Application of new target of bone metabolism ncstn and its agonist in preparation of drugs for preventing or treating osteoporosis

Abstract

The present application belongs to the field of medicine, and particularly relates to a new target point NCSTN for regulating bone metabolism and application of an agonist of the target point in preparation of a medicine for preventing or treating osteoporosis. One object of the present application is to provide a new target point NCSTN for regulating bone metabolism and application of the target point in promoting osteogenic activity, inhibiting osteoclast activity and treating osteoporosis. The present application first discovers a mechanism of treating osteoporosis by using an agonist of NCSTN, and has the effect of inhibiting mature differentiation of osteoclasts and promoting bone formation function of osteoblasts. This is very different from the previously reported mechanism of single inhibition of bone resorption or single promotion of bone formation, and the treatment of osteoporosis by simultaneously acting on bone resorption and bone formation can greatly reduce adverse reactions of the body to the medicine.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of the novel target for regulating bone metabolism, NCSTN (Nicastrin), and its agonists in the preparation of drugs for the prevention or treatment of bone metabolic diseases. Background Technology

[0002] Bone metabolic diseases are a group of diseases caused by an imbalance between bone formation and decomposition. Common types include osteoporosis, rickets, osteomalacia, parathyroid dysfunction, bone loss due to ovarian absence, rheumatoid arthritis, or bone destruction due to tumor metastasis. The main manifestations are bone pain, easy fractures, and skeletal deformities.

[0003] Osteoporosis is one of the most common bone metabolic diseases, affecting over 200 million people worldwide, seriously impacting the elderly and postmenopausal women. Osteoporosis is characterized by decreased bone mass, reduced bone density, and increased bone fragility. This condition primarily stems from the disruption of bone homeostasis maintained by osteoblasts and osteoclasts, manifesting as increased osteoclast-mediated bone resorption and decreased osteoblast-mediated bone formation. Clinically, first-line anti-bone resorption drugs include bisphosphonates and denosumab. However, inhibiting bone resorption alone can disrupt physiological bone remodeling, leading to adverse reactions such as jaw necrosis and atypical fractures. Furthermore, drugs that solely enhance osteoblast activity, such as triparatide, can induce hypercalcemia and other adverse reactions with long-term use.

[0004] Therefore, discovering novel targets and drugs that simultaneously promote bone formation and inhibit bone resorption is of great value for the treatment of osteoporosis. NCSTN is a type I transmembrane glycoprotein that plays an important role in cell growth, differentiation, and signal transduction. As a core component of the Notch signaling pathway, NCSTN (Nicastrin) regulates the maturation and activation of the Notch receptor. Notch activation inhibits the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and cathepsin K (CTSK), thereby inhibiting RANKL-induced osteoclastogenesis. However, there are currently no reports on the relationship between NCSTN and bone metabolism.

[0005] Traditional Chinese medicine (TCM) is characterized by its green and low-toxicity properties. For example, patent document CN115919838A describes schisandrin B, an extract of Schisandra chinensis, which regulates the NF-κB signaling pathway to alleviate osteoporosis. Patent document CN114588176A describes ophiopogon japonicus extract, ophiopogon saponin D, as a TCM extract that avoids the side effects of Western medicine while maintaining the high bioavailability of TCM, showing good therapeutic effects on osteoporosis. 2M4V (vinyl guaiacol) is a traditional TCM extract. Studies have shown that it has anti-tumor and antibacterial medicinal value, but its target and regulatory effect on bone homeostasis are still unclear. Summary of the Invention

[0006] One object of the present invention is to provide a novel target for regulating bone metabolism, NCSTN, and its application in products that promote osteogenic activity while inhibiting osteoclast activity, and for the prevention and / or treatment of bone metabolism.

[0007] The application of NCSTN as a target in the preparation of drugs for the prevention and / or treatment of bone metabolic diseases, wherein the drugs have the effect of inhibiting osteoclast maturation and differentiation while promoting osteoblast bone formation.

[0008] Application of NCSTN as a target in the development, design, and screening of products for the prevention and / or treatment of bone metabolism.

[0009] The drug is a preparation that inhibits osteoclast maturation and differentiation or regulates bone homeostasis and improves bone density and bone microstructure.

[0010] The bone metabolic diseases mentioned are osteoporosis, bone loss due to ovarian absence, rheumatoid arthritis, or bone destruction due to tumor metastasis.

[0011] This invention first demonstrates the application of NCSTN in the treatment of bone metabolic diseases through in vitro and in vivo knockdown and overexpression experiments. Furthermore, NCSTN can be used as a drug target in the development, design, and screening of drugs to alleviate or treat bone metabolic diseases.

[0012] Another object of the present invention is to provide NCSTN agonists and their applications.

[0013] Application of NCSTN agonists in the preparation of drugs for the prevention and / or treatment of bone metabolic diseases.

[0014] The NSTTN agonist is a chemical drug, peptide drug, or protein drug that targets and promotes the expression level or activity of NSTTN.

[0015] Furthermore, the NCSTN agonist is 2M4V or a derivative thereof, as shown in compounds 1-4 of Formula I.

[0016]

[0017] Compound 1 is 2M4V (vinylguaiacol), compound 2 is 4-methylguaiacol, compound 3 is 4-aminomethylguaiacol, and compound 4 is vanillin acetal.

[0018] The present invention provides a pharmaceutical composition for treating osteoporosis by targeting the NSTN, comprising one or more of 2M4V and its derivatives (compounds 1-4).

[0019] The pharmaceutical or pharmaceutical composition described in this application also includes pharmaceutically acceptable excipients.

[0020] Preferably, the excipients include carriers or excipients, such as lactose hydrate, microcrystalline cellulose, mannitol, sodium citrate, calcium phosphate, glycine, and starch; disintegrants such as crospovidone, copovidone, sodium glycolate starch, crospovidone carboxymethyl cellulose, and specific composite silicates; and binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin, and gum arabic.

[0021] The present invention also provides the application of 2M4V and its derivatives in the preparation of preparations that inhibit osteoclast maturation and differentiation, wherein the in vivo drug concentration of 2M4V and its derivatives is 1-10 mg / kg.

[0022] In particular, 2M4V, by strictly targeting and activating NSTN, significantly improved bone loss in ovariectomized mice.

[0023] This invention provides the application of 2-methoxy-4-vinylphenol (2M4V) and its derivatives as agonists of Nicastrin (NCSTN), a novel target for regulating bone metabolism, in the preparation of drugs for the prevention and / or treatment of bone metabolic diseases. The derivatives include, but are not limited to, compounds with structures as shown in general formula I: 4-methylguaiacol (derivative 2), 4-aminomethylguaiacol (derivative 3), and vanillin acetal (derivative 4). This invention discloses for the first time NCSTN as a novel target for the treatment of bone metabolic diseases, demonstrating that knockdown of NCSTN expression exacerbates bone loss, while upregulation of NCSTN significantly promotes bone formation. In vivo experiments demonstrate that 2M4V precisely targets and agonizes NCSTN, effectively regulating bone homeostasis and improving bone mineral density and bone microstructure in ovariectomized mice. This invention not only expands the novel applications of NCSTN and its targeting agonist 2M4V derivatives in the treatment of bone metabolic diseases but also provides a breakthrough drug development strategy for the prevention and / or treatment of osteoporosis.

[0024] In addition, the present invention also provides reagents for detecting NCSTN and their applications.

[0025] Application of reagents for detecting NCSTN in the preparation of products for diagnosing bone metabolic diseases.

[0026] The reagents are primers for specifically amplifying NCSTN and / or antibody probes for specifically recognizing NCSTN; the products are kits, chips, or nucleic acid membrane strips.

[0027] The kit contains a monoclonal or polyclonal antibody against the NCSTN protein.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention is the first to discover a novel target for regulating bone metabolism, NCSTN, and its application as a target in the development, design, and screening of products for the prevention and / or treatment of bone metabolism. The mechanism of NCSTN agonists in treating osteoporosis involves inhibiting osteoclast maturation and differentiation while simultaneously promoting osteoblast bone formation. This differs significantly from previously reported mechanisms that either solely inhibit bone resorption or solely promote bone formation. This simultaneous action on both bone resorption and bone formation in treating osteoporosis greatly reduces adverse drug reactions in the body. Attached Figure Description

[0030] Figure 1 The effects of knockdown / overexpression of NSTN on osteoblast function in vitro were investigated. In this study, a represents the osteoblast proliferation rate in Example 1, b represents the formation and activity level of alkaline phosphatase (ALP) in osteoblasts in Example 2, and c represents the level of bone mineralization deposition in osteoblasts in Example 3.

[0031] Figure 2 The effects of knockdown / overexpression of NSTN on osteoclast function in vitro were investigated. In this study, a represents osteoclast proliferation rate in Example 4, b represents the osteoclast tartrate-resistant acid phosphatase (TRAP) formation level in Example 5, c represents osteoclast bone resorption level in Example 6, and d represents osteoclast peripheral cytoskeletal protein (F-actin) formation level in Example 7.

[0032] Figure 3 This section describes the effect of in vivo knockdown / overexpression of NSTN on mouse bone morphology in Example 8. Image a shows a micro-CT image of the femur of mice 6 weeks after modeling, and image b shows the quantitative results of bone mineral density (BMD), bone volume / tissue volume (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone separation (Tb.Sp).

[0033] Figure 4 For the affinity study of NSTN and 2M4V. a) Molecular docking in Example 9. b) Molecular dynamics simulation in Example 10. c) Surface plasmon resonance (SPR) of NSTN and 2M4V in Example 11. d) Cellular thermal transfer (CETSA) of NSTN and 2M4V in Example 12.

[0034] Figure 5 Figure a shows the chemical structures of 2M4V and its three derivatives 2, 3, and 4, where compound 1 is 2M4V, compound 2 is 4-methylguaiacol (derivative 2), compound 3 is 4-aminomethylguaiacol (derivative 3), and compound 4 is vanillin acetal (derivative 4). Figure b shows the osteoblast proliferation activity levels of osteoblasts in Example 13 after treatment with or without a specified concentration of 2M4V and its derivatives for 48 hours.

[0035] Figure 6 The changes in alkaline phosphatase expression levels in osteoblasts of Example 14 after 7 days of treatment with or without a specified concentration of 2M4V and its derivatives under the intervention of osteogenic induction solution.

[0036] Figure 7 In Example 15, osteoblasts were treated with osteogenic induction solution, with or without specified concentrations of 2M4V and its derivatives 2, 3, and 4 for 7 days. The alkaline phosphatase activity of the osteoblasts was then detected using an ALP activity kit.

[0037] Figure 8 For example, osteoblasts were treated with osteogenic induction solution with or without specified concentrations of 2M4V and its derivatives 2, 3, and 4 for 21 days. The level of osteoblast bone mineralization nodule formation was detected by alizarin red staining.

[0038] Figure 9 The osteoclast proliferation activity level of RAW264.7 cells in Example 17 after treatment with osteoclast induction solution, with or without specified concentrations of 2M4V and its derivatives 2, 3, and 4 for 48 h.

[0039] Figure 10 RAW264.7 cells from Example 18 were treated with osteoclast-inducing solution, with or without specified concentrations of 2M4V and its derivatives 2, 3, and 4, for 7 days. The TRAP formation level of osteoclasts was detected by TRAP staining.

[0040] Figure 11 RAW264.7 cells from Example 19 were treated with osteoclast-inducing solution, with or without specified concentrations of 2M4V and its derivatives 2, 3, and 4, for 7 days. The TRAP activity level of osteoclast cells was detected by TRAP activity assay.

[0041] Figure 12 The F-actin ring formation level of RAW264.7 cells in Example 20 after 7 days of treatment with or without specified concentrations of 2M4V and its derivatives 2, 3, and 4 under osteoclast induction solution intervention.

[0042] Figure 13 The results of Example 21 are shown in Figure a. a is a micro-CT image of the femur of OVX mice treated with 2M4V and OVX+siNCSTN mice after 6 weeks. b is the result of BMD, BV / TV, Tb.N, Tb.Th and Tb.Sp.

[0043] Appendix Figure 5-12 The numbers in the table are: 1 for 2M4V, 2 for 4-methylguaiacol, 3 for 4-aminomethylguaiacol, and 4 for vanillin acetal. Detailed Implementation

[0044] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0045] Example 1: Experiment on the effect of knockdown or overexpression of NSTN on osteoblast proliferation activity

[0046] Mouse embryonic osteoblast precursor cells MC3T3-E1 were treated for 24 h with NSTDN siRNA reagent (SEQ ID NO:1 GCATCAACCCAGAAATCGT) and lentiviral overexpression transfection reagent, respectively, to obtain NSTDN knockdown (siNCSTN) and OE-NCSTN overexpression (OE-NCSTN) osteoblasts. The proliferation activity of MC3T3-E1 cells with siNCSTN and OE-NCSTN was measured using a CCK-8 assay kit according to the instructions. After modeling, MC3T3-E1 cells were cultured at a rate of 1 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured in α-complete medium (blank α-medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 5% CO2 and 37°C for 24 hours. After treating cells with 2M4V and its derivatives 2, 3, and 4 at different concentrations (0.01, 0.1, 1, and 10 μM) for 48 hours, blank α-medium containing 10% CCK-8 reagent was added to each well, and the plates were incubated at 37°C for another 30 minutes. The optical density (OD value) at 450 nm was then measured using a microplate reader.

[0047] The results are as follows Figure 1 As shown in a, siNCSTN interference reduced NSTN expression and inhibited osteoblast proliferation, while NSTN overexpression treatment increased osteoblast proliferation.

[0048] Example 2: Effects of NSTN knockdown or overexpression on osteoblast alkaline phosphatase expression and activity levels.

[0049] Osteoblasts of siNCSTN / OE-NCTN modeled in Example 1 were 1 × 10⁻⁶ 4Cells were seeded at a density of 100 cells / well in 96-well plates and induced for 7 days in osteogenic induction medium (α-complete medium + 0.01 M β-glycerophosphate sodium + 0.01 μM dexamethasone + 50 μg / mL ascorbic acid). The medium was changed after 3 days of culture. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and stained with alkaline phosphatase staining reagent for 30 min. The blue-purple areas of MC3T3-E1 cells were observed under a microscope. Osteoblasts modeled as siNCSTN / OE-NCTN in Example 1 were seeded at a density of 5 × 10⁶ cells / well. 4 Cells were seeded at a density of 100 cells / well in 48-well plates and induced with osteogenic induction medium for 7 days, followed by medium change after 3 days of culture. Cells were washed with PBS, lysed on ice, and the supernatant was collected. ALP activity was detected using an alkaline phosphatase activity assay kit, and the 405 nm OD value was measured using a microplate reader.

[0050] The results are as follows Figure 1 As shown in b, siNCSTN interference reduced NSTN expression and inhibited ALP expression and activity levels in osteoblasts, while NSTN overexpression treatment increased ALP expression and activity levels in osteoblasts.

[0051] Example 3: Effects of NSTN knockdown or overexpression on osteoblast bone mineralization deposition.

[0052] Osteoblasts of siNCSTN / OE-NCTN modeled in Example 1 were 1 × 10⁻⁶ 4 Cells were seeded at a density of 100 cells / well in 96-well plates and induced in osteogenic induction medium for 21 days, with medium changes every 3 days. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, washed 3 times with PBS, stained with alizarin red staining reagent for 2 hours, and observed under a microscope.

[0053] The results are as follows Figure 1 As shown in c, siNCSTN interference reduced NSTN expression and inhibited osteoblast bone mineralization deposition, while NSTN overexpression treatment increased osteoblast bone mineralization deposition.

[0054] Example 4: Experiment on the effect of knockdown or overexpression of NSTN on osteoclast proliferation activity

[0055] RAW264.7 cells were treated with NSTN's siRNA reagent and lentivirus overexpression transfection reagent for 24 hours according to the instructions. RAW264.7 cells were transfected at a concentration of 1 × 10⁻⁶ cells / cells. 4Cells were seeded at a density of 10 cells / well in 96-well plates and induced for 5 days at 37°C in α-complete medium containing 50 ng / mL receptor activator of nuclear factor-κB ligand (RANKL) and 25 ng / mL macrophage colony stimulation factor (M-CSF). After 2 days of culture, the medium was changed to obtain NCSTN knockdown (siNCSTN) and OE-NCSTN overexpression (OE-NCSTN) osteoclasts. On day 6, 10 μL of CCK-8 reagent was added to each well, and osteoclast proliferation activity was measured using the CCK-8 kit according to the manufacturer's instructions. The cells were then incubated at 37°C for another 30 minutes. The OD value was measured at 450 nm using a microplate reader.

[0056] The results are as follows Figure 2 As shown in a, siNCSTN interference reduced NSTN expression, which increased osteoclast proliferation activity, while NSTN overexpression treatment inhibited osteoclast proliferation activity.

[0057] Example 5: Experiment on the effect of knockdown or overexpression of NSTN on the expression level of tartrate-resistant acid phosphatase in osteoclasts.

[0058] The RAW264.7 of the siNCSTN / OE-NCTN model, as described in Example 4, was 1 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and induced to undergo osteoclastosis for 5 days. After 2 days of culture, the medium was changed. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 10% Triton X-100 for 5 min, washed 3 times with PBS, stained with TRAP staining reagent for 30 min, counterstained with hematoxylin, and observed under a microscope.

[0059] The results are as follows Figure 2 As shown in b, siNCSTN interference reduced NSTN expression and promoted TRAP production in osteoclasts, while NSTN overexpression treatment inhibited TRAP production in osteoclasts.

[0060] Example 6: Experiment on the effect of knockdown or overexpression of NSTN on osteoclast bone resorption capacity

[0061] Bovine cortical bone slices soaked in 75% ethanol were removed and immersed in PBS containing penicillin and antibiotics, with the medium changed three times for 10 minutes each time. The bone slices were then removed and irradiated under a UV lamp for 1 hour on each side. They were then transferred to a 96-well plate and subjected to RAW264.7 microarrays (sim NCT / OE-NCTN model) at 1 × 10⁻⁶. 4Bone sections were seeded at a density of 10 cells / well in 96-well plates and cultured in osteoclast-inducing medium for 9 days. The sections were then collected, washed twice with PBS, fixed with 2.5% glutaraldehyde for 7 min, washed three times with 0.25M ammonia for 5 min each time, and air-dried. They were then stained with 1% toluidine blue for 10 min, rinsed with distilled water, observed under a microscope, and photographed.

[0062] The results are as follows Figure 2 As shown in c, siNCSTN interference reduced NSTN expression, which promoted osteoclast bone resorption, while NSTN overexpression treatment inhibited osteoclast bone resorption.

[0063] Example 7: Knockdown or overexpression of NSTN on F-actin ring staining in osteoclasts

[0064] RAW264.7 of siNCSTN / OE-NCTN, modeled in Example 4, was obtained at 5×10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 48-well plates, induced with osteoclast-inducing medium for 5 days, and cultured for 2 days before changing the medium. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 10% Triton X-100 for 5 min. FITC-labeled phalloidin was stained with F-actin and incubated at 37°C for 40 min, followed by washing with PBS. Cell nuclei were stained with DAPI for 5 min, followed by washing with PBS. Laser confocal microscopy was used for detection.

[0065] The results are as follows Figure 2 As shown in d, siNCSTN interference reduced NSTN expression, which promoted the formation of F-actin loops in osteoclasts, while NSTN overexpression treatment inhibited the formation of F-actin loops in osteoclasts.

[0066] Example 8: Effects of NSTN knockdown or overexpression on mouse bone tissue morphology

[0067] Following the instructions, NSTTN knockdown (siNCSTN) and NSTTN overexpression (OE-NCSTN) mice were constructed from normal 8-week-old C57 / BL6 mice by intravenous injection of siNCSTN or NSTTN overexpression plasmid transfected with lentivirus. Mice in each group were sacrificed after 6 weeks of normal feeding. Femurs were collected for micro-CT analysis. Figure 3 In the image, 'a' represents a micro-CT image of the femur of a mouse that has been modeled for 6 weeks.

[0068] The results are as follows Figure 3As shown in b, compared with the normal control group, the siNCSTN group mice showed significantly decreased bone mineral density (BMD), bone volume / tissue volume (BV / TV), trabecular bone number (Tb.N), and trabecular cortical thickness (Tb.Th), while the OE-NCSTN group mice showed significantly reduced trabecular bone separation and significantly increased other bone tissue parameters. This indicates that NCSTN is an important regulatory protein for bone metabolism in vivo.

[0069] Example 9: NCSTN Molecular Docking Experiment on 2M4V

[0070] The NCSTN (4R12) protein structure was obtained from the PDB database, and the compound structure in 2M4Vmol2 format was obtained from the TCMSP database. Molecular docking was performed using the Glide module in Schrödinger and then visualized.

[0071] The results are as follows Figure 4 As shown in a, NCSTN and 2M4V have a good space pocket, and their binding energy of -7.2 kJ / mol indicates that they have good affinity.

[0072] Example 10: NCSTN simulation experiment on 2M4V molecular dynamics

[0073] Molecular dynamics simulations were performed using Amber12. First, the topology and coordinate files for NCSTN were generated using the Tleap function in the Leap program. Then, the parameters of the corresponding biomolecules were calculated using different force fields. The complex was enclosed and filled using the TIP3P water model (water boundary box set to X = 10 Å, Y = 10 Å, Z = 10 Å). Counterions were added to neutralize the complex system, and the energy was optimized using the Sandel program. The molecular dynamics simulation run time was set to 10 ns, the recording interval to 50 ps, ​​and the energy to 1.2 ps. The results were analyzed based on the root mean square deviation (RMSD).

[0074] The results are as follows Figure 4 As shown in b, the RMSD of NSTN and 2M4V is < 3Å, indicating that they have good dynamic binding stability.

[0075] Example 11 NCSTN and 2M4VSPR Experiment

[0076] The binding affinity of NCSTN to 2M4V was determined using a BIAcore T200. Active NCSTN protein was dissolved in sodium acetate solution to a final concentration of 50 μg / mL, and then immobilized on a COOH sensor chip (GE) via amine coupling to achieve a target density of 9100 RU. The running buffer was PBS containing 5% DMSO. Six different concentrations of 2M4V were injected at a flow rate of 30 μL / min at 25°C, with contact and dissociation times of 60 seconds each. The sensor plot was analyzed using IgorProversion 6.1 (WaveMatrix). The KD affinity constant was calculated using kinetic analysis or steady-state affinity methods.

[0077] The results are as follows Figure 4 As shown in c, the KD value of NCSTN and 2M4V is 3.55 μM, indicating that they have good binding affinity.

[0078] Example 12: CETSA Experiment with NCSTN and 2M 4V

[0079] Osteoblasts were collected, washed with PBS, and then lysed in lysis buffer (containing 20 mM HEPES, 138 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) through three freeze-thaw cycles in liquid nitrogen. The lysates were centrifuged at 25,000 × g at 4°C for 20 min to separate the soluble protein fraction from the cell debris. After determining the total protein concentration, the soluble fraction was diluted to a final concentration of 4 mg / mL and divided into two equal aliquots. Each aliquot was incubated for 10 min at room temperature with either 100 μM 2M4V or 0.5% DMSO (control group). The incorporated lysis buffer was divided into 10 aliquots and heat-treated at different temperatures (45, 49, 53, 57, 61, 65, 76, 73, 77, 81°C) for 3 min, followed by cooling at 4°C for 3 min. The lysis buffer was centrifuged at 25,000 g at 4°C for 20 minutes to remove denatured proteins, and the supernatant proteins were used for Western blot analysis. The obtained protein samples were processed by sodium dodecyl sulfate gel electrophoresis and transferred to a PDVF membrane. The membrane was then incubated with 0.5% BSA solution for 1 hour, followed by overnight incubation with NCSTN primary antibody at 4°C. After incubation with secondary antibody, the protein bands were visualized using an ECL detection kit.

[0080] The results are as follows Figure 4As shown in d, compared with the DMSO group, the thermostability of NSTN was significantly increased after incubation of cell lysate with 2M4V, indicating that the two bound together.

[0081] Example 13 Experiment on the proliferative activity of vinyl guaiacol derivatives against osteoblasts

[0082] According to the instructions for use, the proliferation activity of mouse embryonic osteoblast precursor cells MC3T3-E1 was measured using a CCK-8 assay kit. MC3T3-E1 cells were proliferated at a rate of 1 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured in α-complete medium (blank α-medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 5% CO2 and 37°C for 24 hours. Cells were cultured without or with different concentrations (0.01, 0.1, 1, and 10 μM) of 2M4V and its derivatives 2, 3, and 4. Figure 5 (a) After treating the cells for 48 hours, add α-blank medium containing 10% CCK-8 reagent to each well and incubate at 37°C for another half hour. Then measure the optical density (OD value) at 450 nm using a microplate reader.

[0083] The results are as follows Figure 5 As shown in b, 2M4V and its derivatives 2, 3, and 4 can increase the proliferation activity of MC3T3-E1 in a dose-dependent manner.

[0084] Example 14 Experiment on the effect of vinyl guaiacol derivatives on osteoblast alkaline phosphatase expression levels

[0085] MC3T3-E1 with 1× 10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and induced for 7 days with different concentrations (0.01, 0.1, 1 μM) of 2M4V and its derivatives and osteogenic induction medium (α-complete medium + 0.01 M β-glycerophosphate sodium + 0.01 μM dexamethasone + 50 μg / mL ascorbic acid). After 3 days of culture, the medium was changed. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, stained with alkaline phosphatase staining reagent for 30 min, and the blue-purple areas of MC3T3-E1 cells were observed under a microscope.

[0086] The results are as follows Figure 6 As shown, treatment with 2M4V and its derivatives 2, 3, and 4 resulted in a dose-dependent increase in ALP expression levels in MC3T3-E1 cells.

[0087] Example 15: Experiment on the activity of vinyl guaiacol derivatives on osteoblast alkaline phosphatase

[0088] MC3T3-E1 with 5×10 4Cells were seeded at a density of 100 cells / well in 48-well plates and induced for 7 days with 2M4V and its derivatives at different concentrations (0.01, 0.1, 1 μM) and osteogenic induction medium. After 3 days of culture, the medium was changed. Cells were washed with PBS, lysed on ice, and the supernatant was collected. ALP activity was detected using an alkaline phosphatase activity kit, and the OD value at 405 nm was measured using a microplate reader.

[0089] The results are as follows Figure 7 As shown, treatment with 2M4V and its derivatives 2, 3, and 4 resulted in a dose-dependent increase in ALP activity in MC3T3-E1 cells.

[0090] Example 16: Alizarin Red staining experiment on osteoblasts using vinyl guaiacol derivatives

[0091] MC3T3-E1 with 1× 10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and induced for 21 days with different concentrations (0.01, 0.1, 1 μM) of 2M4V and its derivatives and osteogenic induction solution, with medium changes every 3 days. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, washed 3 times with PBS, stained with alizarin red staining reagent for 2 hours, and observed under a microscope.

[0092] The results are as follows Figure 8 As shown, treatment with 2M4V and its derivatives 2, 3, and 4 resulted in a dependent increase in calcium salt deposition in MC3T3-E1 cells.

[0093] Example 17 Experiment on the proliferative activity of vinyl guaiacol derivatives against osteoclasts

[0094] Osteoclast proliferation activity was determined using the CCK-8 assay kit according to the instructions for use. RAW264.7 cells were inoculated at 1×10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and induced for 5 days at 37°C in α-complete medium containing 50 ng / mL receptor activator of nuclear factor-κB ligand (RANKL) and 25 ng / mL macrophage colony stimulation factor (M-CSF). The medium was changed after 2 days of culture. On day 6, the cells were treated with 2M4V and its derivatives at different concentrations (0.01, 0.1, 1, and 10 μM) for 48 hours, followed by the addition of 10 μL of CCK-8 reagent to each well and incubation at 37°C for another 30 minutes. OD values ​​were measured at 450 nm using a microplate reader.

[0095] The results are as follows Figure 9 As shown, 2M4V and its derivatives 2, 3, and 4 can reduce the proliferative activity of RAW264.7-induced osteoclasts in a dose-dependent manner.

[0096] Example 18 Experiment on the expression level of tartrate-resistant acid phosphatase in osteoclasts by vinyl guaiacol derivatives

[0097] RAW264.7 at 1× 10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and induced for 5 days with 2M4V and its derivatives at different concentrations (0.01, 0.1, 1 μM) for 2, 3, and 4, as well as osteoclast induction. The medium was changed after 2 days of culture. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 10% Triton X-100 for 5 min, washed three times with PBS, stained with acid phosphatase staining reagent for 30 min, counterstained with hematoxylin, and observed under a microscope.

[0098] The results are as follows Figure 10 As shown, treatment with 2M4V and its derivatives 2, 3, and 4 resulted in a dose-dependent decrease in TRAP expression levels in osteoclasts.

[0099] Example 19: Experiment on the activity of vinyl guaiacol derivatives against tartrate acid phosphatase in osteoclasts

[0100] RAW264.7 with 5 × 10 4 Cells were seeded at a density of 100 cells / well in 48-well plates and induced for 5 days with 2M4V and its derivatives at different concentrations (0.01, 0.1, 1 μM) and osteoclast induction. The medium was changed after 2 days of culture. Cells were washed with PBS, lysed on ice, and the supernatant was collected. ALP activity was detected using an alkaline phosphatase activity assay kit, and the OD value at 405 nm was measured using a microplate reader.

[0101] The results are as follows Figure 11 As shown, treatment with 2M4V and its derivatives 2, 3, and 4 resulted in a dose-dependent decrease in osteoclast TRAP activity.

[0102] Example 20: Vinyl guaiacol derivative F-actin ring staining experiment on osteoclasts

[0103] RAW264.7 with 5×10 4Cells were seeded at a density of 100 cells / well in 48-well plates and induced for 5 days with osteoclast induction using 2M4V and its derivatives at different concentrations (0.01, 0.1, 1 μM). The medium was changed after 2 days of culture. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 10% Triton X-100 for 5 min. FITC-labeled phalloidin was stained for F-actin and incubated at 37°C for 40 min, followed by washing with PBS. Cell nuclei were stained with DAPI for 5 min, followed by washing with PBS. Laser confocal microscopy was used for detection.

[0104] The results are as follows Figure 12 As shown, treatment with 2M4V and its derivatives resulted in a dose-dependent decrease in F-actin thickness in osteoclasts.

[0105] Example 21: Therapeutic effect of vinyl guaiacol-targeted NSTN on postmenopausal osteoporosis

[0106] Except for the sham group, which had a small amount of periovarian adipose tissue removed, all other groups underwent bilateral ovariectomy in mice to simulate postmenopausal osteoporosis in women. Two groups of OVX mice were randomly selected for siNCSTN modeling as described in Example 8. Ovariectomized mice and one group of ovariectomized mice with reduced NSTN expression were treated daily with 2M4V (10 mg / kg), while the other groups were administered the same volume of physiological saline by gavage. After 6 weeks, the mice were sacrificed, and their femurs were analyzed. The results are as follows: Figure 13 .

[0107] Micro-CT analysis of mouse femurs revealed that, 6 weeks after ovariectomy, the OVX model group showed significant decreases in BMD, BV / TV, Tb.N, and trabecular cortical thickness (Tb.Th) compared to the sham group, indicating successful model establishment. Furthermore, 2M4V treatment significantly improved these indicators, suggesting that 2M4V can improve postmenopausal osteoporosis in vivo. Importantly, the therapeutic effect of 2M4V disappeared when siNCSTN expression was inhibited, indicating that 2M4V exerts its therapeutic effect through strict targeting of the NSTN.

[0108] In summary, this invention provides a novel target for regulating bone metabolism, the NCSTN, and its tightly targeted agonist, vinylguaiacol. This invention has further research value and broad development prospects.

[0109] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. The use of an NCSTN agonist in the preparation of a medicament for the prevention and / or treatment of bone metabolic diseases, wherein the NCSTN agonist is 2M4V with the structural formula shown in Compound 1, and the bone metabolic disease is postmenopausal osteoporosis. 。 2. The application according to claim 1, characterized in that: The drug has the effect of inhibiting osteoclast maturation and differentiation while promoting osteoblast bone formation.

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