Use of low acylated lipopolysaccharide from bacteroides ovatus in the preparation of a medicament for the treatment of osteoporosis

Abstract

The application discloses application of low-acylated lipopolysaccharide from Bacteroides ovatus in preparation of a drug for treating osteoporosis. Bacteroides ovatus It is proved in the application that low-acylated LPS from Bacteroides ovatus can target and inhibit sclerostin (SOST) secreted by bone cells, reduce bone loss, and play a role in treating osteoporosis. In-vivo experiments show that oral administration of low-acylated LPS from Bacteroides ovatus can significantly improve the bone mineral density of osteoporosis model mice carrying human ApoE4 and improve the bone microstructure. The application provides a brand-new candidate substance for clinically developing a safe, effective and orally-taken anti-osteoporosis drug targeting SOST, and has a wide application scenario in the clinic.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the medicinal uses of active substances derived from intestinal flora, particularly Bacteroides ovalis ( Bacteroides ovatus Application of low-acylated lipopolysaccharide (LPS) from [source name] in the preparation of drugs for treating osteoporosis. Background Technology

[0002] Senile osteoporosis (SOP) is a major cause of high disability rates and increased fracture risk in the elderly, with its core pathology lying in the age-related imbalance between bone formation and bone resorption. Genetic factors play a crucial role in the pathogenesis of osteoporosis. In recent years, numerous epidemiological studies and meta-analyses have shown that elderly individuals carrying the ApoE4 gene have lower bone mineral density and a higher risk of fracture compared to those carrying the ApoE2 and ApoE3 genes. Current clinical treatments for osteoporosis (anti-resorption drugs and anti-sclerostin (SOST) antibodies) can, to some extent, slow bone loss, but there are still significant bottlenecks to overcome in promoting bone formation. Furthermore, long-term injection administration, low patient compliance, and severe side effects also limit their clinical benefits.

[0003] The gut-bone axis, as a novel paradigm and core mechanism for regulating bone metabolism, offers a new perspective and control strategies for elucidating bone homeostasis imbalances during aging. Studies have found that aging is often accompanied by gut microbiota dysbiosis and impaired gut barrier function. This leads to the proliferation of bacteria such as *Escherichia coli* (E. coli). E. coli Highly acylated lipopolysaccharide (LPS) produced by pathogenic bacteria, such as [specific bacteria name], translocates into the bloodstream. This enterogenic endotoxemia strongly activates inflammatory pathways, abnormally upregulates the expression of osteoschizoprotein (SOST), and thus exacerbates bone loss.

[0004] SOST is a negative regulator of bone formation secreted by osteocytes. While existing anti-SOST antibodies are direct targets, they only provide terminal blockade at the protein level and pose cardiovascular risks. Osteocytes, as the most abundant cell population in bone tissue (comprising 90%–95%), secrete SOST, which is a regulatory target for maintaining the balance between bone formation and resorption. Therefore, developing novel, orally administered, safe strategies that target upstream regulatory molecules of SOST is urgently needed for the treatment of SOP. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide a targeted drug delivery strategy based on intestinal flora active substances, that is, to provide a drug use for preventing and / or treating osteoporosis by inhibiting SOST expression.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides the use of low-acylated lipopolysaccharides derived from Bacteroides ovalis in the preparation of medicaments for the prevention and / or treatment of osteoporosis.

[0007] This invention does not impose any particular limitation on the *Bacteroides ovalis* strain used; any common *Bacteroides* strain known in the art can be used to implement this invention, such as the type strain ATCC 8483, which can be obtained from public depositories. The low-acylated lipopolysaccharide derived from *Bacteroides ovalis* can be extracted and purified from *Bacteroides ovalis* cells using conventional methods in the art, such as the Westphal method, or using a commercially available bacterial lipopolysaccharide extraction kit and following the kit's instructions.

[0008] In this invention, prevention and / or treatment refer to delaying disease progression, preventing disease progression, and / or reducing the severity of symptoms that will or are expected to develop. Therefore, these terms include improving existing disease symptoms, preventing additional symptoms, improving or preventing underlying metabolic causes of symptoms, inhibiting disorders or diseases, for example, preventing the development of disorders or diseases, alleviating disorders or diseases, regressing disorders or diseases, reducing symptoms caused by diseases or disorders, or stopping the symptoms of diseases or disorders. In a specific embodiment of this invention, the disease is osteoporosis.

[0009] Furthermore, the osteoporosis mentioned is osteoporosis mediated by the ApoE4 gene.

[0010] In this invention, ApoE4 gene-mediated osteoporosis refers to bone loss driven by the ApoE4 genotype. The subjects include patients carrying the human APOE ε4 allele (including ε3 / ε4 heterozygous and ε4 / ε4 homozygous), and animal models carrying the human ApoE4 transgene. The inventors demonstrated in a mouse model carrying the human ApoE4 transgene that low-acylated lipopolysaccharide derived from *Bacteroides ovalis* significantly increases bone mineral density and improves bone microstructure. Given the high carrier frequency of the APOE ε4 allele in the population, this application provides a targeted treatment option for osteoporosis patients carrying the ε4 allele.

[0011] Furthermore, the drug exerts its osteogenic effect by inhibiting the expression of the SOST gene in bone cells and / or reducing the level of SOST protein secreted by bone cells.

[0012] SOST-encoded sclerosing protein is a key negative regulator of the Wnt / β-catenin signaling pathway. Inhibition of high SOST expression can relieve Wnt signal inhibition and promote osteoblast differentiation and bone formation. This is the molecular mechanism by which the hypoacylated lipopolysaccharide from Bacteroides ovalis exerts its anti-osteoporosis effect.

[0013] Furthermore, the drug can increase bone density and / or improve bone microstructure.

[0014] Furthermore, the improvement in bone microstructure includes an increase in one or more of the following indicators: bone volume fraction, trabecular thickness, and trabecular number, and / or a decrease in trabecular separation.

[0015] Furthermore, the drug can suppress inflammatory responses.

[0016] Furthermore, the inhibition of the inflammatory response includes a reduction in the levels of one or more of the pro-inflammatory factors IL-1β, IL-6, and TNF-α.

[0017] Furthermore, the drug can also inhibit inflammatory responses and / or bone loss induced by highly acylated lipopolysaccharides.

[0018] Furthermore, the highly acylated lipopolysaccharide is derived from Escherichia coli.

[0019] In this invention, the dosage form of the drug is not particularly limited. In some embodiments, the drug is prepared as an oral formulation or an enteric formulation. An oral formulation refers to a formulation that releases the active ingredient in the gastrointestinal tract after oral administration, and is selected from tablets, capsules, granules, powders, oral solutions, and oral suspensions; an enteric formulation refers to a formulation that releases or administers the drug at a specific site in the intestine, and is selected from enteric-coated formulations and rectal formulations. Those skilled in the art can select a suitable dosage form known in the art according to actual needs (e.g., improving drug bioavailability, improving patient medication adherence, etc.).

[0020] The dosage of the hypoacylated lipopolysaccharide derived from Bacteroides ovalis described in this invention depends on many factors, such as the nature and severity of the disease to be treated and / or prevented (especially osteoporosis in this invention), the sex, age, weight, and individual response of the patient or animal, the specific compound used, the route of administration, and the frequency of administration. The dosage can be administered as a single dose or in several doses, such as two, three, or four doses.

[0021] Furthermore, various prescriptions can be made based on factors such as formulation method, administration route, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate, and responsiveness. Skilled physicians can usually easily determine the prescription and the effective dosage for the desired treatment and / or prevention. In a specific embodiment of the invention, the dosage of the low-acylated lipopolysaccharide derived from *Bacteroides ovalis* is 1 mg / kg / Day.

[0022] Furthermore, the lipid A portion of the low-acylated lipopolysaccharide derived from *Bacteroides ovalis* has a tetraacylated structure.

[0023] A second aspect of the present invention provides a pharmaceutical composition.

[0024] Furthermore, the pharmaceutical composition comprises a low-acylated lipopolysaccharide derived from Bacteroides ovalis and pharmaceutically acceptable excipients.

[0025] In some embodiments, the pharmaceutically acceptable excipients are those recognized in the art and include, for example, pharmaceutically acceptable materials, compositions, or excipients, such as liquid or solid fillers, diluents, solvents, or encapsulation materials, that participate in carrying or transporting any subject composition from one organ or part of the body to another organ or part of the body. Each excipient must be "acceptable" in the sense of compatibility with other components of the subject composition and harmless to the patient.

[0026] In some implementations, pharmaceutically acceptable excipients are pyrogen-free. Some examples of materials that can be used as pharmaceutically acceptable excipients include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and... Soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0027] Furthermore, the pharmaceutically acceptable excipients include diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and / or disintegrants.

[0028] Furthermore, the pharmaceutical composition also includes a second therapeutic agent.

[0029] In this invention, the second therapeutic agent is not particularly limited; any drug that can be used to prevent and / or treat osteoporosis, or to assist in the prevention and / or treatment of osteoporosis, falls within the scope of the second therapeutic agent. Since this invention has experimentally confirmed the osteoporosis therapeutic effect of low-acylated lipopolysaccharides derived from *Bacteroides ovalis*, those skilled in the art can combine the low-acylated lipopolysaccharides from *Bacteroides ovalis* with other drugs for the treatment of osteoporosis as needed. Therefore, using low-acylated lipopolysaccharides from *Bacteroides ovalis* as the sole active ingredient or combining low-acylated lipopolysaccharides from *Bacteroides ovalis* with the other drugs as active ingredients in the preparation of osteoporosis treatment drugs both fall within the protection scope of this invention.

[0030] Furthermore, the second therapeutic agent is selected from one or more of bisphosphonates, denosumab, parathyroid hormone analogs, sclerosing protein antibodies, calcium preparations, and vitamin D and its analogs.

[0031] A third aspect of the present invention provides a method for regulating the expression level of SOST in osteocytes.

[0032] Furthermore, the method includes treating bone cells with low-acylated lipopolysaccharides derived from Bacteroides ovalis.

[0033] Furthermore, the hypoacylated lipopolysaccharide derived from *Bacteroides ovalis* negatively regulates the expression level of SOST.

[0034] Furthermore, the bone cells are selected from MLO-Y4 cells.

[0035] Advantages and beneficial effects of the present invention: (1) Definite in vivo efficacy and clear intervention mechanism: This invention reveals a novel pharmaceutical application of low-acylated lipopolysaccharides naturally produced by Bacteroides ovalis. Animal experiments showed that, compared with D-galactose (D-gal)-induced osteoporosis mouse models, it significantly increased bone mineral density (BMD) and improved bone microstructure in mouse models carrying the human ApoE4 transgenic gene. Mechanistic studies confirmed that this substance can target and inhibit the high expression of SOST in osteocytes, antagonizing the bone loss-promoting effect induced by pathogenic bacteria (hyperacylated LPS in Escherichia coli), providing a new material basis for the clinical development of anti-osteoporosis drugs.

[0036] (2) High medication safety and compliance: Unlike existing injectable antibody drugs that directly block the terminal SOST protein, the hypoacylated lipopolysaccharide of this invention targets the upstream regulation of SOST. Furthermore, because this substance is suitable for intestinal or oral administration, it effectively improves long-term medication adherence in middle-aged and elderly patients with osteoporosis.

[0037] (3) Unexpected technical results were achieved: The inventors identified the structure of the extracted Bacteroides ovalis lipopolysaccharide using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), confirming its low-acylation structural characteristics. Further comparative studies showed that not all low-acylated lipopolysaccharides from enteric bacteria possess the effect of improving osteoporosis. Specifically, when using other enteric bacteria (such as Bacteroides vulgaris, ...), ... Bacteroides vulgatus When known low-acylated lipopolysaccharides from *Bacteroides ovalis* were used as a control in in vivo intervention, no significant improvement in bone mineral density and bone microstructure parameters was observed in mice. This comparative result confirms that the low-acylated lipopolysaccharides from *Bacteroides ovalis* do not exert their efficacy solely through the general characteristic of "low acylation," but rather through their specific molecular structure, which produces a highly specific inhibitory effect on SOST expression in osteocytes. This technical approach overcomes conventional understanding in the field and possesses outstanding substantive features and significant progress. Attached Figure Description

[0038] Figure 1 for B.ovatus The MALDI-TOF-MS mass spectrometry identification of LPS shows the characteristic peak of its low-acylated lipid A.

[0039] Figure 2 Displaying representative micro-CT images B.ovatus LPS for three-dimensional reconstruction of the distal femoral trabeculae in ApoE4 mice (scale bar = 1 mm).

[0040] Figure 3 For Micro-CT quantitative analysis images B.ovatus The effect of LPS on bone microstructure improvement in ApoE4 mice; Figure A shows the quantitative results of bone mineral density (BMD), Figure B shows the quantitative results of bone volume fraction (BV / TV), Figure C shows the quantitative results of trabecular bone number (Tb.N), Figure D shows the quantitative results of trabecular bone separation (Tb.Sp), and Figure E shows the quantitative results of trabecular bone thickness (Tb.Th).

[0041] Figure 4 Displaying representative micro-CT images B.ovatu Three-dimensional reconstruction of distal femoral trabeculae in D-galactose-induced osteoporotic mice using s LPS (scale bar = 1 mm).

[0042] Figure 5 For Micro-CT quantitative analysis images B.ovatusEffects of LPS on bone microstructure in D-galactose-induced osteoporotic mice; Figure A shows the quantitative results of bone mineral density (BMD), Figure B shows the quantitative results of bone volume fraction (BV / TV), Figure C shows the quantitative results of trabecular bone number (Tb.N), Figure D shows the quantitative results of trabecular bone separation (Tb.Sp), and Figure E shows the quantitative results of trabecular bone thickness (Tb.Th).

[0043] Figure 6 Displaying ELISA results B.ovatus The effects of LPS on bone metabolism indicators in the serum of ApoE4 mice; Figure A shows the SOST detection results, Figure B shows the osteocalcin (BGP) detection results, Figure C shows the CTX-I crosslinking results of type I collagen carboxyl-terminal peptide (CTX-I), Figure D shows the osteoprotegerin (OPG) detection results, and Figure E shows the RANKL detection results.

[0044] Figure 7 Displaying ELISA results B.ovatus The effect of LPS on the levels of confirmatory factors in the serum of ApoE4 mice; Figure A shows the results of TNF-α detection, Figure B shows the results of IL-1β detection, and Figure C shows the results of IL-6 detection.

[0045] Figure 8 show B.ovatus LPS E. coli The effect of induced SOST expression in osteocytes; among which, Figure 8 A represents the ELISA test result. Figure 8 B represents the results of Western blot analysis. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention. Unless otherwise specified in the following embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available conventional products.

[0047] Example 1: Extraction and structural identification of low-acylated lipopolysaccharides from Bacteroides ovalis 1.1 Culture method of Bacteroides ovalis ATCC 8483: Bacteroides ovalis ATCC 8483 (purchased from BeNa Culture Collection) was inoculated onto Columbia blood agar medium and cultured in anaerobic culture bags at 37°C. Columbia blood agar medium (catalog number: jx601) was purchased from Qingdao Haibo Biotechnology. Anaerobic culture bags (C-43) and oxygen indicators (C-22) were purchased from Mitsubishi Corporation, Japan. Anaerobic gas generators (AN0025A) were purchased from Thermo Fisher Scientific.

[0048] 1.2 Extraction method of lipopolysaccharide: Bacterial lipopolysaccharide (LPS) extraction kit, catalog number: BB-31302, purchased from Shanghai Beibo Biotechnology Co., Ltd. The procedure was strictly followed according to the instructions. Collected bacterial cells were resuspended in 500 μl of solution A, centrifuged at 8000g for 3 min, and the supernatant was discarded. The precipitate was resuspended in 500 μl of extraction solution B, and sonicated at 300 W for 15 min (10 s intervals). After centrifugation at 10000g for 10 min, the supernatant was collected. 500 μl of solution C was added to the supernatant, vortexed, and then incubated at 68℃ for 30 min. The mixture was then allowed to stand overnight at 4℃. After centrifugation at 3000g for 20 min, the supernatant was collected to obtain LPS.

[0049] 1.3 Method for detecting lipopolysaccharide concentration: The Total Carbohydrate Assay Kit – Quantification (AB155891), purchased from Abcam, was used to determine LPS concentration. Appropriate amounts of standards and extracted LPS samples were added to 96-well plates, and the volume was adjusted to 30 μl per well using dH2O. 150 µl of concentrated H2SO4 (98%) was added to both the standard and sample wells, and the mixture was stirred on a shaker for 1 min, followed by incubation at 90°C for 15 min. 30 µl of developer was added, and the mixture was stirred on a shaker for 5 min. The OD value was measured at 490 nm.

[0050] 1.4 MALDI-TOF mass spectrometry identification: Weigh 2,5-dihydroxybenzoic acid (DHB) solid powder and dissolve it in a chloroform-methanol mixture (e.g., a 1:1 volume ratio) to prepare a matrix solution with a concentration of 10–20 mg / mL. Take the dried free lipid A sample obtained in the above steps and redissolve it in the chloroform-methanol solvent. Using the dry-drop method, mix 1 μL of the sample solution with 1 μL of the DHB matrix solution on a stainless steel target plate and allow it to evaporate naturally at room temperature until a co-crystallized film is formed. The detection equipment used is a Shimadzu Biotech Axima Performance MALDI-TOF mass spectrometer. To obtain high-abundance and baseline-smooth lipid A negative ion signals, the key instrument parameters were set as follows: Test mode: negative ion, linear mode (Linear_neg); Laser power: set to 111; Pulsed extraction (P.Ext.): optimized and set around 3000 Da (bin 74) to maximize sensitivity in the target mass range; Mass scan range: m / z 700~2100.

[0051] 1.5 Mass spectrometry identification results of Bacteroides ovalis LPS: like Figure 1 As shown, MALDI-TOF-MS analysis revealed that the main peak mass-to-charge ratio (m / z) of lipopolysaccharide lipid A from Bacteroides ovalis was concentrated at 1323.37. This mass number highly matches the deprotonated ions of monophosphorylated and tetraacylated lipid A. The theoretical molecular weight.

[0052] Example 2: Effect of low-acylated LPS from Bacteroides ovalis on bone microstructure improvement in ApoE4 mice To verify the specific therapeutic effect of the hypoacylated LPS of Bacteroides ovalis in this invention, and to confirm whether this therapeutic effect is a universal characteristic of "hypoacylation," this embodiment selected human ApoE4 transgenic mice (purchased from Shanghai Southern Model Biotechnology Co., Ltd.) as the research subjects. Based on our micro-CT image results, their osteoporotic characteristics are consistent with the phenotype of elderly osteoporosis patients carrying the ApoE4 gene, making them suitable as a corresponding model for research. In the same batch of animal experiments, we introduced known studies demonstrating... Bacteroides vulgatus Low-acylated LPS was used as a control for comparison.

[0053] 2.1 Experimental animals and grouping for drug administration: Nine-month-old male ApoE4 mice were randomly divided into the following three groups (n=6 per group): Control group: Daily oral gavage with an equal volume of sterile PBS; Intervention group ( B.ovatus LPS): 1 mg / kg / day via gavage. B.ovatus LPS.

[0054] Bacteroides vulgatus LPS group ( B. vulgatus LPS): 1 mg / kg / day via gavage. Bacteroides vulgatus LPS.

[0055] The intervention lasted for 4 weeks, and the gavage volume was strictly calculated as 0.2 mL / 10 g.

[0056] 2.2 Micro-CT Detection: After the experiment, the right femurs of mice in each group were fixed in 4% paraformaldehyde for 24 hours before undergoing MicroCT (BrukerSkyScan 1176) scanning to assess the microstructural features of the bone. During the scanning process, the tube voltage was set to 90 kV, the source-to-scan distance to 60 mm, and the scanning accuracy to 9 μm. After the scanning, data acquisition was performed using Cruiser software, and image reconstruction was performed using Recon software. Finally, image display and analysis were performed using DataViewer software to calculate bone and trabecular bone parameters, including bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone separation (Tb.Sp), and trabecular bone thickness (Tb.Th).

[0057] 2.3 Statistical Analysis: All data were statistically analyzed using GraphPad Prism 10.0 software (GraphPad Software, USA). Normally distributed quantitative data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for overall comparisons among multiple groups, and Dunnett's test was used for pairwise comparisons between groups. P <0.05 indicates a statistically significant difference (*) P <0.05,** P <0.01).

[0058] 2.4 Experimental Results: like Figure 2 As shown, the structure of the femoral trabeculae in ApoE4 mice was significantly damaged, bone mass was significantly reduced, and the trabeculae were loose and had increased gaps. B.ovatus LPS intervention significantly increased bone mass and reduced the gaps between bone trabeculae in ApoE4 mice. Bacteroides vulgatus LPS intervention did not significantly alter bone mass or the gaps between trabeculae in ApoE4 mice.

[0059] like Figure 3 As shown, compared with the blank control group, B.ovatus The BMD and bone volume fraction (BV / TV) of mice in the LPS group were significantly increased. P <0.05), the trabecular microstructure was significantly improved. However, Bacteroides vulgatus Key bone microstructural parameters such as femoral BMD and BV / TV in LPS group mice showed no statistically significant differences compared with the blank control group. P >0.05).

[0060] The above experimental results show that the Bacteroides ovalis of the present invention ( B. ovatus Hypoacylated LPS can significantly reverse ApoE4 gene-mediated osteoporosis, restoring bone mass and bone microstructure. More importantly, as a hypoacylated lipopolysaccharide, B. vulgatus LPS, however, did not have this effect. This proves... B. ovatus The effect of LPS on improving bone metabolism is not a common feature of all "low-acylated LPS", but rather an unexpected and significant effect of the specific substance of this invention.

[0061] Example 3: B.ovatus Effects of LPS on bone microstructure in D-gal-induced aging C57 mice 3.1 Animal Models: Twenty-four healthy male SPF-grade C57BL / 6N mice, aged 8 weeks, were randomly divided into 4 groups: Control group: Intraperitoneal injection of sterile PBS; Model group (D-gal): Daily intraperitoneal injection of 200 mg / kg of D-gal + gavage of an equal volume of sterile PBS; Intervention group (D-gal+) B.ovatus LPS): D-gal was administered intraperitoneally at a dose of 200 mg / kg daily for four weeks, and simultaneously by gavage at a dose of 1 mg / kg / Day B. ovatus LPS daily until week 8.

[0062] Bacteroides vulgatus LPS group (D-gal + B. vulgatus LPS): D-gal was administered intraperitoneally at a dose of 200 mg / kg daily for four weeks, and simultaneously by gavage at a dose of 1 mg / kg / Day Bacteroides vulgatus LPS daily until week 8 in week 5.

[0063] The volume of gavage should be strictly calculated as 0.2 mL / 10 g.

[0064] 3.2 Micro-CT Detection: Same as Example 2.

[0065] 3.3 Statistical Methods: All data were statistically analyzed using GraphPad Prism 10.0 software (GraphPad Software, USA). Normally distributed quantitative data are expressed as mean ± standard deviation (Mean ± SD). Welch's t-test was used to compare means between two groups; one-way ANOVA was used for overall comparisons among multiple groups, and Dunnett's test was used for pairwise comparisons between groups. P <0.05 indicates a statistically significant difference (*) P <0.05,** P <0.01).

[0066] 3.4 Experimental Results: like Figure 4 As shown, the structure of femoral trabeculae in C57 mice was significantly damaged after D-gal modeling, with loose trabecular morphology and increased gaps. B.ovatus After LPS intervention, there was no significant change in the gaps between bone trabeculae. Bacteroides vulgatus LPS intervention did not significantly alter bone mass or the gaps between trabeculae in mice.

[0067] like Figure 5 As shown, compared with the modeling group, B.ovatus LPS group mice showed significant increases only in BMD and BV / TV ( P <0.05), and other trabecular microstructure indicators showed no significant improvement. B. vulgatus Key bone microstructural parameters such as femoral BMD and BV / TV in LPS group mice showed no statistically significant differences compared with those in the model group. P >0.05).

[0068] A comparison of the Micro-CT data from Examples 2 and 3 shows that the present invention... B.ovatus LPS significantly improves the osteoporotic microstructure mediated by ApoE4, but has no significant reversal effect on D-gal-induced conventional age-related osteoporosis models.

[0069] This comparative result demonstrates that B. ovatus LPS can specifically target bone metabolic imbalance mediated by the ApoE4 genotype. This provides clear experimental evidence for the subsequent development of precision anti-osteoporosis drugs targeting specific genotype populations.

[0070] Example 4: B.ovatus The effect of LPS on bone metabolism in ApoE4 mice 4.1 Laboratory animals and grouping for drug administration: Same as 2.1, 9-month-old male ApoE4 mice were selected and randomly divided into the following two groups (6 mice in each group): Control group: Daily oral gavage with an equal volume of sterile PBS; Intervention group ( B.ovatus LPS): 1 mg / kg / day via gavage. B.ovatus LPS.

[0071] 4.2 Measurement of serum bone metabolism markers: The levels of osteosclerosing protein SOST (E-EL-M2435, Wuhan Yilairuit), osteocalcin (BGP, E-EL-M0864, ​​Wuhan Yilairuit), type I collagen carboxyl-terminal cross-linked peptide (CTX-I, E-EL-M3023, Wuhan Yilairuit), nuclear factor κB receptor activator ligand (RANKL, E-EL-M0644, Wuhan Yilairuit), and osteoprotegerin (OPG, E-EL-M0081, Wuhan Yilairuit) in mouse serum were determined using the ELISA kit according to the instructions.

[0072] 4.3 Measurement of serum inflammatory factor markers: The ELISA kit was used to measure the levels of IL-6 (091206M-48T, Sinobestbio), IL-1β (E20533-48T, Sinobestbio), and TNF-α (2014601M-48T, Sinobestbio) in mouse serum.

[0073] 4.4 Statistical Methods: All data were statistically analyzed using GraphPad Prism 10.0 software (GraphPad Software, USA). Normally distributed quantitative data are expressed as mean ± standard deviation (Mean ± SD). Independent samples t-tests were used to compare means between two groups. P <0.05 indicates a statistically significant difference (*) P <0.05,** P <0.01).

[0074] 4.5 Experimental Results: like Figure 6 ELISA results showed that, compared with the control group, B.ovatus LPS intervention significantly reduced the level of SOST in the serum of ApoE4 mice. Simultaneously, the levels of BGP and OPG, indicators of bone formation, significantly increased, while the levels of CTX-I and RANKL, indicators of bone resorption, significantly decreased.

[0075] like Figure 7 ELISA results showed that B.ovatus LPS intervention significantly reduced serum levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in ApoE4 mice.

[0076] Example 4 further clarifies the situation from a serological perspective. B.ovatus LPS improves bone metabolism through various pathways. Results showed that... B.ovatus LPS significantly inhibited the expression of SOST, a core negative regulatory target of bone metabolism. By downregulating SOST and improving the OPG / RANKL ratio, it effectively corrected the abnormally elevated bone resorption state in ApoE4 mice. Simultaneously... B.ovatus LPS can significantly reduce the level of systemic inflammation in the body. The above data confirms that... B.ovatus LPS exerts its anti-osteoporosis effect through a clear pathway of "inhibiting SOST expression and regulating bone metabolism balance".

[0077] Example 5: B.ovatus The effect of LPS on E. coli-induced SOST expression in osteocytes To further validate the targeting mechanism of the substances of this invention at the molecular and cellular level, this embodiment evaluated in vitro... B.ovatus LPS E. coli The effect of LPS-induced SOST expression.

[0078] 5.1 Experimental Methods: 1) Cell culture and grouping: MLO-Y4 osteoblasts were routinely cultured in vitro, and the experiment was divided into four groups: Blank control group: No stimulants were added; Model group: Add E. coli Cells were stimulated with LPS (1 μg / mL) for 2 hours, then cultured in regular medium for another 24 hours. B.ovatus LPS group: After adding an equal volume of culture medium and culturing for 2 h, add B.ovatus LPS (1 μg / mL) was cultured for another 24 h; Intervention group: Join E. coli Treat with LPS (1 μg / mL) for 2 hours, then add B.ovatus Incubate with LPS (1 μg / mL) for 24 h.

[0079] 2) ELISA detection of SOST levels: Collect cell culture supernatant, centrifuge at 300 g for 5 min at 4°C to remove cell debris, and keep the supernatant for later use. The SOST level in the cell supernatant was detected using ELISA (Mouse SOST ELISA Kit, Wuhan Yilairuit Biotechnology Co., Ltd.).

[0080] 3) Western blot detection of SOST protein: Total protein was extracted from cells using RIPA lysis buffer (SW104, Beijing Seven Innovations), quantified using a BCA kit (Beijing Solarbio), and denatured by boiling. Equal amounts of protein were separated by SDS-PAGE (SW109, Beijing Seven Innovations), transferred semi-dry to a PVDF membrane, and blocked at room temperature for 1 hour. SOST primary antibody (Proteintech, 21933-1-AP, 1:500) and β-actin internal control antibody were added, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed with TBST, and then incubated with secondary antibody at room temperature in the dark for 1 hour. After washing again, images were acquired using an Odyssey near-infrared laser imaging system, and homogenized quantitative analysis of β-actin was performed using ImageJ software.

[0081] 5.2 Statistical Methods: All data were statistically analyzed using GraphPad Prism 10.0 software (GraphPad Software, USA). Normally distributed quantitative data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for overall comparisons among the four groups, and Tukey's test was used for pairwise comparisons between groups. P <0.05 indicates a statistically significant difference (*) P <0.05,** P <0.01).

[0082] 5.3 Experimental Results: like Figure 8 As shown, ELISA ( Figure 8 A) and Western blot ( Figure 8 B) The results show that, E. coli LPS stimulation significantly upregulated SOST expression levels in MLO-Y4 osteoblasts. P <0.01); however, after adding B. ovatus LPS intervention, it was able to significantly antagonize E. coli LPS-induced a significant decrease in SOST expression in osteocytes.

[0083] The above in vitro experimental results show that B.ovatus LPS can directly act on MLO-Y4, effectively antagonizing the effects of... E. coli LPS-induced aberrant SOST expression. This result confirms the findings of the serological assay in Example 4 at the cellular and molecular levels, further demonstrating... B.ovatus One of the important in vitro mechanisms by which LPS exerts its osteoprotective effect is by targeting and inhibiting the expression of SOST, a key negative regulator of osteocytes.

[0084] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of low-acylated lipopolysaccharides derived from Bacteroides ovalis in the preparation of drugs for the prevention and / or treatment of osteoporosis.

2. The application according to claim 1, characterized in that, The osteoporosis mentioned is osteoporosis mediated by the ApoE4 gene.

3. The application according to claim 1, characterized in that, The drug exerts its osteogenic effect by inhibiting the expression of the SOST gene in bone cells and / or reducing the level of SOST protein secreted by bone cells; Preferably, the drug can increase bone density and / or improve bone microstructure; Preferably, the improvement in bone microstructure includes an increase in one or more of the following indicators: bone volume fraction, trabecular thickness, and trabecular number, and / or a decrease in trabecular separation.

4. The application according to claim 1, characterized in that, The drug can suppress the inflammatory response; Preferably, the inhibition of the inflammatory response includes a reduction in the levels of one or more of the pro-inflammatory factors IL-1β, IL-6, and TNF-α; Preferably, the drug can also inhibit the inflammatory response and / or bone loss induced by highly acylated lipopolysaccharides; Preferably, the highly acylated lipopolysaccharide is derived from Escherichia coli.

5. The application according to claim 1, characterized in that, The dosage form of the drug includes oral formulations or enteral formulations; Preferably, the oral preparation includes tablets, capsules, granules, powders, oral solutions, or oral suspensions; Preferably, the intestinal administration formulation includes an enteric-coated formulation or a rectal administration formulation.

6. The application according to claim 1, characterized in that, The lipid A portion of the low-acylated lipopolysaccharide derived from *Bacteroides ovalis* has a tetraacylated structure.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a low-acylated lipopolysaccharide derived from Bacteroides ovalis and pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, Pharmaceutically acceptable excipients include diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and / or disintegrants.

9. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition further includes a second therapeutic agent; Preferably, the second therapeutic agent is selected from one or more of bisphosphonates, denosumab, parathyroid hormone analogs, sclerosing protein antibodies, calcium preparations, and vitamin D and its analogs.

10. A method for regulating the expression level of SOST in osteocytes, characterized in that, The method includes treating bone cells with low-acylated lipopolysaccharides derived from Bacteroides ovalis; Preferably, the hypoacylated lipopolysaccharide derived from Bacteroides ovalis negatively regulates the expression level of SOST; Preferably, the bone cells are selected from MLO-Y4 cells.

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