Application of a wolfberry glycopeptide in improving the function of osteoporosis products

Abstract

The application discloses application of a wolfberry glycopeptide in an osteoporosis improvement functional product and belongs to the technical field of biological medicine; the application first finds that the wolfberry glycopeptide can play an anti-osteoporosis role by bidirectional regulation of the functional balance of osteoblasts and osteoclasts; experiments show that the wolfberry glycopeptide promotes proliferation, migration and osteogenic differentiation of osteogenic precursor cells MC3T3-E1 in a concentration-dependent manner, significantly up-regulates the expression of key osteogenic genes Runx2, ColI alpha 1 and protein beta-catenin; simultaneously, the wolfberry glycopeptide inhibits the proliferation and osteoclast differentiation process of osteoclast precursor cells BMMs, and down-regulates the expression of osteoclast-related genes NFATc1, c-Fos, CTSK and proteins RANK and Dkk-1; the mechanism may be closely related to synergistic regulation of two classical signal pathways of Wnt / beta-catenin and RANKL-RANK.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a goji berry glycopeptide in a product for improving osteoporosis. Background Technology

[0002] Osteoporosis is a systemic bone metabolic disease characterized by bone loss and deterioration of bone microstructure, leading to decreased bone strength, bone fragility, and increased risk of fractures.

[0003] The pathogenesis of osteoporosis is complex, involving multiple factors such as genetics, environment, and endocrine function. Its core pathological mechanism is the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. When osteoclast activity is enhanced or osteoblast activity is weakened, bone resorption exceeds bone formation, ultimately leading to bone loss and osteoporosis (OP). The dynamic balance between the two is crucial for maintaining normal bone remodeling and bone homeostasis.

[0004] Currently, clinical treatments for osteoporosis (OP) mainly include calcitonin, bisphosphonates, and estrogen replacement therapy. While these can slow down bone loss to some extent, they also have significant side effects and raise concerns about the safety of long-term use. Therefore, screening safe and effective anti-OP active ingredients from natural products and elucidating their mechanisms of action has become a hot topic in the field of bone metabolism research.

[0005] Lycium barbarum polysaccharides, as the main active components of Lycium barbarum, have the effects of improving bone density, regulating osteoblast and osteoclast activity, and inhibiting osteoporosis development. Lycium barbarum glycopeptides are a collective term for glycoconjugates obtained through further isolation and purification from Lycium barbarum polysaccharides. Previous studies have shown that LbGP has various pharmacological effects, including antioxidant, immunomodulatory, anti-inflammatory, and neuroprotective effects; however, its regulatory role in bone metabolism and its molecular mechanism remain unclear. Summary of the Invention

[0006] This invention proposes the application of wolfberry glycopeptide in products that improve osteoporosis function, in order to solve the problems mentioned in the background art.

[0007] This invention proposes the application of a wolfberry glycopeptide in a product for improving osteoporosis function.

[0008] Preferably, the product is a pharmaceutical, health food, or food for special medical purposes.

[0009] Preferably, the dosage form of the drug includes tablets, capsules, granules, oral liquids, injections, ointments, or gels.

[0010] Preferably, the dosage form of the health food includes tablets, capsules, granules, oral liquids, or powders.

[0011] Preferably, the wolfberry glycopeptide plays a role in improving osteoporosis by promoting osteoblast proliferation, migration and / or osteogenic differentiation, and / or inhibiting osteoclast proliferation and / or osteoclast differentiation.

[0012] Preferably, the wolfberry glycopeptide promotes osteoblast proliferation, migration and / or osteogenic differentiation by upregulating the expression of the key osteogenic genes Runx2 and / or ColⅠα1, and / or upregulating the expression of the key osteogenic protein β-catenin.

[0013] Preferably, the wolfberry glycopeptide inhibits osteoclast proliferation and / or osteoclast differentiation by downregulating the expression of osteoclast-related genes NFATc1, c-Fos and / or CTSK, and / or downregulating the expression of osteoclast-related proteins RANK and / or Dkk-1.

[0014] Preferably, the wolfberry glycopeptide is a glycoconjugate obtained by isolating and purifying wolfberry polysaccharides.

[0015] Preferably, the effective concentration of the wolfberry glycopeptide is 50-250 μg / mL.

[0016] The present invention also provides a pharmaceutical composition for improving osteoporosis, comprising an effective amount of lycine glycopeptide and pharmaceutically acceptable excipients.

[0017] The application of a goji berry glycopeptide proposed in this invention in products for improving osteoporosis has the following beneficial effects: (1) This invention is the first to discover that wolfberry glycopeptide has an anti-osteoporosis effect, which broadens the application field of wolfberry glycopeptide and provides a new source of natural active substances for the development of anti-osteoporosis drugs.

[0018] (2) This invention confirms that Lycium barbarum glycopeptides can bidirectionally regulate bone metabolism balance, promoting osteoblast proliferation, migration, and osteogenic differentiation to enhance bone formation capacity, while inhibiting osteoclast proliferation and osteoclast differentiation to reduce bone resorption activity. This bidirectional regulatory mode has better therapeutic effects and higher safety compared to single-target anti-OP drugs.

[0019] (3) Compared with Lycium barbarum polysaccharides, Lycium barbarum glycopeptides have the advantages of smaller molecular weight, better water solubility and higher bioavailability. They can be directly absorbed through intestinal peptide transporters and quickly reach the target site to exert regulatory effects.

[0020] (4) This invention elucidates the mechanism of action of Lycium barbarum glycopeptide against osteoporosis through an in vitro cell experiment system, providing sufficient experimental basis for its development and application in anti-osteoporosis products. Attached Figure Description

[0021] Figure 1 The relative proliferation rate of cells in the MC3T3-E1 group.

[0022] Figure 2 The cell proliferation rate of BMMs in the LbGP-treated group.

[0023] Figure 3 The scratch healing rate is denoted as LbGP treatment group.

[0024] Figure 4 The migration rate of the LbGP processing group.

[0025] Figure 5 This represents the number of mineralized nodules in the LbGP treatment group.

[0026] Figure 6 The number of mature multinucleated osteoclasts in the LbGP-treated group.

[0027] Figure 7 LbGP promotes osteoblast differentiation and mineralization.

[0028] Figure 8 The expression levels of RANK, an upstream receptor for osteoclast differentiation, c-Fos, a core transcription factor, NFATc1, and CTSK, a key protein in bone resorption, were all significantly downregulated in the LbGP-treated group (P<0.05). Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments.

[0030] Main materials and reagents used in the examples: Osteoblast cell line MC3T3-E1 and osteoclast precursor cell line BMMs were purchased from Apex Biotech (Shanghai) Co., Ltd.; Lycium barbarum glycopeptide powder (LbGP) was purchased from Ningxia Tianren Lycium Barbarum Biotechnology Co., Ltd. (purity 98%, batch number 20231203)-6; α-MEM medium and penicillin / streptomycin solution were purchased from VivaCell (Shanghai, China); fetal bovine serum (FBS) was purchased from Gibco (USA); macrophage colony-stimulating factor (M-CSF) and nuclear factor κB receptor activator ligand (RANKL) were purchased from Abbkine (Wuhan, China); MTT assay kit and TRAP staining kit were purchased from Elabscience (Wuhan). Alizarin Red S staining solution was purchased from Qingdao Jiesheng Biotechnology Co., Ltd. (China); RIPA lysis buffer (containing protease inhibitor PMSF) was purchased from Solarbio (Beijing, China); SDS-PAGE gel preparation kit and chemiluminescence colorimetric kit were purchased from Beyotime (Shanghai, China); β-catenin, Runx2, ColⅠα1, Dkk-1, NFATC1, c-Fos, CTSK, RANK, Gapdh, and Tubulin primary antibodies were purchased from Abcam (USA) or Affinity (China); horseradish peroxidase (HRP) labeled secondary antibody was purchased from Affinity (China).

[0031] The main instruments and equipment used in the embodiments are as follows: CO2 incubator (Thermo Fisher Scientific, USA); inverted microscope (Olympus, Japan); microplate reader (Bio-Rad, USA); protein electrophoresis apparatus and transfer apparatus (Bio-Rad, USA); chemiluminescence imaging system (Tanon, China); low-temperature high-speed centrifuge (Eppendorf, Germany).

[0032] Example 1: Cell Culture MC3T3-E1 cells were cultured in α-MEM complete medium containing 10% FBS and 1% penicillin / streptomycin; BMMs cells were cultured in α-MEM complete medium containing 10% FBS, 1% penicillin / streptomycin and 10 ng / mL M-CSF. Both cell types were cultured routinely in a 37°C, 5% CO2 incubator, with the medium changed every 2-3 days.

[0033] Example 2: Effects of Lycium barbarum glycopeptides on osteoblast and osteoclast proliferation MC3T3-E1 cells and BMMs cells were seeded into 96-well plates, respectively. MC3T3-E1 cells were cultured in complete medium, while the M-CSF concentration in the BMMs cell culture medium was adjusted to 20 ng / mL. When the cell confluence reached 50%-60%, corresponding mediums containing different concentrations of LbGP (0, 10, 50, 100, 250, 500, 1000 μg / mL) were added to each well, with 6 replicates per group, and cultured for another 24 h. After culture, 20 μL MTT solution (5 mg / mL) was added to each well, and the cells were incubated at 37°C for 4 h. The supernatant was discarded, and 150 μL LDMSO was added to each well, shaken for 10 min to fully dissolve the crystals. The absorbance (OD value) at 570 nm was measured using a microplate reader, and the relative cell proliferation rate was calculated.

[0034] Experimental results: Compared with the control group (0 μg / mL LbGP), the relative proliferation rate of MC3T3-E1 cells in the 50-250 μg / mL LbGP treatment groups was significantly increased (P<0.05). Figure 1 Compared with the control group, the proliferation rate of BMMs in the 250-1000 μg / mL bGP treatment group was significantly reduced (P<0.05). Figure 2 The inhibitory effect of the 250 μg / mL LbGP group was already quite significant. Therefore, 250 μg / mL LbGP was selected for subsequent experiments.

[0035] Example 3: Effects of Lycium barbarum glycopeptides on osteoblast migration (scratch assay) MC3T3-E1 cells were seeded in 12-well plates and cultured in complete medium until the cell confluence reached 90%-100%. A straight line was drawn in the center of the bottom of each well using a 200 μL sterile pipette tip. The cells were washed three times with PBS to remove floating cells. The control group was given basal medium containing 1% FBS, while the experimental group was given basal medium containing 1% FBS and 250 μg / mL LbGP. The cells were then incubated. The scratched areas were observed and photographed under an inverted microscope at 0 h and 24 h. The scratch width was measured using ImageJ software, and the scratch healing rate was calculated using the formula: Scratch healing rate = [(0 h scratch width - 24 h scratch width) / 0 h scratch width] × 100%.

[0036] Experimental results: Compared with the control group, the scratch healing rate of MC3T3-E1 cells treated with 250 μg / mL LbGP was significantly increased at 24 h (P<0.01), indicating that LbGP can significantly promote osteoblast migration. Figure 3 .

[0037] Example 4: Effect of Lycium barbarum glycopeptides on osteoclast migration (Transwell migration assay) After digestion and centrifugation, BMM cells were resuspended in α-MEM medium containing 1% FBS and the cell concentration was adjusted to 2 × 10⁶ cells / year. 5 Cells were cultured at 12 h to synchronize the cell cycle. Transwell chambers were placed in 24-well plates, with 200 μL of cell suspension added to the upper chamber and 600 μL of culture medium to the lower chamber. In the control group, the lower chamber contained α-MEM medium with 1% FBS and 10 ng / mL L-CSF; in the experimental group, the lower chamber contained α-MEM medium with 1% FBS, 10 ng / mL L-CSF, and 250 μg / mL LbGP. After culturing at 37°C in a 5% CO2 incubator for 24 h, the upper chamber medium was discarded, and the upper surface of the chamber was gently washed with PBS to remove unmigrated cells. Cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 20 min. The number of cells that had migrated through the membrane was counted in five randomly selected fields of view under an inverted microscope, and the cell migration rate was calculated.

[0038] Experimental results: Compared with the control group, there was no significant difference in the migration rate of BMMs cells in the 250 μg / mL LbGP treatment group at 24 h, indicating that LbGP had no significant effect on the migration ability of osteoclast precursor cells. Figure 4 .

[0039] Example 5: Effects of Lycium barbarum glycopeptides on osteoblast differentiation MC3T3-E1 cells were seeded in 24-well plates and cultured in complete medium for 24 hours until cell adhesion. The old medium was discarded. The control group was treated with osteogenic induction medium (α-MEM basal medium containing 10 mM β-glycerophosphate, 50 μg / mL vitamin C, 100 nM dexamethasone, 10% FBS, and 1% penicillin / streptomycin), while the experimental group was treated with osteogenic induction medium containing 250 μg / mL LbGP. The medium was changed every 3 days. After 21 days of osteogenic induction, cells were stained with Alizarin Red S and observed and photographed under an inverted microscope. 10% acetic acid was added to each well, and the cells were incubated at room temperature for 30 min, centrifuged at 1000 rpm for 10 min, and the supernatant was collected. The absorbance (OD value) at 562 nm was measured using a microplate reader to reflect the relative content of mineralized nodules.

[0040] Experimental results: Alizarin Red S staining showed that after 21 days of osteogenic induction, the control group had a small number of scattered light red mineralized nodules, while the LbGP-treated group had a significantly increased number and size of mineralized nodules; quantitative analysis using acid dissolution method showed that the OD of the LbGP-treated group was significantly higher. 562 The value was 0.82±0.07, significantly higher than that of the control group (0.35±0.04) (P<0.01). This indicates that LbGP can significantly promote the mineralization and maturation of osteoblasts. Figure 5 .

[0041] Example 6: Effects of Lycium barbarum glycopeptides on osteoclast differentiation BMMs were seeded in 24-well plates and cultured in α-MEM complete medium containing 10 ng / mL L-CSF for 24 h to allow cell adhesion. The old medium was discarded. The control group was treated with osteoclast induction medium (α-MEM basal medium + 10 ng / mL L-CSF + 50 ng / mL RANKL + 10% FBS + 1% penicillin / streptomycin), while the experimental group was treated with osteoclast induction medium containing 250 μg / mL LbGP. The induction medium was replaced with fresh medium for each group every 3 days, and the cells were incubated at 37°C with 5% CO2 for 7 days. Osteoclasts were identified by TRAP staining with wine-red cytoplasm and ≥3 nuclei. Cells were observed and photographed under an inverted microscope, and five random fields of view were used to count positive cells and calculate the positive cell rate.

[0042] Experimental results: TRAP staining showed that at 7 days of osteoclast induction, the control group exhibited a large number of TRAP-positive osteoclasts (≥3 nuclei), with deep purplish-red cytoplasm, large cell volume, and irregular morphology; while the LbGP-treated group showed only a small number of weakly positive mononuclear or binuclear cells, and the number of mature multinucleated osteoclasts was significantly reduced (P<0.01). This indicates that LbGP can significantly inhibit osteoclast formation. Figure 6 .

[0043] Example 7: Western blot detection of protein expression MC3T3-E1 cells and BMMs cells were seeded into 6-well plates and cultured in their respective complete culture media until the cell confluence reached approximately 70%. The control group received the corresponding complete culture media, while the experimental group received the corresponding complete culture media containing 250 μg / mL LbGP. Cells were cultured for another 24 h. Cells were washed twice with pre-chilled PBS, and RIPA lysis buffer containing PMSF was added. Cells were lysed on ice for 30 min. Cells were scraped off with a cell scraper and collected in centrifuge tubes. Centrifugation at 12000g, 4℃ for 10 min was performed, and the supernatant was collected as the total protein solution. These proteins were subjected to 12% SDS-PAGE electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with 5% skim milk at room temperature for 1 h, and washed three times with PBST for 5 min each time. The membranes were then placed in a 4°C freezer and incubated overnight with primary antibodies (β-catenin, Runx2, ColⅠα1, Dkk-1, NFATC1, c-Fos, CTSK, and RANK, all diluted 1:1000) and internal control GAPDH (diluted 1:5000). The next day, the membranes were washed three times with PBST for 5 min each time, and HRP-labeled secondary antibody was added. The membranes were incubated at room temperature for 1 h with gentle shaking, the secondary antibody was discarded, and the membranes were washed three times with PBST for 5 min each time. Chemiluminescence immunoassay was then added, and protein bands were detected using a chemiluminescence imaging system. The relative expression levels of each protein group were quantitatively analyzed using ImageJ software.

[0044] Experimental results: The results of osteogenic-related protein detection showed that, compared with the control group, the LbGP treatment group significantly downregulated the expression of Dkk-1, a specific inhibitor of the Wnt / β-catenin pathway (P<0.05), while significantly upregulating the expression of β-catenin, a core molecule of the pathway, the osteogenic transcription factor Runx2, and ColⅠα1, a key protein in bone matrix synthesis (P<0.01). This suggests that LbGP may promote osteoblast differentiation and mineralization by activating the Wnt / β-catenin signaling pathway and upregulating the expression of osteogenic-related proteins. Figure 7 .

[0045] The results of osteoclast-related protein detection showed that, compared with the control group, the expression levels of upstream osteoclast differentiation receptor RANK, early transcription factor c-Fos, core transcription factor NFATc1, and the key bone resorption protein CTSK were significantly downregulated in the LbGP-treated group (P<0.05). This suggests that LbGP may inhibit osteoclast precursor cell differentiation by blocking the RANKL-RANK signaling pathway, inhibiting the activation of the c-Fos / NFATc1 transcriptional axis, reducing the expression of downstream functional proteins, and thus inhibiting the differentiation of osteoclast precursor cells. Figure 8 .

[0046] Example 8: Statistical Analysis All experiments were repeated at least three times. Statistical analysis was performed using GraphPadPrism 9.0 software. Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0047] As can be seen from the above, (1) this invention is the first to discover that wolfberry glycopeptide has an anti-osteoporosis effect, which broadens the application field of wolfberry glycopeptide and provides a new source of natural active substances for the development of anti-osteoporosis drugs.

[0048] (2) This invention confirms that Lycium barbarum glycopeptides can bidirectionally regulate bone metabolism balance, promoting osteoblast proliferation, migration, and osteogenic differentiation to enhance bone formation capacity, while inhibiting osteoclast proliferation and osteoclast differentiation to reduce bone resorption activity. This bidirectional regulatory mode has better therapeutic effects and higher safety compared to single-target anti-OP drugs.

[0049] (3) Compared with Lycium barbarum polysaccharides, Lycium barbarum glycopeptides have the advantages of smaller molecular weight, better water solubility and higher bioavailability. They can be directly absorbed through intestinal peptide transporters and quickly reach the target site to exert regulatory effects.

[0050] (4) This invention elucidates the mechanism of action of Lycium barbarum glycopeptide against osteoporosis through an in vitro cell experiment system, providing sufficient experimental basis for its development and application in anti-osteoporosis products.

[0051] The above description is only a preferred embodiment of the present invention, but 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.

Claims

1. The application of a goji berry glycopeptide in a product for improving osteoporosis function.

2. The application according to claim 1, characterized in that, The product is a pharmaceutical, health food, or food for special medical purposes.

3. The application according to claim 2, characterized in that, The dosage forms of the medicine include tablets, capsules, granules, oral liquids, injections, ointments, or gels.

4. The application according to claim 2, characterized in that, The dosage forms of the health food include tablets, capsules, granules, oral liquids, or powders.

5. The application according to claim 1, characterized in that, The wolfberry glycopeptide plays a role in improving osteoporosis by promoting osteoblast proliferation, migration and / or osteogenic differentiation, and / or inhibiting osteoclast proliferation and / or osteoclast differentiation.

6. The application according to claim 5, characterized in that, The wolfberry glycopeptide promotes osteoblast proliferation, migration, and / or osteogenic differentiation by upregulating the expression of key osteogenic genes Runx2 and / or ColⅠα1, and / or upregulating the expression of key osteogenic protein β-catenin.

7. The application according to claim 5, characterized in that, The wolfberry glycopeptide inhibits osteoclast proliferation and / or osteoclast differentiation by downregulating the expression of osteoclast-related genes NFATc1, c-Fos and / or CTSK, and / or downregulating the expression of osteoclast-related proteins RANK and / or Dkk-1.

8. The application according to claim 1, characterized in that, The goji berry glycopeptide is a glycoconjugate obtained by isolating and purifying goji berry polysaccharides.

9. The application according to claim 1, characterized in that, The effective concentration of the wolfberry glycopeptide is 50-250 μg / mL.

10. A pharmaceutical composition for improving osteoporosis, characterized in that, It contains an effective amount of goji berry glycopeptides and pharmaceutically acceptable excipients.

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