Use of weissella cibaria in alleviating estrogen-deficiency osteoporosis

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of Weissella cibaria in relieving estrogen deficiency osteoporosis. The application screens a strain of Weissella cibaria CCUG57177, the strain can realize safe and effective intervention on estrogen deficiency osteoporosis by regulating bone metabolism balance and an intestinal-bone axis pathway. On one hand, the strain can significantly improve bone microstructure, improve bone density, up-regulate osteogenic protein expression, inhibit osteoclastic activity, and effectively delay bone mass loss; on the other hand, the strain can repair intestinal mucosa damage caused by estrogen deficiency, up-regulate intestinal tight junction protein expression, and improve intestinal barrier function. In addition, the strain has no long-term intervention organ toxicity, has good biological safety, and can be used as a new probiotic preparation for preventing and treating postmenopausal osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of *Westernella esculenta* in alleviating estrogen deficiency osteoporosis. Background Technology

[0002] Estrogen deficiency-induced postmenopausal osteoporosis (PMOP, also known as estrogen deficiency osteoporosis) is characterized by decreased bone mass, bone microarchitectural deterioration, increased bone fragility, and elevated fracture risk. It is a prevalent disease among postmenopausal women and a significant cause of fragility fractures, chronic bone pain, and decreased quality of life in middle-aged and elderly women. With the increasing aging of the population, the prevalence of this disease continues to rise, attracting widespread attention. Currently, clinical interventions mainly focus on bone resorption inhibitors and bone formation promoters. While these can delay bone loss to some extent, long-term use suffers from numerous adverse reactions, limited applicability, and poor patient compliance. In recent years, with the gradual confirmation of the gut-bone axis regulatory mechanism, probiotic intervention has provided new research ideas for the prevention and treatment of osteoporosis. Probiotics possess advantages such as high safety, mild effects, and the ability to regulate the body's overall metabolic state, and have now become a research hotspot in the field of bone metabolism regulation.

[0003] *Weissella cibaria* is a Gram-positive facultative anaerobic lactic acid bacterium widely distributed in naturally fermented foods, animal mouths, intestines, and fermentation environments. Belonging to the genus *Weissella*, it is a recognized safe probiotic resource. *Weissella cibaria* plays an important regulatory role in maintaining the homeostasis of the host's intestinal microecology, possessing various beneficial activities such as antibacterial, anti-inflammatory, antioxidant, immunomodulatory, and intestinal barrier function improvement. It has good development potential in the fields of food fermentation and health intervention.

[0004] However, there are currently no reports on the use of Weissella esculenta to alleviate estrogen deficiency osteoporosis or regulate bone metabolism balance. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a strain of Weissella cibaria CCUG57177 that can alleviate estrogen deficiency osteoporosis, thus filling the gap in the application of Weissella cibaria in the field of bone metabolism regulation. This strain can achieve safe and effective intervention for estrogen deficiency osteoporosis by regulating bone metabolism balance and the gut-bone axis pathway.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides the use of Weissella cibaria CCUG57177 in the preparation of medicaments for relieving and / or treating estrogen deficiency osteoporosis.

[0007] Preferably, the *Weissella cibaria* CCUG57177 alleviates estrogen deficiency osteoporosis by regulating bone metabolism balance and the gut-bone axis pathway. This regulation of bone metabolism balance and the gut-bone axis pathway refers to: improving bone microstructure, increasing bone density, upregulating osteoblast protein expression, inhibiting osteoclast activity, delaying bone loss, repairing intestinal mucosal damage caused by estrogen deficiency, upregulating intestinal tight junction protein expression, and improving intestinal barrier function.

[0008] Preferably, the effective concentration of *Weissella cibaria* CCUG57177 in the drug is 1 × 10⁻⁶. 9 ~1×10 11 CFU / mL.

[0009] Preferably, the estrogen deficiency osteoporosis is postmenopausal osteoporosis.

[0010] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0011] More preferably, the excipients include at least one of fillers, disintegrants, binders, lubricants, solubilizers, stabilizers, and flavoring agents. All excipients are pharmaceutically acceptable excipients that do not inhibit or kill *Westernella esculenta* activity and are suitable for oral live bacterial preparations.

[0012] Further, the filler includes at least one of starch, lactose, microcrystalline cellulose, mannitol, and dextrin; the disintegrant includes at least one of sodium carboxymethyl starch, crospovidone, and low-substituted hydroxypropyl cellulose; the binder includes at least one of hydroxypropyl methylcellulose, polyethylene glycol, and starch paste; the lubricant includes at least one of magnesium stearate, talc, and micronized silica gel; the solubilizer includes at least one of propylene glycol, glycerin, polyethylene glycol, Tween-80, and hydroxypropyl-β-cyclodextrin; the stabilizer includes at least one of skim milk powder, trehalose, sucrose, mannitol, sorbitol, β-cyclodextrin, and sodium ascorbate; and the flavoring agent includes at least one of steviol glycosides, xylitol, sucrose, fructose, lemon flavor, strawberry flavor, peppermint flavor, and maltitol.

[0013] More preferably, the dosage form of the drug is an oral liquid preparation, including at least one of suspension and oral liquid.

[0014] Compared with the prior art, the beneficial effects of the present invention are: (1) Gut-bone axis targeted intervention strategy: Unlike traditional estrogen replacement therapy and chemical anti-bone resorption drugs, which have many adverse reactions and high risks of long-term use, this invention uses Weissella cibaria CCUG57177 as the core functional component. It plays a bone-protective role by orally intervening in the targeted regulation of the gut-bone axis pathway. It has the advantages of high safety, mild effect and long-term intervention, and expands the strain resource boundary of probiotics in the field of bone health.

[0015] (2) Achieving bidirectional coupling regulation of osteogenic and osteoclast: Compared with ordinary commercial probiotic strains, the Weissellacibaria CCUG57177 strain screened in this invention can significantly promote osteogenic differentiation and inhibit excessive activation of osteoclasts. In the OVX rat osteoporosis model, this strain can effectively alleviate estrogen deficiency-induced bone loss, increase femoral bone density, increase the number of trabeculae, and significantly improve bone microstructure.

[0016] (3) The Weissella cibaria CCUG57177 used in this invention can be prepared by high-density fermentation, with high cell yield and good formulation stability, making it suitable for clinical and daily application scenarios for oral administration.

[0017] (4) After continuous gavage intervention of estrogen-deficient osteoporosis rats with the Weissella cibaria CCUG57177 of the present invention for 11 weeks, no pathological changes were observed in important organs such as heart, liver, spleen, lungs and kidneys. The strain has no hemolytic activity and is sensitive to commonly used clinical antibiotics, and has good long-term biosafety.

[0018] (5) This invention uses a single Weissella cibaria CCUG57177 as the core functional strain to achieve effective intervention for estrogen deficiency osteoporosis. It does not require multiple strains or other anti-osteoporosis drugs. The fermentation and preparation process is simple and controllable, and the production cost is low. It can be widely used in the development of probiotic preparations, functional health care preparations and other products, and has good prospects for industrial application. Attached Figure Description

[0019] Figure 1 Culture and characterization of Weissella cibaria CCUG57177; where AB represents the morphological characteristics of CCUG57177 strain under transmission electron microscopy; C represents the average Zeta potential of CCUG57177 strain; and DF represents the culture and growth curve of CCUG57177 strain.

[0020] Figure 2 The experimental procedure and validation results of the regulatory effect of Weissella cibaria CCUG57177 on bone metabolism were presented. Specifically, AC shows the experimental procedure diagram of CCUG57177 intervention, OVX surgical modeling, and oral gavage operation diagrams; D shows the weight measurement analysis during the entire experimental period of CCUG57177 gavage intervention; and E shows the changes in bone parameters assessed by Micro-CT after 11 weeks of CCUG57177 gavage, validating the successful model construction. Figure 3 To verify the bone metabolism regulation effect and in vivo biosafety of the edible Weissella cibaria strain CCUG57177; A is the effect of HE and Masson staining on alleviating osteoporosis in OVX rats; B is the effect of IHC staining on osteogenic and osteoclastogenic effects of CCUG57177 on OVX rats; C is the in vivo biosafety of CCUG57177 strain after 11 weeks of gavage.

[0021] Figure 4 This study validates that intervention with Weissella cibaria CCUG57177 can effectively alleviate intestinal barrier dysfunction caused by estrogen deficiency osteoporosis. Detailed Implementation

[0022] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0023] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0024] The *Weissella cibaria* used in this invention is the publicly disclosed strain CCUG57177 deposited at CCUG (Culture Collection, University of Gothenburg, Sweden). The strain used in the experiment was purchased from BeNa Culture Collection, trade number BNCC358279.

[0025] Example 1: Culture and characterization of Weissella cibaria CCUG57177 (1) Activation of strain: Weissella cibaria CCUG57177 was a purchased standard purified strain. Single colonies were obtained by streaking MRS medium plates. The stable third generation strain was selected for subsequent experiments.

[0026] (2) Fermentation culture and preparation of bacterial agent: Activated Weissella cibaria CCUG57177 was transferred to MRS liquid medium at an inoculation rate of 1-2% (volume percentage) and statically cultured in a CO2 incubator at 37℃ for 24-28 h until the end of the logarithmic growth phase. After the culture was completed, the bacterial cells were collected by centrifugation at 3000g at 4℃ for 10 min, washed twice with sterile PBS, and resuspended in 200μL of sterile PBS. The bacterial count was determined by plate count method. The obtained bacterial agent can reach 1×10⁻⁶. 10 CFU / mL, which is the amount of Weissella cibaria CCUG57177 bacterial agent prepared by oral gavage.

[0027] (3) Characterization of the strain: Transmission electron microscopy revealed that *Weissella cibaria* CCUG57177 had a short, elliptical rod-like structure, and its average Zeta potential was measured to be -20.99 ± 0.18 mV. By measuring its growth curve, it was determined that *Weissella cibaria* CCUG57177 reached its growth plateau phase in approximately 24 hours. Figure 1 ).

[0028] Example 2: Bone metabolism regulation and in vivo biosafety of Weissella cibaria CCUG57177 To determine whether *Weissella cibaria* CCUG57177 possesses in vivo efficacy in alleviating estrogen deficiency osteoporosis, a bilateral ovariectomized (OVX) SD rat model was used. Specifically, SPF-grade female SD rats were selected to establish the OVX osteoporosis model. One week after modeling, the rats were randomly divided into a sham-operated group (only some reproductive fat near the ovary was removed, without ovarian removal), an OVX model group, and a *Weissella cibaria* intervention group. The intervention group received 200 μL of 1×10⁻⁶ bacteria via gavage. 10CFU / mL Weissella cibaria CCUG57177 bacterial agent was administered twice weekly for 11 consecutive weeks. During this period, rat body weight and general survival status were recorded, completing the grouping intervention procedure for the in vivo animal experiment. After the intervention, rat femurs were harvested for MicroCT scanning and 3D reconstruction to quantitatively analyze bone mineral density and bone microstructure parameters. Bone tissue sections were stained with HE, Masson's trichrome, and IHC to observe bone morphology and the expression of osteogenic and osteoclast-reactive proteins. Major organs such as the heart, liver, spleen, lungs, and kidneys were harvested, sectioned, and stained with HE. The tissue morphology, cell arrangement, and inflammatory infiltration of each organ were observed under an optical microscope.

[0029] The results show that ( Figure 2 and Figure 3 Compared with the OVX model group, the femoral bone mineral density of rats in the *Westernella esculenta* intervention group was increased, the number of trabeculae was increased, and bone microstructure damage was significantly improved. The expression of osteoblast protein OPN and osteoclast protein TRAP in bone tissue was upregulated, confirming that this strain can effectively alleviate bone loss in estrogen deficiency osteoporosis. The organs and tissues of rats in the *Westernella esculenta* intervention group were intact, and the cells were arranged in a regular manner. No obvious inflammatory cell infiltration, cell necrosis, or pathological damage was observed, which was not significantly different from other groups. This confirms that long-term oral administration of *Westernella esculenta* does not cause damage to major organs and has good long-term biocompatibility in vivo.

[0030] Example 3: Intervention with Weissella cibaria CCUG57177 can effectively alleviate intestinal barrier dysfunction caused by estrogen deficiency osteoporosis. To clarify the restorative effect of *Weissella cibaria* CCUG57177 on estrogen deficiency-induced intestinal barrier dysfunction, an evaluation was conducted based on an OVX rat osteoporosis model, focusing on intestinal tissue morphology and tight junction protein expression. Specifically, the animal grouping and intervention protocol was consistent with that in Example 2, with the following groups: sham-operated group, OVX model group, and *Weissella cibaria* intervention group (200 μL 1×10⁻⁶). 10 (CFU / mL, twice a week, for 11 consecutive weeks); after the intervention, rats were sacrificed, and intestinal tissue was quickly harvested, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and serially sectioned for HE and AB-PAS staining. The integrity of the intestinal mucosal epithelium, crypt structure morphology, inflammatory cell infiltration, and goblet cell number were observed under an optical microscope. At the same time, IHC staining was used to detect the expression and localization of the tight junction core protein Occludin in the colon tissue, and semi-quantitative analysis was performed using ImageJ software.

[0031] The results show that ( Figure 4Compared with the sham-operated group, the OVX model group rats showed varying degrees of defects in the intestinal mucosal epithelium, disordered crypt arrangement, inflammatory cell infiltration in the lamina propria, reduced goblet cells, and significantly downregulated Occludin protein expression. After intervention with Weissella cibaria CCUG57177, the continuity of the intestinal mucosal epithelium was restored, the number of goblet cells increased, the crypt structure became regular, inflammatory infiltration was significantly reduced, and the Occludin protein expression level significantly rebounded compared with the model group. This indicates that Weissella cibaria CCUG57177 can effectively improve the morphological damage of colonic tissue caused by estrogen deficiency and repair the intestinal barrier function.

[0032] In summary, strain CCUG57177 can safely and effectively intervene in estrogen deficiency osteoporosis by regulating bone metabolism balance and the gut-bone axis pathway. On the one hand, it can significantly improve bone microstructure, increase bone density, upregulate osteoblast protein expression, inhibit osteoclast activity, and effectively delay bone loss. On the other hand, it can repair intestinal mucosal damage caused by estrogen deficiency, upregulate intestinal tight junction protein expression, and improve intestinal barrier function. In addition, this strain has no organ toxicity with long-term intervention and has good biosafety, making it a potential novel probiotic preparation for the prevention and treatment of postmenopausal osteoporosis.

[0033] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Use of Weissella cibaria CCUG57177 in the preparation of a drug for alleviating and / or treating estrogen-deficient osteoporosis.

2. Use according to claim 1, characterized in that, The Weissella cibaria CCUG57177 alleviates estrogen-deficient osteoporosis by regulating bone metabolism balance and "gut-bone axis" pathway; the regulation of bone metabolism balance and "gut-bone axis" pathway refers to: improving bone microstructure, improving bone density, up-regulating osteogenic protein expression, inhibiting osteoclast activity, delaying bone mass loss, and repairing intestinal mucosa damage caused by estrogen deficiency, up-regulating intestinal tight junction protein expression, and improving intestinal barrier function.

3. Use according to claim 1, characterized in that, The effective concentration of the said Weissella cibaria CCUG57177 in a medicament is 1 x 10 9 ~ 1 x 10 11 CFU / mL.

4. Use according to claim 1, characterized in that, The estrogen-deficient osteoporosis is postmenopausal osteoporosis.

5. The use according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable excipient.

6. Use according to claim 5, characterized in that, The excipient includes at least one of a filler, a disintegrant, a binder, a lubricant, a solubilizer, a stabilizer, and a flavoring agent.

7. Use according to claim 5, characterized in that, The dosage form of the drug is an oral liquid preparation, including at least one of a suspension and an oral liquid.

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