and osteoclastosis / inflammation-related proteins (receptor activator of nuclear factor Kappa-B ligand (RANKL), inducible nitric oxide synthase (iNOS), NOD-like receptor family pyrin domain-containing 3 (NLRP3), interleukin-1β (IL-1β), and tumor necrosis factor-α) in alveolar bone tissue. Expression levels of TNF-α and interleukin-6 (IL-6) were determined. 16S rRNA gene sequencing was performed on the oral and gut microbiota, and key differentially expressed bacteria were identified using linear discriminant analysis effect size (LEfSe) and correlation analysis.

[0058] Results and Analysis:

[0059] A high-fat diet successfully induced a hyperlipidemia phenotype. Hyperlipidemia and hyperlipidemic periodontitis mice showed significantly increased body weight, fasting blood glucose, and serum TC, TG, and LDL-C levels. Figure 4 In the central BC region, there was proliferation of adipose tissue (iWAT, eWAT, pWAT) and hepatic steatosis (detected by Oil Red O staining), along with a significant increase in liver and fat indices. Figure 4 The middle jiao (DE) showed no significant pathological changes, while other major organs (heart, spleen, kidney, ileum) showed no significant pathological changes. Figure 5 (AC). Micro-CT analysis showed that alveolar bone loss was more severe in hyperlipidemic periodontitis mice than in periodontitis mice, with no significant difference between the hyperlipidemic group and the control group. Figure 6 (A). Compared with periodontitis mice, hyperlipidemic periodontitis mice showed a significantly increased CEJ-ABC distance, significantly decreased BMD, BV / TV, Tb.Th, and Tb.N, and a significantly increased Tb.Sp. Figure 6(B) H&E staining results were consistent with quantitative imaging results. At the molecular level, the expression of osteogenic-related proteins such as OPG, OPN, RUNX2, Osterix, and OCN in the alveolar bone of hyperlipidemic periodontitis mice showed a decreasing trend, while the expression of osteoclast-promoting / inflammation-related proteins such as RANKL, iNOS, NLRP3, IL-1β, TNF-α, and IL-6 showed the opposite trend. Figure 5 C in the middle Figure 6 (C). The oral microbiome in hyperlipidemic periodontitis mice showed dysregulation, with enrichment of pathogenic bacteria (such as Bacteroides and Streptococcus) and depletion of beneficial bacteria (such as Lactobacillus and Akkermansia). Figure 6 (D). LEfSe analysis showed that *Ostomyces viviparus* was enriched in the intestines of control mice, but its abundance was significantly reduced in hyperlipidemic periodontitis mice. Figure 7 Correlation analysis showed that the abundance of *Ostomyces spp.* in the gut was positively correlated with beneficial oral bacteria (e.g., *Akkermansia*, *Lactobacillus*) and negatively correlated with pathogenic *Streptococcus*. Figure 7 (C). Furthermore, its abundance was also significantly correlated with various intestinal metabolites (C). Figure 7 (D). These animal model results are consistent with clinical observations and support the causal role of gut microbiota in the pathogenesis of hyperlipidemic periodontitis.

[0060] Example 3: Pathogenicity of hyperlipidemic periodontitis-associated gut microbiota transplantation on periodontal damage and metabolic disorders.

[0061] method:

[0062] The experimental design for fecal microbiota transplantation (FMT) is as follows: Figure 8A. Fresh fecal suspensions from donor mice with periodontitis and hyperlipidemic periodontitis were prepared. SPF C57BL / 6J recipient mice were treated with an antibiotic cocktail (vancomycin (0.5 g / L), neomycin (1 g / L), metronidazole (1 g / L), and ampicillin (1 g / L)) in drinking water for 4 weeks to establish a gut microbiota depletion model. Subsequently, recipient mice were randomly assigned to two groups (n=6 per group): the periodontitis fecal microbiota transplantation group (PD-FMT group), receiving fecal suspensions from PD donors; and the hyperlipidemic periodontitis fecal microbiota transplantation group (HPD-FMT group), receiving fecal suspensions from HPD donors. Mice in each group were orally administered 200 μL three times weekly by gavage for 8 weeks. At week 7 of gavage, periodontitis was induced in all recipient mice using a ligation method. The extent of periodontal tissue damage, bone microstructure parameters, bone metabolism, and expression of inflammation-related proteins were assessed (using Western blot and immunofluorescence staining). Tartrate-resistant acid phosphatase (TRAP) staining was used to observe osteoclast infiltration. Oral and intestinal flora structure were analyzed, and Oil Red O staining of the liver was performed to assess hepatic steatosis.

[0063] Results and Analysis:

[0064] Recipient mice receiving donor microbiota from hyperlipidemic periodontitis (HPD-FMT group) showed more severe periodontal tissue destruction than recipient mice receiving donor microbiota from periodontitis (PD-FMT group). Figure 9 In mice with hyperlipidemic periodontitis and fecal microbiota transplantation, the CEJ-ABC distance and Tb.Sp were significantly increased, while BMD, BV / TV, Tb.Th, and Tb.N were significantly decreased. Figure 9 (B) Histological observation results were consistent with quantitative imaging results. There were no significant differences in body weight or fasting blood glucose levels among the recipient mice. Figure 8 At the molecular level, the expression of OPG, RUNX2, and OCN in the alveolar bone of mice with hyperlipidemic periodontitis after fecal microbiota transplantation was significantly downregulated, while the expression of RANKL, NLRP3, and IL-6 was upregulated. Figure 8 D, Figure 9 (C). Immunofluorescence staining confirmed decreased RUNX2 and increased RANKL and NLRP3 signaling. Figure 8 E, Figure 9 (D). TRAP staining showed increased osteoclast infiltration in mice with hyperlipidemic periodontitis and fecal microbiota transplantation. Figure 9 The composition of the oral microbiota differs significantly between the two groups (E). Figure 8 FG, Figure 9In mice with hyperlipidemia and periodontitis, the relative abundance of the pathogenic bacterium *Prevotella* was increased in fecal microbiota transplantation. Figure 9 The pathogenic characteristics of the G (glucose-containing) are consistent with those of patients with hyperlipidemia-induced periodontitis. The gut microbiota structure is also significantly different. Figure 8 In hyperlipidemic periodontitis mice, fecal microbiota transplantation significantly reduced several known beneficial bacteria while increasing potentially pathogenic bacteria (such as Helicobacter pylori). Notably, the abundance of visceral odor bacilli was low in fecal microbiota transplantation mice with hyperlipidemic periodontitis. Figure 9 In addition, liver Oil Red O staining confirmed the transfer of metabolic phenotypes; mice with hyperlipidemic periodontitis and fecal microbiota transplantation showed more extensive hepatic lipid accumulation, while other organs were unaffected (H). Figure 10 (AD). These results directly demonstrate that the gut microbiota associated with hyperlipidemic periodontitis is sufficient to exacerbate periodontal damage and transmit metabolic disorders.

[0065] Example 4: The therapeutic effect of visceral ozone bacteria intervention on hyperlipidemic periodontitis

[0066] method:

[0067] See the schematic diagram of the visceral ozone bacteria intervention experiment design. Figure 11 Mice were divided into a control group, a hyperlipidemic periodontitis group, a *Aeromonas viscerata* intervention group, and a heat-inactivated *Aeromonas viscerata* intervention group, with n=6 in each group. Except for the normal control group (NC group), the other three groups of mice were fed a high-fat diet (HFD) for 20 weeks to induce hyperlipidemia. Starting from week 21, mice in the three high-fat diet groups underwent oral gavage intervention three times a week for 8 weeks. Specifically, mice in the hyperlipidemic periodontitis group (HPD group) were orally administered an equal volume of sterile PBS, while mice in the *Aeromonas viscerata* intervention group were orally administered live *Aeromonas viscerata* (1×10⁻⁶). 9 CFU / mL), mice in the heat-inactivated *Ostomyces visceratus* intervention group were orally administered heat-inactivated *Ostomyces visceratus* (1×10⁻⁶ CFU / mL). 9CFU / mL). At week 7 of the above gavage intervention, periodontitis was induced in all mice in the hyperlipidemic periodontitis group, the *Ostomyces viscerans* intervention group (OS group), and the heat-inactivated *Ostomyces viscerans* intervention group (OS-I group) using silk suture ligation, with the ligatures maintained for one week. After the 8-week gavage intervention, all mice were sacrificed, and samples were collected. Periodontal bone loss, bone microstructure parameters, inflammatory infiltration, oral microbiota structure, and systemic metabolic indicators (body weight, fasting blood glucose, blood lipids, adipose tissue, and hepatic steatosis) were assessed. The expression of osteogenic-related proteins (OPG, OPN, RUNX2, Osterix) and osteoclast / inflammation-related proteins (RANKL, iNOS, NLRP3, IL-1β, TNF-α, IL-6) in alveolar bone tissue was detected by Western blot and immunofluorescence staining. TRAP staining was used to label osteoclasts.

[0068] Results and Analysis:

[0069] Compared with hyperlipidemic periodontitis mice, mice treated with *Ostomyces viviparus* showed significantly reduced CEJ-ABC distance, improved bone microstructure parameters, and less inflammatory infiltration. Figure 12 (AB). The treatment effect of the visceral odor bacillus group was more significant than that of the heat-inactivated visceral odor bacillus group, and its oral microbial community structure was closer to that of the normal control group in the non-metric multidimensional scaling ordination space, while the improvement of heat-inactivated visceral odor bacillus was limited. Figure 12 (C) Treatment with *Ostomyces viscerans* significantly upregulated osteogenic proteins (OPG, OPN, RUNX2, Osterix) in alveolar bone and downregulated osteoclast-associated protein (RANKL) and pro-inflammatory markers (iNOS, NLRP3, IL-1β, TNF-α, IL-6). Figure 11 B, Figure 12 (D). Immunofluorescence staining detected increased expression of RUNX2 and Osterix, and decreased expression of RANKL, NLRP3, and IL-1β. Figure 11 C in the middle Figure 12 (E), while osteoclasts were significantly reduced ( Figure 13 (A). The therapeutic benefits also extend to the whole body; treatment with *Ostomyces viviparus* reversed key features of metabolic syndrome, including weight loss, improvement of dyslipidemia, reduction of fat accumulation, and reduction of hepatic steatosis, without causing any detectable adverse reactions. Figure 11 (DE). Simultaneously, the treatment significantly reduced fat accumulation and alleviated hepatic steatosis, without causing any detectable adverse reactions to the morphology and indices of major organs such as the heart, spleen, kidneys, and ileum. Figure 14(AD). Non-targeted metabolomics analysis of intestinal contents revealed 522 differentially expressed metabolites between the visceral odor bacterium treatment group and the hyperlipidemic periodontitis group, of which 212 were upregulated and 310 were downregulated. Figure 13 (Middle B). Notably, β-guanidinopropionic acid was significantly elevated in the treatment group mice (fold change = 3.49, p < 0.05) and was a central node in the metabolite-microbe correlation network ( Figure 13 (C). Correlation analysis between differentially expressed gut bacteria and key metabolites upregulated by treatment of *Ostomyces viviparus* confirmed that β-guanidinopropionic acid was significantly associated with multiple microbial species. Figure 13 (D). These results collectively confirm that live *Ostomyces viviparus* effectively alleviates periodontal damage and systemic metabolic disorders caused by hyperlipidemic periodontitis by reshaping the gut microbial ecosystem and upregulating key functional metabolites such as β-guanidinopropionic acid.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of *Visceral Odorbacterium* in the preparation of products for treating hyperlipidemic periodontitis, characterized in that, The preservation number of the visceral ozone bacillus is BNCC359789.

2. The application according to claim 1, characterized in that, The product is a medicine.

3. The application according to claim 2, characterized in that, The drug may also include pharmaceutically acceptable carriers or excipients.

4. The application as described in claim 2, characterized in that, The drug in question is one that can alleviate periodontitis caused by hyperlipidemia.

5. The application according to claim 2, characterized in that, The dosage form of the drug is selected from one or more of the following: emulsion, capsule, pill, tablet, suspension, syrup, ointment, oral liquid, injection, and granule.

6. A product for treating hyperlipidemic periodontitis, characterized in that, The product contains *Visceral Odor Bacterium*. The preservation number of the visceral ozone bacillus is BNCC359789.

7. The product according to claim 6, characterized in that, The product also includes pharmaceutically acceptable carriers or excipients.

8. The product according to claim 6, characterized in that, The dosage form of the product is selected from one or more of the following: emulsion, capsule, pill, tablet, suspension, syrup, ointment, oral liquid, injection, and granule.

9. The use of the product as described in any one of claims 6-8 in the preparation of a medicament for treating hyperlipidemic periodontitis.

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