Application of mace lignan in the preparation of targeted therapeutic drugs for osteoporosis

Abstract

The present invention discloses the use of mace lignans in the preparation of targeted therapeutic drugs for osteoporosis. Through a series of in vitro experimental studies, it was found that mace lignans inhibited the formation and bone resorption function of osteoclasts in a dose-dependent and time-dependent manner. At the protein level, it was found that mace lignans inhibited the expression of ERK and NFATc1 induced by RANKL to inhibit osteoclasts. At the same time, the effect of mace lignans on osteoclasts was further verified in vivo using an ovariectomized osteoporosis model. The results showed that mace lignans significantly reduced the level of bone loss in mice. Based on this, mace lignans have great potential in the targeted treatment of osteoporosis. Therefore, it is expected that the development of mace lignans as a natural product will provide new treatment options for osteoporosis and other bone loss-related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical application of myrislignan (MRL), and in particular relates to the application of myrislignan in the preparation of targeted therapeutic drugs for osteoporosis. Background Art

[0002] With the accelerated aging of my country's population, the incidence of osteoporosis is on the rise. As the most common bone disease, osteoporosis has gradually been recognized as a major global public health problem, especially affecting many postmenopausal women. Osteoporosis is a systemic bone disease whose main pathological characteristics are reduced bone mass, damaged bone microstructure, increased bone fragility, and susceptibility to fractures. Fractures caused by osteoporosis often occur in the vertebra, hip joint, distal forearm and proximal humerus, and are the main cause of disability and death in patients.

[0003] Bone health is strictly regulated by the body, and the development of osteoporosis primarily stems from the disruption of bone homeostasis, which relies on the balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Osteoclasts are the only specialized multinuclear cells in the body derived from mononuclear macrophages. They can be activated by various pathological factors (such as chronic medication, immobilization, malnutrition, and chronic inflammation), accelerating bone resorption and leading to a range of osteoporosis and related diseases, including Alzheimer's disease, diabetes, and cancer. Therefore, inhibiting the formation and function of osteoclasts is a key strategy for the treatment of osteoporosis.

[0004] Primary bone marrow-derived macrophages fuse with mononuclear osteoclast precursors to form multinucleated osteoclasts. Osteoclast precursors express two essential receptors on their surface: colony-stimulating factor 1 receptor (c-Fms) and receptor activator of nuclear factor κB (RANK). Binding of c-Fms to macrophage colony-stimulating factor (M-CSF) promotes the subsequent survival and proliferation of osteoclast precursors and osteoclasts, while the induction of osteoclast differentiation is associated with the binding of RANK to receptor activator of nuclear factor κB ligand (RANKL). The RANKL / RANK signaling pathway then leads to the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6), inducing the activation of downstream pathways such as the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways, which are classical pathways associated with osteoclast differentiation. This in turn activates the expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), a key transcription factor controlling osteoclastogenesis. Finally, the transcription factor NFATc1 induces the expression of osteoclast-related protein genes, including tartrate-resistant acid phosphatase (TRAP), cathepsin (CTSK), ATPase H +It transports V0 subunit d2 (Atp6v0d2) and dendritic cell-specific transmembrane protein (Dcstamp), which promote osteoclast formation and lead to bone resorption.

[0005] Currently, there are multiple methods for treating osteoporosis targeting the above-mentioned mechanisms, with drug therapy being the most common. Drug treatments include basic therapies such as calcium and vitamin D supplements, osteoclast inhibition such as bisphosphonates and zoledronic acid, bone resorption inhibition such as calcitonin, and estrogen replacement therapy. However, all of these drug therapies have varying degrees of side effects, including gastric discomfort, constipation, duodenal ulcers, reflux esophagitis, and breast cancer. Therefore, there is an urgent need to develop new anti-osteoporosis drugs with minimal side effects and that can effectively inhibit osteoclast activity.

[0006] Compounds extracted from animals, plants, and microorganisms are defined as natural compounds, most of which have pharmacological or biological activities and are becoming an important source of inspiration for the development of various potential new drugs. Due to the beneficial properties of active metabolites in natural compounds, their application in a range of diseases such as osteoporosis is expanding. Myrislignan (MRL), a natural product of the aromatic evergreen tree Myristica fragrans, has a wide range of pharmacological activities. Previous studies have shown that myrislignan induces surface contraction and mitochondrial damage in Toxoplasma gondii, leading to its programmed cell death; can inhibit inflammation, inhibit cancer, and suppress the production of nitric oxide; can treat asthma, toothache, relieve rheumatic pain and infection; and has liver protection, lipid-lowering, and anti-lung cancer effects. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an application of mace lignan in the preparation of osteoporosis targeted therapeutic drugs.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] Application of mace lignan in the preparation of medicines for treating diseases caused by excessive osteoclast activation.

[0010] Related diseases are diseases related to bone loss.

[0011] A disease associated with decreased bone mass is osteoporosis.

[0012] Osteoclast overactivity is caused by estrogen depletion.

[0013] Estrogen deficiency results from ovarian removal.

[0014] The medicine is a preparation composed of mace lignan as an active ingredient and a pharmaceutical carrier.

[0015] The preparation is in the form of oral dosage form, intravenous dosage form, intramuscular dosage form, suppository or external dosage form.

[0016] Oral dosage forms are capsules, pills or tablets.

[0017] Treatment is achieved by inhibiting the formation of osteoclasts.

[0018] The inhibition of osteoclast formation was achieved by downregulating RANKL-induced ERK and NFATc1 expression.

[0019] Excessive activation of osteoclasts is the main cause of bone loss in osteoporosis. At present, the inhibitory effect of mace lignans (hereinafter referred to as MRL) on osteoclasts has not been studied. The inventors first found through a series of in vitro experimental studies that MRL inhibits the formation and bone resorption function of osteoclasts in a dose-dependent and time-dependent manner, and found at the protein level that MRL inhibits the expression of ERK and NFATc1 induced by RANKL to inhibit the effect of osteoclasts. At the same time, by establishing an ovariectomized osteoporosis model in mice, the inventors further verified the effect of MRL on osteoclasts in vivo. The results showed that compared with the ovariectomy group, MRL significantly reduced the level of bone loss in mice, which was consistent with the results of in vitro experiments. In summary, MRL inhibits the formation and function of osteoclasts by downregulating the expression of ERK and NFATc1 induced by RANKL. Based on this, mace lignans have great potential in the targeted treatment of bone loss-related diseases caused by excessive osteoclast activation, especially osteoporosis. Moreover, there are no major adverse events during MRL drug treatment. Therefore, it is expected that drugs with new indications for MRL can be developed to provide new treatment options for osteoporosis and other bone loss-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figures 1 and 2 show the results of a study on the effect of MRL on the inhibition of RANKL-induced osteoclastogenesis in vitro. In the figure: Figure A shows the chemical structure and molecular formula of MRL; Figure B shows the proliferation of BMMs cells detected by CCK-8 assay after treatment with different concentrations of MRL for 48 and 96 hours; Figures C and D show representative images and quantitative analysis of the inhibition of osteoclast differentiation by MRL; Figures E and F show representative images and quantitative analysis of the inhibition of actin ring formation by MRL.

[0021] Figure 2 These are the research results on the effect of MRL on the bone resorption function of mature osteoclasts in vitro. In the figures: Figures A and B show representative images and quantitative analysis of MRL inhibiting osteoclast differentiation at different stages; Figure C shows the situation of MRL inhibiting osteoclast-related genes; Figures D to F show representative images and quantitative analysis of MRL inhibiting the bone resorption function of osteoclasts.

[0022] Figure 3 These are the research results of MRL inhibiting RANKL-induced NFATc1 expression in vitro. In the figure: Figure A shows the inhibition of osteoclast-related genes by different concentrations of MRL; Figures B to F show the inhibition of osteoclast-related protein expression by MRL and quantitative analysis.

[0023] Figure 4 These are the results of MRL inhibiting the occurrence of osteoclasts by reducing the phosphorylation of ERK 1 / 2 proteins. In the figure: Figure A shows that MRL inhibits the expression of osteoclast-related upstream proteins, including ERK, JNK, P38, P65 and IκBα; Figures B to F show the quantitative graphs of MRL inhibiting the expression of the above proteins.

[0024] Figure 5 Figure 3 is a graph showing the research results that LM22B-10 promotes the phosphorylation of ERK 1 / 2 proteins, while mace lignan inhibits this effect. In the graph: Figure A shows that after BMMs were stimulated with different concentrations of the ERK 1 / 2 agonist LM22B-10 for 48 hours, the cell proliferation was detected by the CCK-8 method; Figure B shows that after BMMs were stimulated with LM22B-10 and MRL for 48 hours, the cell proliferation was detected by the CCK-8 method; Figures C and D show representative images and quantitative analysis of the effects of MRL and LM22B-10 on osteoclast differentiation; Figures E to G show the effects of MRL and LM22B-10 on the expression of osteoclast-related genes, including Fos, Ctsk, and Atp6v0d2.

[0025] Figure 6 These are the research results showing that mace lignans inhibit osteoclast function by targeting ERK 1 / 2 protein expression in vitro. In the figures: Figures A and B show the effects and quantitative analysis of MRL and LM22B-10 on the osteoclast-related upstream protein ERK; Figure C shows the effects of MRL and LM22B-10 on osteoclast-related downstream proteins, including c-FOS, NFATc1, CTSK, and Atp6v0d2; Figures D to G show the quantitative graphs of the expression of related downstream proteins.

[0026] Figure 7 These are the results of a study on the effect of mace lignans on the inhibition of osteoporosis in ovariectomized mice in vivo. Figure A shows a three-dimensional reconstruction of representative images of the tibiae from the different treatment groups; Figures B to E show quantitative analysis of bone tissue parameters, including the ratio of bone volume to total volume, the number of trabeculae, the degree of trabecular separation, and the trabecular thickness.

[0027] Figure 8 It is the chemical structure and molecular formula diagram of MRL. DETAILED DESCRIPTION

[0028] 1. Materials and Methods

[0029] 1) Reagents

[0030] MRL was purchased from CHENGDU MUST BIO-TRCHNOLOGY CO., LTD (Chengdu, Sichuan, China) with a purity of 99.24% and a molecular weight of 374.43. MRL was dissolved in dimethyl sulfoxide (DMSO) to make a 100 mM stock solution. MRL was then diluted to a 1 mM working solution with minimal essential medium α-MEM before subsequent experiments. LM22B-10 (LM) was purchased from MedChemExpress (MCE, Shanghai, China) with a purity of 99.72% and a molecular weight of 485.01. LM was dissolved in DMSO to make a 50 mM stock solution, and then diluted to a 500 μM working solution with α-MEM. In cell culture, α-MEM and fetal bovine serum (FBS) were from Gibco-Technology (Thermo Fisher Institute of Biotechnology, MD, United States). Recombinant mouse M-CSF and recombinant mouse RANKL were provided by R&D Biotechnology Company (Minneapolis, MN, United States), and penicillin / streptomycin was provided by Thermo Fisher Scientific (Scoresby, Victoria, Australia). Cell counting kit (CCK-8) and estrogen (E2) were provided by MedChemExpress (MCE, Shanghai, China). Rever Aid RT Kit was from Thermo Fisher Scientific (Scoresby, Victoria, Australia). Rhodamine-labeled phalloidin and 4'6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (St. Louis, MO, USA). Western blotting protein gradient marker was from Thermo Fisher Scientific (Thermo Fisher Scientific, Shanghai, China). Primary antibodies against NFATc1 (#sc-7294) and CTSK (#sc-48353) were from Santa Cruz Biotechnology (Dallas, CA, United States), and antibodies against Atp6v0d2 (#ab236375) and c-FOS (#ab134122) were from Abcam (Cambridge, England).Using CellSignaling Technology (Danvers, MA, United States), the inventors obtained primary antibodies (mouse and rabbit) and secondary antibodies (mouse and rabbit) against phospho-NF-κB p65 (#3033), NF-κB p65 (#8242), IκBα (#4814), β-actin (#4970), p-ERK 1 / 2 (#4370), ERK 1 / 2 (#4695), p-JNK 1 / 2 (#4668), JNK 1 / 2 (#9252), p-P38 (#4511), and P38 (#8690).

[0031] 2) In vitro extraction and culture of primary bone marrow-derived macrophages (BMMs)

[0032] C57BL / 6J mice were obtained from the Animal Center of Guangxi Medical University (Nanning, Guangxi, China). First, the femurs and tibias of 4-6-week-old C57BL / 6J mice were separated. The femurs and tibias were then stripped of muscle and dissected to completely expose the bone marrow cavity. The bone marrow cavity was flushed with α-MEM until it turned white. The fluid was transferred to a centrifuge tube using a strainer to filter out impurities such as muscle. The fluid in the centrifuge tube was thoroughly mixed and resuspended in a T-75 cell culture flask containing complete medium consisting of 10% fetal bovine serum, 25 ng / ml M-CSF, and 1% penicillin-streptomycin. The cells were cultured at 37°C, 95% O₂, and 5% CO₂ for 48 hours. The flask was then rinsed with phosphate-buffered saline (PBS) and replaced with fresh complete medium. The next day, the adherent cells in the cell culture flask were ready for use. The medium in the cell culture flask was discarded and the flask was rinsed twice with PBS. 3 ml of digestive enzyme was added to the flask and incubated in a CO₂ incubator for 3 minutes. Then, 4 ml of complete culture medium was added to the bottle to terminate the digestion, the cells were scraped off, centrifuged, and transferred to a centrifuge tube and mixed with fresh complete culture medium to form a cell mother solution for use.

[0033] 3) Cytotoxicity assay

[0034] BMMs were placed at 7 × 10 per well. 3The cells were seeded into 96-well plates at a density of 100 cells / well and cultured in a CO2 incubator at 37°C, 95% O2, and 5% CO2 for 36 hours to ensure cell attachment. The supernatant was then replaced with different concentrations of drugs (MRL / LM) every 48 hours and the cells were incubated. 10 μL of CCK-8 solution was then added to each well and cultured in a CO2 incubator for 2 hours. The cell plates were then shielded from light and measured for absorbance or optical density (OD) at a wavelength of 450 nm. All data were performed on a TriStar2LB 942 multi-module microplate reader (Berthold Technologies Gmbh & Co. KG, Baden-Wurttemberg, Germany), and the cell number was then analyzed to verify whether the drug was toxic.

[0035] 4) BMMs differentiation and tartrate-resistant acid phosphatase (TRAP) staining experiments

[0036] Approximately 7 × 10 3 Cells were seeded into 96-well plates and placed in a CO2 incubator to ensure cell attachment. After 36 hours, the supernatant was replaced with different doses of drugs (MRL / LM) and 50 ng / ml of RANKL. This was repeated every 48 hours for 7 days until mature multinuclear osteoclasts were formed in the control group. All wells were then fixed with 100 μL 4% paraformaldehyde (PFA) for 2 hours and gently rinsed twice with PBS. 70 μL of TRAP dye was then added to each well. Finally, the plates were dried using Cytation 5 (Bio TekInstruments Inc., Winooski, VT, USA) and photos of the entire well were taken. Osteoclasts with 3 or more nuclei were counted and quantified using ImageJ 1.53 software.

[0037] 5) Pseudopodial body formation assay

[0038] To investigate whether MRL could impair the formation of pseudopods and F-actin-mediated cytoskeleton structure, 7×10 3BMMs were implanted into 96-well plates for subsequent experiments. BMMs were cultured with different concentrations of drugs (MRL) and 50ng / ml RANKL for 7 days until mature osteoclasts were formed. The cells were then fixed with 4% PFA for 2 hours and washed twice with PBS. They were treated with 0.1% Triton X-PBS at room temperature for 10 minutes and 3% BSA-PBS at room temperature for 60 minutes. After washing twice with PBS containing 0.2% bovine serum albumin, they were incubated with 1:200 rhodamine-phalloidin in the dark for 2 hours. The cells were washed twice with PBS and 0.2% BSA respectively, and stained with 1:100 DAPI in the dark for about 10 minutes. The cells were washed twice with PBS, and after the culture dish was dried, a full-well fluorescence image was captured using Cytation 5.

[0039] 6) Bone resorption experiment

[0040] Osteoclasts are the only cells in the body that have bone resorption function. Therefore, bovine bone slices were used to study the bone resorption function of osteoclasts in vitro. The bovine bone slices were dried under ultraviolet light for 40 minutes and serially numbered on the back. They were then treated with anhydrous ethanol, PBS, and α-MEM for 48 hours respectively. The bovine bone slices, which had been sterilized by ultraviolet light, were placed in a 96-well plate with the numbered side facing down. 7×10 cells were then seeded on each well of the bone slices. 3 BMMs were cultured at a high density and placed in an incubator for attachment. The adherent cells were treated with 50 ng / ml RANKL for 4 days to allow them to grow into small osteoclasts. The supernatant was then replaced with various concentrations of MRL containing 50 ng / ml RANKL for 2 days to allow the control BMMs to become mature multinucleated osteoclasts. The bovine bone slice group was treated identically to the TRAP staining control group (no bovine bone slices).

[0041] Each group of bovine bone slices were washed twice with PBS and fixed with 4% PFA for 1 hour. All bovine bone slices were then collected and all photos were recorded using a scanning electron microscope (SU 8100, 3.0KV). Recording the bone pit area reflects the bone resorption capacity of osteoclasts. After the cells in each well of the TRAP staining control group were fixed with 4% PFA for 1 hour, they were gently washed twice with PBS. TRAP dye was then added to each well to stain mature osteoclasts, and all images were recorded using Cytation 5. Osteoclasts were stained red, and osteoclasts with 3 or more nuclei were counted for quantitative analysis.

[0042] 7) Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0043] qRT-PCR was used to detect the RNA expression of osteoclasts. 5BMMs were seeded into 6-well plates and cultured for 36 hours to ensure cell attachment. The cell culture supernatant was replaced once every 2 days, and the main component was different concentrations of drugs (MRL / LM) containing 50ng / ml RANKL. After mature multinuclear osteoclasts were formed, TRIzol (Thermo Fisher Scientific) was added to the 6-well plate to split the cells for 1 hour. The lysis buffer was then mixed with chloroform, isopropanol and 75% ethanol in sequence, and the extracted RNA was reverse transcribed into complementary DNA (cDNA) in the presence of a reverse transcription kit. After determining the cDNA concentration, qRT-PCR was performed using Syber Green (Thermo Fisher Scientific) and RNA-related primers. All operations were performed in The experiment was performed in a 96-well system (Roche, Basel, Switzerland) with a cycle of 60 min at 42°C, 5 min at 70°C, and a continuous low temperature of 4°C. -ΔΔCt The following are the primer sequences for different genes:

[0044] Table 1 Primer sequences for different genes

[0045]

[0046]

[0047] 8) Western blotting

[0048] All expressed proteins were detected by Western blotting. 5 BMMs were seeded into 6-well plates and osteoclast-related short-term proteins were extracted. 5BMMs were seeded into 6-well plates to extract long-acting osteoclast-associated proteins. BMMs were cultured for 7 days with different doses of drugs (MRL / LM) containing 50 ng / ml RANKL to allow the formation of mature osteoclasts, from which long-acting proteins were extracted. Alternatively, BMMs were incubated with drugs (MRL / LM) and 50 ng / ml RANKL for 1 hour to extract short-acting proteins. After drug and RANKL treatment, BMMs were treated with RIPA lysis buffer containing 1% phosphatase inhibitors, 1% protease inhibitors, and 1% phenylmethylsulfonyl fluoride (PMSF). The extracted proteins were scraped and stored at -80°C until further use. Proteins mixed with loading buffer were then separated by SDS-PAGE and transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Shanghai, China). The membrane was then soaked in skim milk for 1 hour at room temperature to prevent nonspecific immunobinding. The membrane was then gently washed three times with TBST and incubated with the primary antibody for approximately 14 hours at 4°C. The next day, the membranes were washed three times with TBST and incubated with IRDye fluorescently labeled secondary antibodies for 1 hour in the dark at room temperature. Finally, the membranes were placed in an ImageQuant LAS-4000 instrument (GE Healthcare, Chicago, Illinois, USA) to acquire protein bands. Band grayscale values ​​were calculated and quantitatively analyzed using ImageJ 1.53 software.

[0049] 9) Micro CT analysis

[0050] CT threshold analysis: The left tibia was scanned using a SCANCO MEDICAL Micro-CT 50 device (SCANCO MEDICAL, AG, Switzerland), and the region of interest (ROI) was defined. Three-dimensional reconstruction was performed using thin slices of the proximal tibia in the ROI (8 mm). Data were analyzed using CT Analyser 1.15.2.2, CTvol 2.3.2.0, and CTvox 3.3.0 rl403. Bone parameters such as bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular spacing (Tb.Sp), and trabecular thickness (Tb.Th) were analyzed and quantified at a depth of 0.5 mm below the growth plate.

[0051] 10) Data Analysis

[0052] All experiments were performed three or more times, and all results are presented as mean ± standard deviation (SD) or as graphs. Quantitative analysis was performed using Student's t-test or analysis of variance. Unless otherwise stated, p < 0.05 was considered statistically significant.

[0053] 2. Experimental results

[0054] 1) MRL inhibits RANKL-induced osteoclastogenesis in vitro

[0055] The chemical structure and molecular formula of MRL are as follows Figure 1 As shown in A. In order to clarify whether MRL has a killing effect on BMMs, the inventors conducted a CCK-8 experiment. Figure 1 As shown in B, after 48 and 96 hours of treatment with 80 μM MRL, there was no change in the cell activity of BMMs. To clarify how MRL affects osteoclast differentiation, the inventors seeded a certain number of BMMs into 96-well plates and incubated them with 50 ng / ml RANKL and different concentrations of MRL for 7 days. When multinuclear osteoclasts matured, they were fixed with paraformaldehyde and stained with tartrate-resistant acid phosphatase (TRAP) staining solution. Figure 1 C and Figure 1 D shows that with the increase of MRL concentration, osteoclast differentiation is significantly inhibited. After 7 days of stimulation with 50ng / ml RANKL, mature osteoclasts were stained with rhodamine combined with phalloidin. Figure 1 E and Figure 1 As shown in Figure F, the intact pseudopod ring, associated with actin cytoskeleton formation and morphology, is stained red, while the cell nucleus is stained blue. However, compared with the 50 ng / ml RANKL group, 15 μM and 30 μM MRL significantly inhibited F-actin ring formation. Taken together, these results demonstrate that MRL inhibits osteoclast differentiation.

[0056] 2) MRL affects the bone resorption function of mature osteoclasts in vitro

[0057] To determine the duration of MRL inhibition of osteoclast differentiation, BMMs were treated with 30 μM MRL and 50 ng / ml RANKL in a time-dependent manner. Figure 2 A and Figure 2 B confirmed that MRL has the strongest inhibitory effect on osteoclastogenesis in the middle stage of osteoclast differentiation (day 3-4), when the number of TRAP-positive osteoclasts is the least. The inventors also extracted mRNA at different stages of osteoclast differentiation and found that MRL mainly inhibited the expression of osteoclast-related genes in the middle stage of osteoclast differentiation, including Fos, Nfatc1, Ctsk, Atp6v0d2, Mmp9 and Dcstamp, such as Figure 2 As shown in Figure C. Osteoclasts are the only cells with bone resorption function in vitro, so bovine bone slices were used to study whether MRL could inhibit the bone resorption function of osteoclasts. Figure 2 D. Figure 2 E and Figure 2Results from F showed that high concentrations of MRL reduced the area of ​​bone pits formed by osteoclasts on bovine bone slices. However, stimulation of osteoclasts with 50 ng / ml RANKL alone resulted in the formation of numerous bone pits. Furthermore, the number and morphology of TRAP-positive osteoclasts remained consistent across all groups.

[0058] 3) MRL inhibits RANKL-induced NFATc1 expression in vitro

[0059] As mentioned above, MRL inhibits the expression of osteoclast-related RNA and protein. Figure 3 As shown in Figure 2, BMMs were treated with 50 ng / ml RANKL and different concentrations of MRL until they differentiated into mature multinucleated osteoclasts. After cDNA was extracted, the inventors reverse transcribed it into mRNA. Figure 3 As shown in A, compared with the RANKL group, high concentrations of MRL significantly inhibited the relative expression of osteoclast-related gene markers, including Nfatc1, Ctsk, Atp6v0d2, Mmp9, Dcstamp, and Acp5. Mature multinucleated osteoclasts were treated with 50 ng / ml RANKL for 7 days, and long-term proteins were extracted after 0, 1, 3, and 5 days of treatment with 30 μM MRL. Compared with the RANKL group, the osteoclast-related long-term proteins c-Fos, NFATc1, CTSK, and Atp6v0d2 were significantly inhibited in the MRL group, as shown in Figure 4. Figure 3 B- Figure 3 As shown in F.

[0060] 4) MRL inhibits osteoclastogenesis by reducing the phosphorylation of ERK 1 / 2 proteins

[0061] The MAPK pathway is a classic pathway for osteoclast differentiation, including the proteins ERK 1 / 2, c-Jun N-terminal kinase (JNK) 1 / 2, and P38. It is downstream of the RANK pathway and is an early osteoclast-related protein. Therefore, the inventors studied the role of MRL in the MAPK pathway of osteoclast differentiation. Figure 4 A- Figure 4 D shows that MRL significantly inhibited the phosphorylation of ERK 1 / 2, but did not affect the phosphorylation of JNK 1 / 2 or P38. Osteoclasts were treated with 50ng / ml RANKL for 60min, and MRL (0μM or 30μM) was treated for 0, 5, 10, 20, 30 and 60min, and short-term proteins were extracted. It is well known that the NF-κB pathway is composed of P65 and IκBα proteins, both of which play an indispensable role in the process of osteoclast differentiation. Figure 4 E- Figure 4As shown in Figure F, 30 μM MRL had no effect on the phosphorylation of P65 and the degradation of IκBα. The results of Western blotting experiments suggested that MRL has a specific association with ERK 1 / 2.

[0062] 5) LM promotes the phosphorylation of ERK 1 / 2 proteins to promote osteoclastogenesis, while MRL inhibits this effect

[0063] To further investigate the inhibitory effect of MRL on ERK 1 / 2, the inventors added LM, an activator of ERK 1 / 2, to the experiment. First, the inventors confirmed that LM alone and in combination with MRL had no killing effect on BMMs proliferation. Figure 5 A- Figure 5 As shown in B. BMMs were treated with 30μM MRL, 30μM LM alone, and 30μM MRL and 30μM LM in combination, and then stimulated with 50ng / ml RANKL for 7 days until multinuclear osteoclasts were formed. After staining with TRAP dye, the results were as follows Figure 5 C- Figure 5 As shown in D. As mentioned above, after 7 days of treatment with 30 μM LM, the number of osteoclasts was significantly greater than that of the RANKL group treated with only RANKL for 7 days. However, BMMs treated with 30 μM MRL failed to differentiate into mature multinucleated osteoclasts. The number of BMMs treated with 30 μM MRL and 30 μM LM was between that of the 30 μM MRL group and the 30 μM LM group. Osteoclast-related genes, including Fos, Ctsk, and Atp6v0d2, were detected at the transcriptional level. Figure 5 E- Figure 5 G. The results of quantitative reverse transcription polymerase chain reaction (qRT-PCR) were consistent with the results of TRAP staining. LM promoted the expression of osteoclast-related RNA, while MRL inhibited the expression of osteoclast-related RNA. 6) MRL inhibited osteoclast function by targeting ERK 1 / 2 protein expression in vitro

[0064] BMMs were treated with 30 μM MRL alone, 30 μM LM alone, and 30 μM MRL and 30 μM LM combined, and stimulated with 50 ng / ml RANKL for 7 days until mature multinuclear osteoclasts were formed, and long-acting proteins were extracted. Cells were treated with the same drugs for 60 minutes to extract short-acting proteins. Figure 6 A- Figure 6As shown in Figure (G), 30 μM LM promoted ERK 1 / 2 phosphorylation, while MRL inhibited it. When 30 μM MRL and 30 μM LM were used together, ERK 1 / 2 phosphorylation levels were intermediate between those observed with LM and MRL alone. Expression trends of the downstream proteins c-FOS, NFATc1, CTSK, and Atp6v0d2 were similar. Western blotting results were similar to those of TRAP staining and qRT-PCR, confirming that MRL inhibits osteoclast function by targeting ERK 1 / 2 proteins.

[0065] 7) MRL inhibits osteoporosis in ovariectomized mice in vivo

[0066] To further explore the potential therapeutic effects of MRL, the inventors used an ovariectomized mouse model to simulate systemic osteoporosis. Starting one week after ovariectomy, mice in each group received intraperitoneal injections of saline or MRL (15 mg / kg or 30 mg / kg) every two days. No major adverse events (including death, infection, and weight loss) occurred during ovariectomy and MRL treatment. Figure 7 A Micro CT results showed that MRL has a good protective effect on bone loss in mice after ovariectomy. Quantitative CT analysis of the proximal tibia Figure 7 B- Figure 7 E demonstrated that the extent of trabecular bone loss in mice treated with MRL was significantly reduced compared to the untreated OVX group. Therefore, as expected, in vivo treatment of mice with MRL significantly protected them from estrogen deficiency-induced bone loss.

[0067] Discussion

[0068] Osteoporosis is an osteolytic bone disease characterized by disrupted bone homeostasis, bone loss, and bone microarchitectural degeneration. It is one of the most common diseases in middle-aged and elderly people worldwide. It is caused by overactivation of osteoclast differentiation, which results from strong RANK / RANKL signaling. Long-term use of drugs that inhibit osteoclast function and production is associated with various adverse reactions. For example, antiresorptive bisphosphonates effectively block the bone resorption function of osteoclasts, but they disrupt normal bone remodeling, leading to mandibular osteonecrosis and atypical femoral fractures. Therefore, the development of new treatments for osteoporosis is highly desirable in recent years. Natural products exhibit multiple pharmacological effects, are readily available, have low manufacturing costs, and lack adverse reactions, and are becoming an important source for new drug development. In the search for new bioactive natural anti-osteoclastogenic drugs, the inventors discovered that MRL exhibits anti-inflammatory effects. However, the specific mechanisms of MRL in osteoclast formation and function have not been fully investigated. The present invention investigates the anti-osteoclast effects of MRL using in vitro bone marrow mesenchymal stem cells (BMMs) and in vivo ovariectomized mouse models. The results of this study demonstrate that MRL inhibits osteoclast formation and function in vivo by suppressing ERK 1 / 2 phosphorylation and NFATc1 activation, representing a novel and effective strategy for the treatment of osteoporosis. Furthermore, the inventors used an ovariectomized mouse model to further validate the anti-osteoclastogenic effect of MRL in vivo. Therefore, MRL may be a valuable drug for targeting osteoclastogenesis.

[0069] Multinuclear osteoclasts are characteristic cells with bone resorption capacity, originating from the fusion of monocytes and macrophages. Osteoclasts are responsible for clearing damaged bone tissue and initiating bone remodeling, as they are the sole bone-resorbing cells in the body. While osteoclast formation and differentiation are strictly controlled under physiological conditions, various pathological factors can stimulate osteoclast formation, leading to the development of osteolytic bone diseases. RANKL and M-CSF are key cytokines regulating osteoclast formation and differentiation. The survival and proliferation of osteoclast precursors are closely linked to M-CSF and c-fms, and the RANKL-RANK interaction is essential for osteoclastogenesis. RANKL binding to RANK recruits TRAF6, subsequently activating downstream signaling pathways such as MAPK and NF-κB, stimulating osteoclast formation. The canonical NF-κB signaling pathway involves the IKK complex, which degrades IκBα, leading to the translocation of NF-κB to the nucleus and initiation of transcriptional activity. The MAPK pathway, including ERK 1 / 2, JNK 1 / 2 and P38, is closely related to the formation and differentiation of osteoclasts.

[0070] Among them, the ERK 1 / 2 signaling pathway is crucial for osteoclast survival and differentiation, while the P38 signaling pathway is essential for osteoclast differentiation, but not fusion. Subsequently, when the NF-κB or MAPK signaling pathway is activated, downstream transcription factors such as c-Fos are phosphorylated and activated to control osteoclast differentiation. This key transcription factor plays a crucial role in the early induction of NFATc1. NFATc1 is an essential transcription factor for osteoclast formation and a master regulator of osteoclast differentiation. Embryonic stem cells lacking NFATc1 lack the ability to generate osteoclasts due to their inability to respond to RANKL stimulation. Activated c-Fos activates the NFATc1 promoter, leading to its auto-amplification and nuclear translocation during osteoclastogenesis. Subsequently, NFATc1 activation leads to the coordinated upregulation of a series of osteoclast-related genes, including Mmp9, Atp6v0d2, and Ctsk, which contribute to the bone resorption function of osteoclasts and contribute to the formation of mature osteoclasts. When osteoclasts perform bone resorption, Mmp9 participates in the early migration of osteoclasts and the bone resorption of mature osteoclasts, and promotes the formation of the wrinkled edge of osteoclasts. + CTSK is a key protease in osteoclast bone resorption, primarily responsible for the degradation of collagen and other matrix proteins. Mice lacking CTSK may exhibit hypermineralization and abnormal bone remodeling.

[0071] The experimental results of the inventors showed that MRL inhibited RANKL-induced osteoclast formation in a dose-dependent and time-dependent manner. Among them, 30μM MRL had the greatest inhibitory effect on osteoclast formation, and its inhibitory effect was mainly exerted in the middle stage of osteoclast formation. On the other hand, MRL reduced the formation of actin rings in mature osteoclasts in a dose-dependent manner, destroyed the formation of osteoclast skeletons, and inhibited bone resorption function. At the protein level, MRL inhibited RANKL-induced osteoclast formation by inhibiting ERK 1 / 2 phosphorylation, inhibiting the MAPK signaling pathway and the activation of transcription factor c-Fos. Subsequently, MRL reduced the transcriptional activity of NFATc1 by inhibiting the phosphorylation of c-Fos, and inhibited the expression of osteoclast-related proteins such as MMP-9, Atp6v0d2 and CTSK, making osteoclasts unable to perform bone resorption function. The above results show that MRL reduces the occurrence of osteoclasts by blocking the activation of ERK 1 / 2 in the MAPK signaling pathway. When ERK 1 is deficient, defective osteoclasts form; when the ERK 1 / 2 signaling pathway is blocked, RANKL-induced osteoclastogenesis is reduced. To further elucidate the mechanism by which MRL inhibits ERK 1 / 2, this study incorporated LM22B-10. LM22B-10 is an ERK 1 / 2 agonist and has been previously used in numerous articles and studies. Following the addition of LM22B-10, phosphorylation levels of ERK 1 / 2 and its downstream proteins, c-Fos, NFATc1, CTSK, and Atp6v0d2, were significantly elevated compared to the RANKL group. However, when MRL and LM22B-10 were used simultaneously, phosphorylation levels of the relevant proteins fell between those observed with MRL and LM22B-10 alone.

[0072] Based on these in vitro results, the inventors established an ovariectomized mouse model to investigate in vivo whether MRL could inhibit osteoclast function and improve osteoporosis. The ovariectomized mouse model exhibited bone loss characteristics similar to those observed in postmenopausal women. Similarly, in vivo experiments demonstrated that MRL treatment significantly improved trabecular bone structure compared to the OVX group. Therefore, it can be concluded that MRL has a protective effect against OVX-induced bone loss in mice.

[0073] In summary, the inventors have discovered for the first time that MRL possesses anti-osteoclast activity both in vitro and in vivo. The natural compound MRL acts by inhibiting ERK 1 / 2 phosphorylation and c-Fos and NFATc1 activation, effectively inhibiting osteoclast differentiation and function. Furthermore, the anti-osteoclast effect of MRL may provide valuable insights into the discovery of anti-osteoclast compounds derived from natural products, offering promising prospects for the treatment of osteoporosis.

Claims

1. Application of mace lignan in the preparation of drugs for the targeted treatment of osteoporosis.

2. The use according to claim 1, characterized in that: The medicine is a preparation composed of mace lignan as an active ingredient and a pharmaceutical carrier.

3. The use according to claim 2, characterized in that: The preparation is in the form of oral dosage form, intravenous injection dosage form, intramuscular injection dosage form or external use dosage form.

4. The use according to claim 3, characterized in that: The oral dosage form is capsule, pill or tablet.

5. The use according to claim 1, characterized in that: The treatment is achieved by inhibiting the formation of osteoclasts.

6. The use according to claim 5, characterized in that: The inhibition of osteoclast formation is achieved by downregulating the expression of ERK and NFATc1 induced by RANKL.

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