Application of Lactobacillus intestinalis in the treatment of postmenopausal osteoporosis

Abstract

This invention discloses Enterobacter ( Lactobacillus intestinalis This invention relates to the application of *Lactobacillus* (preferably strain DSM 6629) in the preparation of products for treating postmenopausal osteoporosis, belonging to the fields of biomedicine and microbiology. Clinical trials and ovariectomized rat model experiments have demonstrated that *Lactobacillus* can significantly increase bone mineral density (BMD) in postmenopausal osteoporosis patients, increase the number and thickness of trabeculae in model rats, improve bone microstructure, and reduce bone marrow fat area. This invention provides a safe, effective, and long-term usable probiotic intervention program, which can be prepared into drugs or health products, offering a novel strategy for the treatment of postmenopausal osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and microbiology, and relates to the application of enterobacteria in the treatment of postmenopausal osteoporosis. Background Technology

[0002] Postmenopausal osteoporosis (PMO) is a disease in postmenopausal women caused by declining estrogen levels, leading to bone metabolism imbalance, bone microstructure destruction, decreased bone mineral density, and a significantly increased risk of fractures. It has become a major public health issue. Current clinical treatments primarily focus on inhibiting bone resorption or promoting bone formation, but these methods suffer from limitations such as single-target therapy, varying degrees of side effects, and poor patient compliance. Traditional Chinese medicine (TCM) offers advantages in the prevention and treatment of PMO, including holistic regulation, multi-target therapy, and high safety. Guilu Erxian Jiao, a classic formula for tonifying the kidneys and replenishing essence, and simultaneously nourishing Yin and Yang, has been clinically proven to significantly increase bone mineral density and improve bone metabolism imbalance in PMO patients. Recent studies have shown that gut microbiota and its metabolites participate in bone metabolism regulation through the gut-bone axis.

[0003] Currently, there is no way to include enterobacteria (Lactobacillus) in the treatment of enterobacteria (Lactobacillus) Lactobacillus intestinalis There are no patents or products disclosed for the treatment of postmenopausal osteoporosis, either alone or in combination, nor have standardized intervention programs been developed with this strain as the core. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides the application of *Lactobacillus enterica* in the treatment of postmenopausal osteoporosis. *Lactobacillus enterica* (… Lactobacillus intestinalis As an active ingredient, it can significantly increase bone density and improve bone microstructure in patients, thereby achieving the purpose of treating or adjuvant treatment of postmenopausal osteoporosis.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0006] This invention discloses Enterobacter ( L. intestinalis Its application in the preparation of products for the treatment of postmenopausal osteoporosis.

[0007] Furthermore, the strain of *Lactobacillus enterica* is accession number DSM 6629 or a passaged strain with the same biological function.

[0008] Furthermore, the product is a drug, functional food, or health product.

[0009] Furthermore, the dosage form of the product is an oral preparation, preferably a capsule, tablet, powder, granule or oral liquid.

[0010] Furthermore, the effective dose of enterobacterial lactobacillus in the product is 1×10⁻⁶ per day. 8 ~1×10 10 CFU.

[0011] Furthermore, the product is used to increase bone mineral density (BMD) and / or improve bone microstructure in patients with postmenopausal osteoporosis.

[0012] Preferably, the improvement of bone microstructure includes increasing the number of trabeculae, increasing trabecular bone thickness, increasing bone volume fraction (BV / TV), and / or reducing bone marrow fat area.

[0013] Furthermore, in any of the above applications, the product further comprises pharmaceutically acceptable excipients, including lyophilization protectants (such as maltodextrin, trehalose) and / or enteric coating materials.

[0014] Furthermore, in any of the above applications, the product further comprises one or more active ingredients selected from the group consisting of tortoise shell and deer antler extract, calcium, vitamin D, bisphosphonates, and selective estrogen receptor modulators for the treatment or adjunctive treatment of osteoporosis.

[0015] The applicant's preliminary research found that: Enterobacteriaceae ( L. intestinalis (This is a key target bacterium for the anti-PMO effect of GEG (Guilu Erxian Jiao). Clinical RCT trials and animal studies have found that supplementation with GEG in PMO patients and ovariectomized (OVX) rat models...) L. intestinal It can significantly increase bone density and improve bone microstructure.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] 1) First time clearly stated: L. intestinalis This bacterium is a functional probiotic for treating PMO, filling a gap in the application of bacterial strains.

[0018] 2) Clear mechanism: explained for the first time L. intestinalis The evidence is conclusive that bacteria improve bone structure and increase bone density.

[0019] 3) Clinically applicable: Clinical RCTs and animal validations have been completed, demonstrating definite efficacy and high safety. Attached Figure Description

[0020] Figure 1 : L. intestinalis Bacterial identification report.

[0021] Figure 2 : L. intestinalis Bacterial amplification, formulation preparation, and RCT test results: (A) Photograph of the bacterial culture plate ( L. intestinalis (LI-2023); (B) Detection of bacterial concentration (OD600=1.0, 1×10⁻⁶) 9 (CFU / mL); (C) L. intestinalis Live bacteria capsules (1×10) 10(CFU / particle); (D) Scatter plot of changes in bone mineral density (BMD), inflammatory factors (IL-1, IL-6) and metabolites (MeIAA) before and after treatment in PMO patients (** indicates P<0.01, * indicates P<0.05, ns indicates no significant difference).

[0022] Figure 3 : L. intestinalis Improving bone structure and osteogenic indices in OVX rats: (A) HE staining, Masson staining, and Micro-CT three-dimensional reconstruction results of femoral tissue; (B) Immunohistochemical staining results of OCN, OPG, OSX, and RUNX2 protein expression in bone tissue (scale bar = 50 μm); (C) Statistical analysis of Micro-CT bone microstructure parameters (BMD, Tb.N, Tb.Th, Tb.Sp, BV / TV, SMI) and the ratio of immunohistochemical positive areas in each group (** indicates P<0.01, # indicates P<0.05, ## indicates P<0.01). Note: Sham group, sham operation group; OVX group, ovariectomized model group; Lint+OVX group. L. intestinal Bacterial intervention group.

[0023] Figure 4 : L. intestinalis Western blotting results of the effect of bacteria on RUNX2 protein expression in bone tissue of OVX model rats: (A) Immunoblot images of RUNX2 and internal reference GAPDH proteins in bone tissue (molecular weights 57 kD and 36 kD, respectively); (B) Statistical analysis bar chart of relative RUNX2 protein expression (p-RUNX2 / GAPDH ratio) in each group (** indicates P<0.01, # indicates P<0.05, ## indicates P<0.01). Note: Sham group, sham operation group; OVX group, ovariectomized model group; GEG+OVX group, Guilu Erxian gelatin intervention group; Lint+OVX group, L. intestinalis Bacterial intervention group.

[0024] Figure 5 : L. intestinalis Effects of bacteria on the gut microbiota structure of OVX rats: (A) Alpha diversity index analysis (Chao1, Shannon, Simpson, Pielou_e, Faith_pd, etc.); (B) Principal coordinate analysis (PCoA); (C) Inter-group microbiota similarity analysis; (D) Heatmaps of relative abundance of microbiota at the phylum and genus levels; (E) Stacked bar charts of microbiota composition at the phylum level for each group; (F) Heatmaps predicting microbiota function. Note: SHAM group, sham-operated group; GEG group, turtle and deer jelly group; OVX group, ovariectomized model group.

[0025] Figure 6 : Screening of differentially expressed microbiota and correlation analysis with bone microstructure indicators: (A) Distribution of LDA values ​​of differentially expressed species in LEfSe between OVX and SHAM groups (LDA score>2.0); (B) Distribution of LDA values ​​of differentially expressed species in LEfSe between GEG and OVX groups; (C) Cladogram of differentially expressed microbiota between OVX and SHAM groups; (D) Cladogram of differentially expressed microbiota between GEG and OVX groups; (E) Spearman correlation heatmap of differentially expressed microbiota genera and bone microstructure indicators (BMD, Tb.N, Tb.Th, Tb.Sp, BV / TV, SMI, Conn.D) (** indicates P<0.01, * indicates P<0.05). Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0027] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0028] Example 1: L. intestinalis Intervention of bacteria in the OVX rat PMO model.

[0029] Laboratory animals and main reagents.

[0030] 1. Experimental groups: Sham surgery group (Sham), model group (OVX) L. intestinalis Intervention group (Lint+OVX).

[0031] 2. Experimental Animals: Sixty SPF-grade female SD rats, weighing (300±10) g, were randomly divided into three groups of 20 rats each. All rats were provided by Liaoning Changsheng Biotechnology Co., Ltd. (License No. SCXK(Liaoning)2015-0001). These rats were housed at the Animal Center of Liaoning University of Traditional Chinese Medicine at a room temperature of 21±2°C. The animal experiments were approved by the Ethics Committee of Liaoning University of Traditional Chinese Medicine.

[0032] 3. Main drugs used in the experiment: L. intestinalis 1×10 9 CFU / animal / day, administered via gavage for 8 weeks.

[0033] 4. Establishment and evaluation of the OVX rat model: (1) Model establishment: After weighing, rats were anesthetized by intraperitoneal injection of 0.1% sodium pentobarbital solution (45 mg / kg). The rats were fixed in a prone position. The skin was prepared with a radius of 1.5 cm, 1.5 cm lateral to the midline of the back and 1.5 cm below the costal margin on both sides. The surgical site was disinfected with iodine and alcohol. The skin was incised, the subcutaneous tissue was bluntly dissected, the muscle layer was incised, and the abdominal cavity was exposed. White fat was visible in the incision field. After separating the fat, the ovary, which was pink and mulberry-shaped, was visible. The fallopian tube below the ovary was clamped with tissue forceps and ligated with surgical sutures. The ovary was removed, and the severed fallopian tube was returned to the abdominal cavity and sutured layer by layer. The other ovary was removed in the same way. After surgery, 40,000 IU of sodium penicillin G was injected into the thigh muscle of the rat. (2) Model evaluation: Eight weeks after the model was established, micro-CT was used to detect bone density and microstructural changes in rats.

[0034] II. Experimental Procedure and Related Testing.

[0035] 1. Detect changes in the gut microbiome of OVX rats.

[0036] Metagenomic sequencing technology: Experimental samples are collected from intestinal feces and transported at low temperatures (below 0°C) to the testing company. Qualified DNA samples undergo library construction and testing. Qualified libraries are then sequenced using an Illumina PE150. The resulting raw data will be used for subsequent information analysis (Beijing Novogene Technology Co., Ltd.).

[0037] 2. Detect bone metabolism and bone mineral density in the serum of OVX rats.

[0038] Rat bone tissue collection: After the last administration of medication, rats were anesthetized by intraperitoneal injection of 0.1% sodium pentobarbital solution (45 mg / kg). After anesthesia, the rats were fixed supine on a surgical frame. The abdomen and femur were disinfected with povidone-iodine. The abdominal cavity was opened, and blood was collected to induce shock and death. The femurs of both hind limbs were harvested, and the attached muscles and fascia were removed. The femurs were fixed in formalin, and the portions used for immunostaining were decalcified with EDTA decalcification solution for 2 weeks. The tissues were then embedded in paraffin, sectioned, stained, cleared, mounted, and examined under a microscope.

[0039] (1) Micro-CT scan to detect changes in bone microstructure; femoral specimens were fixed in 10% formalin.

[0040] μCT scans were performed 24 hours later. 3D reconstructions were performed on all scan images, and the bone volume fraction (BV / TV), structural pattern index (SMI), trabecular bone number (Tb.N), and bone mineral density (BMD) (Tb.Sp) of cancellous bone were calculated using system analysis software. Larger cross-sectional areas were continuously sliced, with a thickness of 1.5 mm, and designated as regions of interest (ROIs) for 3D reconstruction. 3D image reconstruction was performed using N-Recon software, and 3D analysis was conducted using CT-AN software.

[0041] (2) Western blotting and immunohistochemistry were used to detect osteogenic related indicators (OCN, OPG, Runx2, Osterix).

[0042] 1) Western blotting detection of Runx2 protein expression: Protein was extracted from bone tissue using lysis buffer and quantified using the BCA method. 20 μg of protein sample was subjected to SDS-PAGE electrophoresis on a 10% polyacrylamide gel. After electrophoresis, the sample was transferred to a PVDF membrane at 100V. The membrane was blocked at 37℃ for 1 h and incubated overnight with primary antibody at 4℃. The sample was then incubated with diluted horseradish peroxidase (HRP)-labeled secondary antibody, and imaged using ECL luminescence. The image was scanned into a computer (n>3). The average grayscale value was measured using ImageJ software. 2) Immunohistochemical detection of OCN, OPG, Runx2, and Osterix protein expression: Routine dewaxing and hydration were performed, followed by antigen retrieval for 10 min. Endogenous peroxidase was blocked with 3% hydrogen peroxide, and the cells were blocked with 5% serum at 37°C for 30 min. The cells were then incubated overnight in a humidified chamber at 4°C in the dark with primary antibody. Horseradish peroxidase-labeled secondary antibody was then incubated at 37°C for 30 min. DAB staining was applied, and the cells were observed under a microscope. Positive signals were brownish-yellow or brownish-red. Hematoxylin counterstaining and fixation were performed, and the cells were analyzed using ImagePro software. Five high-power fields were randomly selected from the bone marrow cavity, and the percentage of positive area in each field was calculated (the percentage of positive cells with brownish-yellow nuclei in each microscopic field is the percentage of the measured field area).

[0043] Experimental results.

[0044] 1. L. intestinalis Strain identification.

[0045] Genomic DNA was extracted from the strain, and 16S rRNA gene was amplified by PCR. The results were detected by 1.0% agarose gel electrophoresis, and clear bands were observed. Figure 1 After bidirectional sequencing of the PCR products, BLAST-N alignment was performed using NCBI, and the results showed that strain number 374668 was consistent with... L. intestinalis The similarity is 100%, confirming that this strain is... L. intestinalis .

[0046] PCR product electrophoresis results (Marker DL2000, 1.0% agarose gel, 100V, 40min); sequencing and BLAST-N alignment results (strain number 374668 and...). L. intestinalis (100% similarity).

[0047] 2. L. intestinalis Improves bone microstructure in OVX rats.

[0048] (1) Observation of organizational morphology, such as Figure 3 As shown in Figure A, HE staining revealed that the trabeculae in the Sham group were tightly arranged and structurally intact; the trabeculae in the OVX group were significantly thinner, broken, and fewer in number, with an enlarged medullary cavity; and the trabecular structure in the Lint+OVX group was significantly improved, with a more dense arrangement. Masson staining showed that the blue collagen fibers in the OVX group were sparsely distributed, while collagen deposition was significantly increased in the Lint+OVX group.

[0049] (2) Micro-CT three-dimensional reconstruction and quantitative analysis of Micro-CT three-dimensional reconstruction results Figure 3 A) Visual observation: In the OVX group, the trabeculae were rod-shaped, sparse, and poorly connected; in the Lint+OVX group, the trabeculae were plate-like, densely connected, and similar to the Sham group. Quantitative statistical analysis ( Figure 3 C) indicates: Bone mineral density (BMD): Compared with the Sham group, the BMD of the OVX group was significantly lower; the BMD of the Lint+OVX group was significantly higher. Bone volume fraction (BV / TV): The BMD of the OVX group was significantly lower than that of the Sham group (P<0.01). The BMD of the Lint+OVX group was significantly higher (P<0.05 vs OVX group). Trabecular bone parameters: Compared with the OVX group, the Tb.N and Tb.Th increased, and the Tb.Sp decreased in the Lint+OVX group. Structural pattern index (SMI): The SMI of the OVX group was significantly higher, indicating that the trabecular bone degenerates from plate-like to rod-like structures; the SMI of the Lint+OVX group was significantly lower, indicating that the plate-like structure was restored.

[0050] .

[0051] 3. L. intestinalis Promotes the expression of osteogenic-related proteins.

[0052] (1) Immunohistochemical results. For example... Figure 3 As shown in B and C, the positive expression areas (brownish-yellow areas) of osteogenic markers OCN, OPG, OSX and RUNX2 in bone tissue of the OVX group were significantly lower than those of the Sham group (P<0.01).

[0053] (2) Western blotting results. For example... Figure 4As shown, the gray value of the RUNX2 protein band in the OVX group was significantly lower than that in the Sham group. Quantitative analysis showed that the RUNX2 / GAPDH ratio decreased in the OVX group, significantly lower than that in the Sham group (P<0.01); it increased in the GEG+OVX group (P<0.05 vs OVX group); and it significantly increased in the Lint+OVX group (P<0.01 vs OVX group), with no significant difference from the Sham group.

[0054] 4. L. intestinalis Remodeling the gut microbiota structure of OVX rats.

[0055] (1) Alpha and Beta diversity analysis. For example... Figure 5 As shown in Figure A, Alpha diversity analysis revealed that compared to the Sham group, the Chao1 and Shannon indices in the OVX group were significantly lower (P<0.05), indicating a decrease in gut microbiota richness and diversity in the model group; the diversity index in the Lint+OVX group significantly rebounded, approaching the level of the Sham group. PCoA analysis ( Figure 5 B) shows that the sample points of the Sham group, OVX group and Lint+OVX group are significantly separated in spatial distribution (P<0.05), indicating that... Lactobacillus intestinal The intervention effectively reversed the gut microbiota imbalance caused by oophorectomy.

[0056] (2) Changes in microbial community composition and function. Species composition analysis ( Figure 5 (D, E) indicates that the gut microbiota of rats in each group is mainly composed of Firmicutes (D, E, E). Firmicutes ) and Bacteroidetes ( Bacteroidetes The composition of the group was Lactobacillus, but the relative abundance differed significantly. The Lint+OVX group contained significantly higher levels of Lactobacillus spp. (…). Lactobacillus The abundance of microbial community increased significantly. (Prediction of microbial community function) Figure 5 F) showed that, compared with the OVX group, the Lint+OVX group had significantly increased abundance in beneficial metabolic pathways such as "fermentation" and "nitrate reduction", while the abundance of "human pathogens gastroenteritis" was significantly decreased.

[0057] (3) LEfSe differential analysis identifies target bacteria such as Figure 6 As shown in A and B, LDA analysis indicates that... Lactobacillus intestinal Significant enrichment was observed in the Lint+OVX group (LDA score > 2.0, P < 0.05). Evolutionary branching diagram ( Figure 6 C and D) confirm this from a taxonomical perspective. Lactobacillus intestinalisand its belonging Lactobacillaceae are distinctive differential species unique to the intervention group.

[0058] (4) Correlation between target bacteria and bone microstructure. Spearman correlation heatmap ( Figure 6 E) shows that: Lactobacillus intestinalis the relative abundance of is significantly positively correlated with BMD, BV / TV and Tb.N (r=0.56-0.68, P<0.01), and significantly negatively correlated with Tb.Sp and SMI (r=-0.52 to -0.61, P<0.05). This confirms from a statistical perspective that Lactobacillus intestinalis the enrichment degree of is highly consistent with the improvement degree of bone microstructure.

[0059] Example 2: Lactobacillus intestinalis Strain preparation and clinical intervention for PMO patients.

[0060] (1) Study subjects and grouping 60 patients (60 PMO patients, aged 50-70 years old) diagnosed and treated at the outpatient department of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine from January 2024 to December 2024 were selected as study subjects. According to the order of outpatient visits, patients were randomly divided into a control group (30 cases) and a treatment group ( L. intestinalis bacterial oral administration group, 30 cases).

[0061] (2) Western medicine diagnostic criteria Refer to the "Guidelines for the Diagnosis and Treatment of Primary Osteoporosis (2017)" formulated by the Chinese Medical Association Society of Osteoporosis and Bone Mineral Research for diagnosis: Dual-energy X-ray absorptiometry (DXA) was used to measure the bone mineral density of the 1st to 4th lumbar vertebrae and femoral neck. A T-score reduction greater than -1 SD is normal; a T-score reduction between -2.5 SD and -1 SD is diagnosed as osteopenia; a T-score reduction less than -2.5 SD is diagnosed as osteoporosis.

[0062] (3) Traditional Chinese medicine diagnostic criteria Refer to the "Guidelines for the Diagnosis and Treatment of Postmenopausal Osteoporosis (Bone Atrophy) with Traditional Chinese Medicine (2019)" [6] the relevant diagnostic criteria therein and combine with clinical symptoms for diagnosis. Main symptoms: low back and back pain, soreness and weakness of waist and knees, deafness and tinnitus; secondary symptoms: lower limb pain, dry mouth and throat, red tongue, scanty coating, thready and rapid pulse.

[0063] (4) Inclusion criteria ① Naturally postmenopausal women, aged between 50 and 70 years, menopausal for more than 1 year, self-reported bone pain symptoms such as low back and joint pain, excluding the possibility of secondary osteoporosis; ② After examination, conforming to the above-mentioned Chinese and Western medicine diagnostic criteria for PMO; ③ Patients with no history of drug allergy, voluntarily participating as test subjects, actively cooperating with treatment, and signing informed consent.

[0064] (5) Exclusion criteria: ① Patients with malignant tumors; ② Patients with serious primary diseases of the heart, brain, liver, kidneys, etc.; ③ Patients with secondary osteoporosis; ④ Patients with serious mental illness or poor compliance and unable to adhere to treatment; ⑤ Patients with digestive tract diseases, thyroid dysfunction, etc.; ⑥ Patients who have been receiving long-term treatment with glucocorticoids, antipsychotic drugs, or bone metabolism drugs; ⑦ Patients with severe scoliosis, bone trauma, or sequelae of orthopedic surgery that affect bone mineral density measurement; ⑧ Patients with severe osteoporosis, i.e., T decrease ≤ -2.5 SD and also with fractures.

[0065] (6) Treatment: (1) Basic treatment: Administered 2 tablets of Dicalcium Carbonate D3 chewable tablets once daily. (2) Control group: Administered basic treatment. (3) Treatment group: L. intestinalis (DSM 6629) was activated, centrifuged, and freeze-dried to produce 1×10 9 CFU / enteric-coated capsule. Take twice daily, for a total daily dose of 1. 0 10 CFU. The placebo product consisted of maltodextrin powder. Study participants were asked to ingest the powder after mixing it with a cold, non-alcoholic food or beverage.

[0066] (7) Observation indicators: (1) Visual analog scale (VAS) score: The pain level of patients was assessed before treatment and 4 weeks after treatment. (2) Stool collection: Stool was collected in the morning at enrollment and 1 month, 6 months and 12 months after treatment and stored in sterile EP tubes at -80℃. (3) At enrollment and 1 month, 6 months and 12 months after treatment, fasting blood samples were collected in the morning and serum was stored in sterile EP tubes at -80℃. (1) Metagenomic sequencing technology was used to observe changes in the gut microbiome; (2) LC-MS method was used to determine the full spectrum of metabolites in plasma samples of each group; (3) The levels of aging-related factors (IL-1, IL-6) in serum were detected.

[0067] (8) Efficacy evaluation criteria: The efficacy criteria for treating osteoporosis with new traditional Chinese medicines were formulated with reference to the "Guiding Principles for Clinical Research of New Traditional Chinese Medicines". Marked effect: Pain completely disappears, and bone density increases by more than 2% compared to before treatment; Effective: Pain is significantly relieved, and bone density increases by 1% to 2% compared to before treatment; Ineffective: Pain does not improve, and bone density increases by <1% compared to before treatment. The treatment period is one month, and only the improvement of the patient's pain is observed.

[0068] (9) Statistical methods: Descriptive statistical analysis: N, Mean (SD), Median, MD, Min, and Max were calculated for continuous data; frequencies and percentages were calculated for categorical data. t-tests and ANOVA were used for continuous data. Wilcoxon's nonparametric method was used when the data did not conform to a normal distribution or had unequal variances. Chi-square, corrected chi-square, and Fisher's exact test were used for categorical data. Ridit analysis and Wilcoxon's rank-sum test were used for ordinal data. All statistical analyses were performed using SPSS 18.0 software.

[0069] Experimental results.

[0070] Clinical efficacy evaluation (changes in bone mineral density (BMD)): After 12 months of treatment, dual-energy X-ray absorptiometry (DXA) was performed on both groups of patients.

[0071] The results show: Treatment group ( L. intestinalis In the oral administration group: Bone mineral density (BMD) of the lumbar spine L1-L4 was significantly increased compared with that before treatment (mean increase of 3.5% ± 0.8%), and femoral neck BMD increased by 2.1% ± 0.5% on average. According to the efficacy evaluation criteria, 18 cases showed significant effect, 9 cases showed effect, and 3 cases showed no effect, with a total effective rate of 90%. The VAS pain score decreased significantly from (6.5±1.2) points before treatment to (2.1±0.8) points (P<0.01). Control group (basic treatment): Lumbar spine BMD increased by an average of 0.5% ± 0.2%, while femoral neck BMD showed no significant change. Two cases showed significant improvement, eight cases showed improvement, and 20 cases showed no improvement, with a total effective rate of 33.3%. The VAS score decreased to (4.5 ± 1.0) points.

[0072] Conclusion: Compared with the control group, L. intestinalis The intervention group significantly improved bone mineral density and relieved bone pain in PMO patients (P<0.01).

[0073] .

[0074] In summary, L. intestinalis (DSM 6629) significantly improved bone microstructure in OVX rats, increased bone mineral density (BMD), and promoted the expression of osteogenic-related proteins (OCN, OPG, OSX, RUNX2). It exerted its anti-osteoporosis effect by reshaping the gut microbiota, enriching beneficial bacteria, and regulating the gut-bone axis. Clinical RCT trials have confirmed this. L. intestinalis Oral administration for 12 months can significantly improve bone density and relieve bone pain in PMO patients. It has a good safety profile and can be used as a novel probiotic preparation for the treatment or adjuvant therapy of PMO.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Enterobacteriaceae ( Lactobacillus intestinalis Its application in the preparation of products for the treatment of postmenopausal osteoporosis.

2. The application according to claim 1, characterized in that, The strain of *Lactobacillus enterica* is preservation number DSM 6629 or a passaged strain with the same biological function.

3. The application according to claim 1, characterized in that, The product is a drug, functional food, or health product.

4. The application according to claim 1, characterized in that, The product is an oral preparation, preferably in the form of capsules, tablets, powders, granules, or oral liquid.

5. The application according to claim 1, characterized in that, The effective dose of *Lactobacillus in the product is 1 × 10⁻⁶ daily. 8 ~1×10 10 CFU.

6. The application according to claim 1, characterized in that, The product is to be used continuously for at least 3 months.

7. The application according to claim 1, characterized in that, The product is intended to increase bone mineral density (BMD) and / or improve bone microstructure in patients with postmenopausal osteoporosis.

8. The application according to claim 7, characterized in that, The improvements in bone microstructure include increasing the number of trabeculae, increasing trabecular thickness, increasing bone volume fraction (BV / TV), and / or reducing bone marrow fat area.

9. The application according to any one of claims 1-8, characterized in that, The product also contains pharmaceutically acceptable excipients, including lyophilization protectants (such as maltodextrin, trehalose) and / or enteric coating materials.

10. The application according to any one of claims 1-8, characterized in that, The product also contains one or more active ingredients selected from the following: tortoise and deer antler extract, calcium, vitamin D, bisphosphonates, and selective estrogen receptor modulators, for the treatment or adjunctive treatment of osteoporosis.

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