Application of Schisandrin B in the preparation of drugs for preventing and treating osteoporosis

Abstract

This invention provides the application of schisandrin ethyl in the preparation of drugs for the prevention and treatment of osteoporosis. Firstly, the basic molecular mechanism was elucidated by detecting the MAPK and NF-κB signaling pathways involved in osteoclast differentiation using Western blotting and PCR experiments. Furthermore, the effects of schisandrin ethyl on osteogenic differentiation and mineralization were investigated. Additionally, an ovariectomized OVX mouse model was constructed to simulate the occurrence of osteoporosis, and the degree of bone destruction and osteoclast number were analyzed using microcomputed tomography (CT) and staining with hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP), thereby clarifying the molecular mechanism by which schisandrin ethyl prevents osteoporosis. The results showed that schisandrin ethyl can effectively inhibit RANKL-induced osteoclast formation, reduce bone resorption and the formation of F-actin loops, and the expression of osteoclast marker genes. It was found that schisandrin ethyl inhibits osteoclast formation by mediating the TRAF6-NF-κB / MAPK signaling pathway through ROS and promoting the expression of antioxidant enzymes to eliminate ROS.

Description

[Technical field]

[0001] The invention belongs to the technical field of bone disease treatment, and relates to the application of schisandra chinensis alcohol B in preparing medicines for preventing and treating osteoporotic diseases. [Background technology]

[0002] Bone remodeling is generally considered to consist of two major events: osteoclasts absorb existing bone and osteoblasts form new bone. During bone remodeling, absorption and formation are balanced, and the two processes are coupled in space and time. Once the bone absorption rate and bone formation rate are unbalanced, various bone-related diseases such as osteoporosis will result. Osteoporosis is a group of bone diseases caused by various reasons. The bone tissue has normal calcification, and the calcium salt and matrix are in a normal ratio. It is a metabolic bone disease characterized by a decrease in the amount of bone tissue per unit volume. Osteoporosis can occur in different genders and at any age, but it is more common in postmenopausal women and elderly men. It is characterized by bone pain and easy fractures.

[0003] The most important aspect of osteoporosis treatment is to inhibit bone resorption, which is accomplished by osteoclasts. Osteoclasts are derived from hematopoietic stem cells and are mainly controlled by regulating the expression of RANKL and macrophage colony stimulating factor M-CSF. In addition, many molecules have been identified as downstream factors of RANK / RANKL signaling in osteoclasts and precursor osteoclasts, including TRAF6, MAPK, and AP-1, which ultimately cascade to the common transcription factor NFATc1 to affect osteoclasts and induce osteoclast gene expression. Considering that osteoclasts play a very important role in bone loss, the development of drugs that effectively inhibit osteoclast activity can provide valuable options for the prevention and treatment of osteoporotic diseases.

[0004] Osteoporosis, that is, the current drugs for the treatment of osteoporosis, are mainly divided into anti-resorptive drugs and anabolic drugs for reducing bone resorption or increasing bone formation. Among them, anti-resorptive treatments, such as bisphosphonates, estrogen and selective estrogen receptor modulators, denosumab, target osteoclasts, while anabolic drugs, such as parathyroid hormone and parathyroid hormone-related protein, aim to improve bone formation. However, since the pathophysiology of osteoporosis varies from person to person, treatment may have serious side effects. Therefore, finding drugs with low toxicity and side effects and affordable and easily available to inhibit the number and activity of osteoclasts has become an important strategy for the treatment of osteoporosis and other osteolytic diseases.

[0005] Schisandrin B is one of the lignans in the dried fruit of Schisandra chinensis. The traditional Chinese medicine Schisandra chinensis has the therapeutic effects of astringency, invigorating qi and promoting body fluid, and tonifying the kidney and calming the mind. One of its main components, Schisandrin A, inhibits osteoclastogenesis by inhibiting reactive oxygen species and activating Nrf2 signaling. Another main component, Schisandrin B, has multiple biological effects including antioxidant, anticancer and anti-inflammatory effects. It is reported that Schisandrin B has a protective effect against acetaminophen-induced hepatotoxicity by regulating the MAPK signaling pathway. In addition, Schisandrin B can also protect mice from acute liver injury induced by acetaminophen by activating the NRF2 / antioxidant element pathway. This protective effect can also be achieved by inhibiting the biological activation of cytochrome regulation and regulating liver regeneration. However, the role of Schisandrin B in osteoclasts and osteoporosis has been little explored.

[0006] In view of this, the present invention provides an application of Schisandrin B in the preparation of a drug for preventing and treating osteoporosis, so as to solve the above problems. [Summary of the invention]

[0007] In order to solve the above problems, the present invention provides an application of Schisandrin B in the preparation of drugs for preventing and treating osteoporotic diseases. Schisandrin B has osteoclast formation and absorption effects.

[0008] In order to achieve the above object, the present invention provides an application of Schisandrin B in the preparation of a drug for preventing and treating osteoporosis, which is specifically described as follows:

[0009] Schisandrin B has an inhibitory effect on RANKL-induced osteoclastogenesis, F-actin ring formation and bone resorption; the basic molecular mechanism was elucidated by detecting the MAPK and NF-κB signaling pathways involved in osteoclast differentiation through WB and PCR experiments; in addition, the effect of Schisandrin B on osteogenic differentiation and mineralization was also detected.

[0010] In order to evaluate the therapeutic effect of Schisandrin B in vivo, the present invention also constructed an ovariectomized OVX mouse model to simulate the occurrence of osteoporosis, and analyzed the degree of bone destruction and the number of osteoclasts by micro-computed tomography (CT), hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining, thereby clarifying the molecular mechanism of Schisandrin B in preventing osteoporosis.

[0011] After a large number of experiments, the results showed that Schisandrin B can effectively inhibit RANKL-induced osteoclast formation, reduce bone resorption and the formation of F-actin rings and the expression of osteoclast marker genes; it was further discovered that Schisandrin B inhibits osteoclast formation by mediating the TRAF6-NF-κB / MAPK signaling pathway through ROS and promoting the expression of antioxidant enzymes to eliminate ROS. In vivo experiments showed that it prevents bone loss in estrogen-deficient mice by inhibiting osteoclast activity and generation.

[0012] Therefore, Schisandrin B is a very promising drug for the prevention and treatment of osteoporosis.

[0013] The present invention found that: in terms of the biological behavior of osteoclasts, schisandrin B in schisandrin lignans inhibits RANKL-induced osteoclastogenesis in a concentration- and time-dependent manner, and shows no toxic effect on the cell activity of BMMs. Functionally, schisandrin B can inhibit the expression of osteoclast-related genes, and at the same time has the ability to weaken the formation of osteoclast actin rings and bone absorption. In terms of the mechanism of inhibiting osteoclastogenesis, schisandrin B inhibits the expression of intracellular Ca2+ and transcription factor NFATc1. At the same time, schisandrin B inhibits osteoclasts by inhibiting RANKL-mediated TRAF6 and related downstream osteoclast formation and bone absorption-related protein expression. For the classical signaling pathways involved in RANKL-mediated, schisandrin B inhibits the phosphorylation of P65 protein in the NF-κB signaling pathway and the degradation of IκBα protein; and reduces the phosphorylation levels of JNK, ERK and p38 proteins in the MAPK signaling pathway. In addition, schisandrin B can reduce ROS production by inhibiting NOX1 expression, while promoting the expression of antioxidant enzymes NQO1, HO-1, SOD1 and SOD2 to clear ROS in cells and mitochondria. In terms of osteoblasts, schisandrin B had no significant promoting or inhibiting effect on the activity, differentiation and mineralization of osteoblasts. In addition, in OVX mice, schisandrin B significantly increased the volume fraction and number of tibial trabecular bone and reduced the content of CTX-1 in serum. It can be seen that schisandrin B significantly reduced the number of osteoclasts, NFATc1 transcription factor and NOX1 protein expression in the tibia of mice. The results of reverse transcription quantitative PCR and protein immunoblotting of bone tissue in vivo showed that schisandrin B inhibited the expression of NFATc1 and CTSK, while increasing the expression of antioxidant enzyme NQO1 to clear ROS, inhibit the number and activity of osteoclasts, and thus protect bone loss.

[0014] In summary, schisandrin B can inhibit RANKL-mediated TRAF6-NF-κB and MAPK signaling pathways to reduce intracellular Ca2+ and transcription factor NFATc1 expression, enhance antioxidant enzyme expression to clear intracellular ROS, thereby inhibiting osteoclastogenesis and bone resorption. Schisandrin B can effectively protect OVX mice from bone loss by inhibiting NOX1 expression, enhancing antioxidant enzyme expression and reducing ROS.

Brief Description of the Drawings

[0015] Figure 1 Figure 1 is a diagram showing that Schisandrin B inhibits RANKL-mediated osteoclastogenesis in vitro; (A) is the structural formula of eight dibenzocyclooctadiene lignans; (BC) is a representative diagram of TRAP staining of mature osteoclasts treated with each compound (10 μm and 20 μm) and the count of osteoclasts with ≥3 nuclei; (D) is the CCK-8 kit results of the eight compounds, scale bar = 200 μm, data are expressed as mean ± SD, NC: negative control; PC: positive control; **p < 0.01, ***p < 0.001;

[0016] Figure 2 Figure 1 is a graph showing that schisandrin B inhibits osteoclast-specific genes, F-actin ring formation and absorption function; (A) is the expression level of osteoclast-specific genes (Acp5, Ctsk, Mmp9, Dcstamp, Nfatc1, Fos, Atp6v0d2 and Calcr) after treatment with schisandrin B (0 μM, 5 μM, 10 μM, 20 μM); (B) is the relative mRNA expression of Acp5, Ctsk, Mmp9, Dcstamp, Nfatc1, Fos, Atp6v0d2 and Calcr on days 0, 1, 3 and 5 after treatment with 20 μM schisandrin B; (CD) is the F-actin ring treated with different concentrations of schisandrin B during osteoclastogenesis stained with TRITC phalloidin And quantify the area, (for example) scan the osteoclast resorption pits on the bovine bone slices with a scanning electron microscope and calculate the resorption area, scale bar = 500 μm, monitor the RTCA-DP system with the cell viability curve of BMMs treated with Schisandrin B at a concentration lower than 30 μM; (FG) are the TRAP staining results and osteoclast differentiation counts treated with Schisandrin B (0 μM, 5 μM, 10 μM, 20 μM); (HI) are representative pictures of TRAP staining and quantification of osteoclastogenesis at different stages (1-6, 1-3, 3-5 and 5-6 days) treated with Schisandrin B (20 μM), scale bar = 200 μm, data are expressed as mean ± SD, NC: negative control, PC: positive control; **p<0.01, ***p<0.001;

[0017] Figure 3It is the expression diagram of schisandrae alcohol B reducing the phosphorylation of NF-κB and MAPK pathway proteins; (A) Raw264.7 cells with NF-κB luciferase reporter gene were treated with or without schisandrae alcohol B and RANKL for 6 hours, and the luciferase activity of NF-κB was detected; (BC) Nuclear localization and mean fluorescence intensity of p65 protein, (DF) phosphorylated P65 and total IκBα in the NF-κB signaling pathway were detected and quantified; (GJ) Phosphorylated JNK, Erk and P38 proteins in the MAPK signaling pathway were detected and quantified, scale bar = 100 μm, data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001;

[0018] Figure 4 Figure 2 is a graph showing that Schisandrin B inhibits intracellular calcium oscillation and expression of proteins related to the NFATc1 signaling pathway; (A) Raw264.7 cells with NFAT luciferase reporter gene were treated with or without Schisandrin B and RANKL for 24 hours to detect the luciferase activity of NFAT; (B) Intracellular calcium oscillation stained with Fluo4-AM; (C) Average fluorescence intensity of Ca2+ within 2 minutes; (D) Immunofluorescence staining of NFATc1 with or without Schisandrin B (20 μM) after stimulation with RANKL for 3 or 5 days; (E) Immunofluorescence staining of NFATc1 after 5 days of treatment with schisandrin B (0, 10, 20 μM); (F) Detection of NFATc1 signaling pathway-related proteins after stimulation with RANKL for 0, 1, 3, and 5 days (with or without schisandrin B (20 μM)); (G) Detection of osteoclasts and corresponding proteins in F under stimulation with increasing concentrations of RANKL and schisandrin B; (HI) Quantification of F and G, respectively, scale bar = 200 μm, data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001;

[0019] Figure 5The figure shows that Schisandrin B attenuates Nox1 expression and reactive oxygen species production; (A) is the result of NFATc1 luciferase reporter gene experiment; (B) is a representative picture of calcium ion fluorescent probe staining (scale bar = 1000 μm); (C) is a curve chart of real-time monitoring of intracellular calcium ion fluorescence intensity for 2 minutes; (D) is a representative picture of NFATc1 fluorescence labeling after 5 days of osteoclast differentiation treated with different doses of Schisandrin B (scale bar = 200 μm); (E) is a representative picture of NFATc1 fluorescence labeling on the 3rd and 5th days of osteoclast differentiation intervened by Schisandrin B (0 and 20 μM) Figure (scale bar = 200 μm), n = 3; (F) is a representative picture of TRAF6 and downstream related protein bands in osteoclasts stimulated by RANKL for 0, 1, 3, and 5 days; (G) is a quantitative statistical analysis of Figure A; (H) is a representative picture of TRAF6 and downstream related protein bands in osteoclasts stimulated by RANKL for 0, 1, 3, and 5 days; (I) Representative pictures of protein bands after RANKL induction for 5 days under the action of different concentrations of Schisandrae Chinensis B, n = 3, **p < 0.01, ***p < 0.001, bar graph data are expressed as mean ± standard deviation;

[0020] Figure 6 Figure 2 shows that Schisandrin B promotes the expression of antioxidant elements and reduces intracellular ROS, inhibits NOX1 and promotes the expression of related antioxidant enzymes; (A) Antioxidant element luciferase reporter gene detection results; (B) Representative images of intracellular and mitochondrial ROS fluorescence (scale bar = 1000 μm); (C-D) Quantitative statistics of intracellular and mitochondrial ROS fluorescence intensity, n = 3, compared with the positive control group; (E) Nox1, Nqo and schisandrin B in BMMs cells treated with or without RANKL and Schisandrin B for 48 h 1. Relative gene expression levels of Hmox1, Sod1 and Sod2; (F) Relative gene expression levels of Nox1, Nqo1, Hmox1, Sod1 and Sod2 in BMMs cells treated with or without RANKL and schisandrin B for 48 h; (GL) Representative images and quantitative statistics of protein bands of NOX1, NQO1, HO-1, SOD1 and SOD2, *p<0.01, **p<0.01, ***p<0.001, data are expressed as mean ± SD;

[0021] Figure 7The figure shows that Schisandrin B can prevent trabecular bone loss and inhibit osteoclast activity in vivo in OVX model mice; (A) is a representative 3D picture of tibial trabecular and cortical bone of OVX model mice; (BF) is a quantitative analysis of tibial trabecular parameters; (G) is a quantitative analysis of cortical bone density; (H) is a representative picture of TRAP staining, NFATc1 and NOX1 immunohistochemical staining after tibia sectioning of mice in each group (scale bar = 500μm); (IK) is the quantitative statistical result of Figure H; (LM) is the CTX-1 and PINP serum content detection results of mice in each group, and the data are expressed as mean ± standard deviation; (N) is a representative picture of liver and kidney HE staining results of mice in each group (scale bar = 200μm) n = 5, *p<0.01, **p<0.01, ***p<0.001, and the data are expressed as mean ± standard deviation. [Specific implementation method]

[0022] In order to make the present invention more clear, the present invention is further explained in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0023] 1. Materials and Methods

[0024] 1.1 Pharmaceuticals and Chemicals

[0025] Schisandrin B was purchased from Munster (Chengdu, Sichuan, China). α-MEM and FBS were purchased from Gibco (USA). RANKL and M-CSF were purchased from R&D Systems (Minneapolis, MN, USA). Specific antibodies were purchased from Cell Signaling Technology (Danvers, MA). TRAP staining kit was purchased from Sigma (St. Louis, MO, USA). 96-well hydroxyapatite plates were purchased from Corning Osteoassay. Hematoxylin and eosin staining kit was purchased from Solebol Technology (Beijing, China).

[0026] 1.2 Cell culture

[0027] Extraction and culture of primary bone marrow macrophages (BMMs): C57BL / 6 mice of about six weeks old that grew well were killed by cervical dislocation in the sampling room. Ethanol disinfectant was sprayed on the mouse body to achieve the purpose of sterilization. Ophthalmic scissors that were sterilized in advance were used to quickly cut the Achilles tendon of the lower limb, peel off the muscles from the Achilles tendon to the femoral head, cut the lower limb at the hip joint and cut off the claws (avoid exposing the bone marrow cavity), put them in a culture dish (the culture dish was placed on ice), and transferred to a biosafety cabinet to further remove the remaining muscle tissue of the tibia and femur. Add 5mL PBS to wash once, then add 3mL MEM Alpha complete medium (M-CSF final concentration is 25ng / mL), clamp the lower limb with forceps and cut the epiphysis, then use a 1mL syringe to absorb the complete medium and repeatedly rinse the medullary cavity until the bone head turns white. Transfer the mixture of bone marrow and culture medium to a 50mL centrifuge tube and blow it away with a Pasteur pipette. Centrifuge at 1200rpm for 5min and discard the supernatant. Resuspend the cells with 3mL MEMAlpha complete medium, filter the cells with a 100μm filter and drop into a T75 culture flask. Make up the medium to 15mL and leave it in a cell culture incubator for 2 days. After that, remove the old medium in the flask, rinse the cells twice with PBS to remove dead cells and impurities, replace with new MEM Alpha complete medium and continue to culture for two days. The adherent cells are considered to be bone marrow-derived macrophages and can be used for subsequent experiments.

[0028] MC3T3-E1 cell culture: MC3T3-E1 Subclone 14 cells were provided by Shanghai Fuheng Biotechnology Co., Ltd. and were correctly identified by STR. After receiving the cells, wipe the outside of the culture flask with 75% alcohol, observe whether the cell state is good under a microscope, and then place it in a cell culture incubator for 3 hours to stabilize the cell state. After 3 hours, take out the culture flask, discard the culture medium in the flask, rinse the cells once with PBS, add 1mL of 0.25% trypsin-EDTA solution to digest until the cells become round, add 2mL of DMEM phenol red-free complete medium to terminate the digestion, gently blow the cells and transfer them to a 15mL centrifuge tube, and centrifuge at 1000rpm for 5min. Discard the supernatant, resuspend the cell pellet in 2mL of complete medium, and divide 1mL / bottle into 2 T25 culture flasks. Repeat the above subculture operation 3 times until the cells reach the logarithmic growth phase and subsequent experiments can be carried out.

[0029] RAW264.7 cell culture: Take out RAW264.7 cells stably transfected with nuclear factor of activated T cells 1 (NFATc1), nuclear factor κB (NF-κB) and antioxidant response element (ARE) luciferase reporter gene elements from the liquid nitrogen tank, place them in a 37°C water bath and shake quickly to thaw, wipe the tube wall with ethanol disinfectant and place it in a biosafety cabinet for operation. Transfer the cells in the tube to 15mL centrifuge tubes, add 2mL DMEM complete medium, and centrifuge at 1000rpm for 5min. Discard the supernatant, resuspend the cell pellet with 2mL complete medium, inoculate it into a T75 culture flask, make up the volume of the culture medium to 12mL, and culture it in a 37°C, 5% CO2 cell culture incubator. The next day, observe the cells under a microscope and replace with fresh DMEM complete medium. Then replace the medium every other day until the cell confluence reaches about 90%, and then passaging or seeding can be performed.

[0030] 1.3 Cell viability assay

[0031] Effects of the main components of Schisandra lignans on the activity of BMMs cells: The counted BMMs cells were adjusted to a density of 6×104 cells / mL, and the volume of 100 μL per well was inoculated in a 96-well plate. An equal amount of PBS solution was added around the wells where the cells were inoculated to reduce the evaporation of the cell culture medium, and then placed in a cell culture incubator for overnight culture. The next day, after the cells were fully attached, the original culture medium was discarded and replaced with MEM Alpha culture medium containing different monomers (concentrations of 10 μM and 20 μM), and a control group without drug was set up, i.e., containing the same volume of DMSO. After the cells were cultured in the incubator for 48 hours, CCK-8 solution (10 μL) was added to the cells in each well, and care was taken to avoid bubbles. After the culture plate was placed in the incubator for another 2 hours, the absorbance at 450 nm was detected by an ELISA instrument and the experimental results were derived.

[0032] Effect of Schisandrin B on MC3T3-E1 cells: MC3T3-E1 cells were seeded in 96-well plates at a density of 6×103 cells / well, with 3 replicates per column, and the cells were cultured in DMEM complete medium without phenol red for 24 hours. Afterwards, the cells were treated with Schisandrin B (0, 5, 10, 20, 30 μM) for 48 hours, and after the above treatment was completed, the operation was the same as above.

[0033] 1.4 Osteoclast differentiation assay and TRAP staining

[0034] Different concentrations of Schisandrin B act on osteoclast differentiation: 6×103 BMMs cells / well were inoculated in a 96-well plate and cultured overnight. The cells were observed under a microscope the next day to confirm whether the cells grew well. Grouping was set up: negative control group (culture medium was MEM Alpha complete culture medium), positive control group (culture medium was MEM Alpha complete culture medium + 50ng / mL of RANKL) drug treatment group (MEM Alpha complete culture medium + 50ng / mL of RANKL + 0 / 5 / 10 / 20μM Schisandrin B). Unless otherwise specified, the working concentration of RANKL used in this example was 50ng / mL. Remove the original culture medium, replace the above culture medium and change the medium every other day until the cells in the positive control group fuse to form giant mature osteoclasts containing multiple nuclei. Take the culture plate out of the incubator, gently aspirate the culture medium, fix it with 4% paraformaldehyde for 1h, rinse the bottom cells 3 times with 1×PBS, add TRAP staining solution to each well, incubate in the dark for 30min, check the degree of cell staining under the microscope, and remove the staining solution when the overall color is pink. Each well was then rinsed three times with double distilled water to remove excess staining solution, dried in a 37°C oven, and scanned with a cell imaging instrument, Cytation5.

[0035] Schisandrin B acts on osteoclast differentiation at different time periods: BMMs were seeded in 96-well plates at a cell number of 6×103 / well and incubated overnight in an incubator to allow the cells to fully adhere to the wall. The groups were set up before changing the medium the next day: negative control group (culture medium was MEM Alpha complete medium), positive control group (culture medium was MEM Alpha complete medium + RANKL) and drug treatment group (1-2, 3-4, 5-6 and 1-6 days drug treatment group, MEM Alpha complete medium + RANKL + 20μM Schisandrin B). The culture medium of the negative control group, positive control group and 1-6 days drug treatment group (20μM Schisandrin B) needs to be replaced with the corresponding fresh culture medium each time the medium is changed. The medium containing 20μM Schisandrin B was replaced in the drug treatment group 1-2 days after the first medium change, and MEM Alpha complete medium was replaced in the other drug treatment groups. The second medium change removed the original medium of the 1-2 day drug treatment group and replaced it with MEM Alpha complete medium. The medium of the 3-4 day drug treatment group was MEM Alpha complete medium + RANKL + 20μM schisandrin B, and the other drug treatment groups were replaced with MEM Alpha complete medium. Similarly, the third medium change replaced the medium containing 20μM schisandrin B for the 5-6 day drug treatment group. When the cells were cultured for 6 days, mature osteoclasts were observed under the microscope. At this time, the medium was aspirated and fixed with 4% paraformaldehyde for 1h. The rinsing and TRAP staining steps were the same as the osteoclast differentiation experiment with different concentrations of schisandrin B.

[0036] 1.5 Bone resorption function experiment

[0037] Bovine cortical bone slices were placed in a 10 cm cell culture dish, and the back was marked with a pencil to divide them into negative control group and drug treatment group (5, 10, 20 μM Schisandrin B). Ethanol disinfectant was added to cover the bone slices, and then the culture dish was sealed with a sealing film and placed in a 4 ° C decolorizing shaker for 24 hours. After 24 hours, it was replaced with MEM Alpha culture medium and continued to shake for 24 hours to replace the remaining ethanol disinfectant. The bone slices were transferred to a 96-well cell culture plate with ophthalmic forceps, and the BMMs cells that had been counted were inoculated on the bone slices at a number of 104 / well. At the same time, 6×103 / well were inoculated in a 96-well plate for TRAP staining control and placed in a cell culture incubator overnight. RANKL stimulation was added the next day, and the culture medium was changed every two days until mature osteoclasts appeared in the 96-well plate used for control. The 96-well plate was removed, the culture medium was aspirated, and 4% paraformaldehyde was added to fix the cells for 1 hour. The 96-well cell culture plate containing bone slices was replaced with a solution, and then cultured for 48 hours before fixing with electron microscopy fixative. 50 μL of TRAP stain was added to each well for 20 minutes, and the excess stain was washed off with double distilled water. After drying, the cells were photographed. Cells with positive staining and ≥3 nuclei were considered mature osteoclasts. The bone slices were removed with ophthalmic forceps, and the cells were brushed off with a brush and dried. After gold spraying, electron microscopy scanning and photography were performed.

[0038] 1.6 Determination of F-actin ring formation

[0039] 6×103 BMMs cells were inoculated in a 96-well plate, and groups were set up: control group (equal volume of DMSO) and schisandra chinensis alcohol B treatment group (5, 10, 20 μM), and cultured overnight. The cells were replaced every two days until most cells in the control group fused and mature osteoclasts appeared. The culture medium was then removed and the cells were fixed with 4% paraformaldehyde for 1 hour. The cells were rinsed twice with 1× PBS buffer, and 100 μL of Triton X-100 was added to each well to permeabilize the cell membrane for 10 minutes. The original liquid was removed, and 100 μL of 3% BSA-PBS was added to each well to block the cells for 1 hour. After calculating the required volume according to the number of wells, tetramethylrhodamine isothiocyanate-labeled phalloidin was diluted in 0.2% BSA-PBS at a ratio of 1:200. After blocking, the cells were washed three times with 0.2% BSA-PBS, and 100 μL of diluted TRITC-labeled phalloidin was added to each well and stained for 30 min in the dark. DAPI was diluted in 0.2% BSA-PBS (1:500 ratio), and 100 μL was added to each well for 5 min in the dark to counterstain the cell nucleus. The excess DAPI stain was washed away with double distilled water, and the cells were photographed using the fluorescence channel of the cell imager Cytation5, and the images were saved.

[0040] 1.7 RNA extraction and quantitative PCR assay

[0041] According to the experimental protocol of the kit manual, total RNA was isolated and extracted from osteoclasts using Trizol reagent. To synthesize cDNA, 1 μg RNA was reverse transcribed with oligo-dT primer (Thermo Fisher kit, USA). Real-time PCR was performed using LightCycler 96 (Switzerland, Basel), and the amplification process was: 94°C pre-denaturation for 10 minutes, followed by 35 cycles, each cycle of 94°C (10 seconds), 60°C (15 seconds), and finally at 72°C (10 seconds), and then the PCR data was read after the reverse transcription was terminated. The specific primers for osteoclast-related genes used in this experiment are listed in Table 1 below:

[0042] Table 1

[0043]

[0044]

[0045] 1.8 Luciferase gene detection experiment

[0046] RAW264.7 cells with good growth status stably transfected with nuclear factor of activated T cells 1 (NFATc1), nuclear factor κB (NF-κB) and antioxidant response element (ARE) luciferase reporter gene elements were taken. All cells with gene elements were set up as negative control group, positive control group and schisandra chinensis alcohol B drug treatment group (5, 10, 20μM), and were inoculated in 48-well plates at a number of 5×104, 1.5×105, and 1.5×105 / well, respectively, and cultured overnight to allow cells to adhere to the wall. The next day, the negative control group replaced the DMEM complete medium, the positive control group replaced the medium with the DMEM complete medium containing RANKL, and the schisandra chinensis alcohol B drug treatment group added 5, 10, and 20μM schisandra chinensis alcohol B on the basis of the DMEM complete medium containing RANKL. For RAW264.7 cells stably transfected with NFATc1 and ARE reporter genes, the treatment time of RANKL and schisandrin B was 24h; for RAW264.7 cells stably transfected with NF-κB reporter genes, the treatment time of RANKL and schisandrin B was 6h. After the treatment time, the culture medium of each well was discarded, and the cells were rinsed twice with 1×PBS buffer to remove the influence of phenol red in the culture medium on the experimental results. The 1×PBS buffer was aspirated, and 100μL of luciferase lysis buffer (2mM DTT was added before use) was added to each well, and the reaction was fully carried out on the ice box for 20min, during which the high-speed centrifuge temperature was set to 4℃. The lysate was transferred to a clean 1.5mL EP tube and centrifuged at 12000rpm for 20min. After the centrifugation, 50μL of supernatant was gently aspirated into the 96-well plate to avoid bubbles that would affect the reading. Then the plate was placed in a multifunctional fluorescence luminescence analyzer, and the luciferase detection kit was mixed evenly according to the instructions, and the injection tube was inserted to start detecting the fluorescence intensity of the corresponding gene element.

[0047] 1.9 Western blot analysis

[0048] BMMs were treated with Schisandrin B for 4 hours in advance, and then stimulated with 50ng / mL mouse RANKL for 0 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes and 60 minutes. Subsequently, cells were washed with 1x PBS and lysed with ice-cold RIPA buffer containing a mixture of phosphatase and protease inhibitors. Protein concentration was then assessed using BCA protein assay reagent (Beyotime, China). Whole cell lysates (15 μL) were subjected to 10% SDS-PAGE and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA).

[0049] 1.10 Construction of ovariectomized OVX mice

[0050] After 25 female C57BL / 6 mice were transported back to the animal experiment center, they were adapted to the environment until they were 12 weeks old. They were deprived of water and food for 4 hours before surgery. After weighing, they were anesthetized by intraperitoneal injection of 2% tribromoethanol and randomly divided into 5 groups, 5 mice in each group. The groups were set as sham surgery (Sham), Vehicle (Ve), estradiol (E2), OVX + 10mg / kg schisandrae alcohol B and OVX + 20mg / kg schisandrae alcohol B. A disposable surgical sheet was laid on the operating table, and the mice were placed on it. The hair on the back of the mice was cut with ophthalmic scissors. After disinfecting the skin with iodine, the skin was clamped at the waist with tweezers and a longitudinal incision of about 1 cm was cut. The muscle layer was cut open, and the ovary was found below it. After tying the knot with sterile suture, the ovary was removed, the tissue was reset, and the incision was sutured. The ovary on the other side was operated in the same way as above. The fat near the ovary was removed in the sham operation group. Penicillin was injected intramuscularly at 50,000 units / kg to prevent infection. Berberine ground into a paste was applied to the incision to prevent the mice from biting the wound after waking up. To provide a warm environment, the mice were placed next to a warm lamp after surgery. The next day, after the mice were active, the Sham group and the Vehicle group were intraperitoneally injected with saline containing 1% DMSO, the estrogen (E2) group was intraperitoneally injected with E2 (100ng / kg), and the OVX+10mg / kg Schisandrin B and OVX+20mg / kg Schisandrin B groups were injected with dissolved Schisandrin B, respectively. The drug was administered once every 2 days, for a total of 21 times. After the modeling was completed, the eyeball blood of each group of mice was collected, placed at room temperature for 30min, centrifuged at 3000rpm for 15min, and the supernatant was transferred to a new 1.5mL EP tube and temporarily stored at -80℃ for ELISA experiments. The mice were then killed by cervical dislocation, and the bones of both lower and upper limbs were extracted, and the attached muscles were removed as much as possible. The kidneys and livers were also taken out. The upper limbs of the mice were directly frozen in a -80°C refrigerator for in vivo detection of relevant indicators. The lower limbs, kidneys and livers of the mice were immersed in 4% paraformaldehyde and fixed for 2 days, and then stored at room temperature for CT scanning and histological experiments.

[0051] 1.11 Statistical analysis

[0052] The experiments involved were completed at least three times. The experimental results were statistically analyzed using GraphPad Prism (9.0), and the data results were presented as mean ± standard deviation. Statistical analysis was performed using one-way / two-way analysis of variance. When the p value was less than 0.05, the difference was considered statistically significant.

[0053] 2. Results

[0054] 2.1 Schisandrin B inhibits RANKL-mediated osteoclastogenesis in vitro

[0055] In order to more accurately detect the effects of different concentrations of Schisandrin B (0, 5, 10, 20, 30 μM) on BMMs cell proliferation, the present invention uses real-time cell analysis technology to continuously monitor the cells. The results show that within 96 hours, Schisandrin B below 30 μM concentration has no significant inhibitory effect on BMMs cell proliferation (such as Figure 2 E). In addition, under the intervention of Schisandrin B (0, 5, 10, 20 μM), the number of mature osteoclasts in the Schisandrin B treatment group was significantly reduced compared with the group treated with RANKL alone (e.g. Figure 2 AB). To examine the effect of Schisandrin B on different stages of osteoclast differentiation, 20 μM Schisandrin B was used to act on days 1-2, 3-4, 5-6 and 1-6 during osteoclast differentiation. Compared with the control group (only RANKL was added), the group treated with 20 μM Schisandrin B on days 1-6 of osteoclast differentiation formed the least osteoclasts (p < 0.001), followed by Schisandrin B only treated on days 1-2, indicating that Schisandrin B may inhibit osteoclast formation in the early stages of osteoclast differentiation and ultimately lead to a decrease in the number of osteoclasts (e.g. Figure 2 CD).

[0056] 2.2 Schisandrin B inhibits osteoclast-specific genes, F-actin ring formation and absorption

[0057] Based on the effect of Schisandrin B on the number of osteoclasts and bone resorption function, the present invention further explored the effect of Schisandrin B on osteoclast-related genes by reverse transcription quantitative PCR. The detected osteoclast-related specific expression genes included the expression of genes mediating osteoclast fusion, absorption and calcitonin receptors (Acp5, Ctsk, Mmp9, Dcstamp, Nfatc1, Fos, Atp6v0d2 and Calcr). After treatment with RANKL and Schisandrin B (0, 5, 10, 20 μM), compared with the positive control group, the expression level of each osteoclast-specific gene decreased significantly with the increase of Schisandrin B concentration (p < 0.01) (such as Figure 3 E). In addition, in order to explore the effect of Schisandrin B on gene expression at different time points of osteoclast differentiation, this example also extracted total RNA of osteoclasts at 0 days, 1 day, 3 days and 5 days of osteoclast differentiation under the action of Schisandrin B at a concentration of 20 μM, and performed reverse transcription quantitative PCR. The results showed that except for Nfatc1 and Fos, whose expression levels were significantly reduced at 1 day, 3 days and 5 days, the expression levels of the remaining genes were significantly reduced at 3 days and 5 days (p < 0.01) (e.g. Figure 3 F)

[0058] Osteoclasts participate in cell-matrix adhesion based on the actin cytoskeleton structure, degrade mineralized bone matrix, and perform bone resorption function. Rhodamine-labeled phalloidin can selectively bind to filamentous actin, so the embodiment of the present invention explores the effect of schisandrin B on the formation of osteoclast actin rings and the absorption of bone to form pits through phalloidin staining and bone resorption experiments. The results of phalloidin staining showed that with the increase of the concentration of schisandrin B, the F-actin ring formed by osteoclasts and the area of ​​osteoclasts were significantly reduced. The results showed that after the action of different concentrations of schisandrin B, there was no significant difference in the number of mature osteoclasts in each group in the control plate, while the area of ​​osteoclast absorption pits on the bovine bone slices gradually decreased with the increase of schisandrin B concentration, and the difference was statistically significant (p < 0.01)

[0059] 2.3 Schisandrin B reduces phosphorylation of NF-κB and MAPK pathway proteins

[0060] The NF-κB signaling pathway includes members such as P65 and IκBα, which play an indispensable role in the RANKL-induced osteoclast differentiation process. Inhibiting the activation of the NF-κB signaling pathway inhibits the differentiation and generation of osteoclasts. Therefore, the present invention firstly tested whether Schisandrin B has an effect on the NF-κB signaling pathway by stably transfecting RAW264.7 cells with the NF-κB luciferase element. The results showed that compared with the control group, the NF-κB fluorescence signal weakened with the increase of Schisandrin B concentration (such as Figure 4 A). Furthermore, the present invention uses cell immunofluorescence to localize the P65 protein in the nucleus. The results show that compared with the control group, the fluorescence intensity of the P65 protein in the nucleus treated with Schisandrin B (20 μM) is significantly weakened (p < 0.05) (e.g. Figure 4 BC). Then, the embodiment detected the expression of phosphorylated P65 protein and IκBα protein at 6 time points within 1 hour of RANKL stimulation in BMMs. Compared with the control group, the phosphorylated P65 protein in the Schisandrin B (20 μM) group was significantly reduced at 5min and 10min of RANKL stimulation (p < 0.01), while the expression of IκBα protein increased at 10min, 20min and 30min (p < 0.05) (as shown in Figure 2). Figure 4 DF)

[0061] MAPK (including JNK, ERK and p38) signaling pathways are activated during RANKL-induced osteoclast differentiation. Therefore, in order to explore whether Schisandrin B inhibits osteoclasts by affecting the MAPK signaling pathway, the present invention extracted total protein in BMMs cells after Schisandrin B (20 μM) treatment for 1 hour, and used protein immunoblotting to detect the phosphorylation expression of JNK, ERK and p38 proteins. Compared with the control group, the expression of phosphorylated JNK and p38 decreased significantly at 10min and 20min (p < 0.01), and the expression of phosphorylated ERK also decreased significantly at 20min (p < 0.01) (as shown in Table 1). Figure 4 GJ).

[0062] 2.4 Schisandrin B inhibits intracellular calcium oscillation and NFATc1 signaling pathway-related proteins

[0063] After RANKL binds to the receptor RANK, it activates the NFATc1 transcription factor to induce osteoclast differentiation. Therefore, the present invention uses a luciferase reporter gene cell line containing the NFATc1 element to detect whether Schisandrin B inhibits the expression of NFAT in cells. The results show that as the concentration of Schisandrin B increases, the fluorescence intensity of the NFATc1 element gradually decreases compared with the control group, and is statistically significant (p < 0.01) (e.g. Figure 5 A). Since calcium ion concentration plays a regulatory role in RANKL-induced osteoclast development, the present invention uses a calcium ion fluorescent probe to detect the intracellular calcium ion situation. The results show that the average fluorescence intensity of calcium ions in the group treated with Schisandrin B is weaker than that in the positive control group (such as Figure 5 BC). Then, the distribution of NFATc1 protein in cells was localized by fluorescence staining. As the cell dose of Schisandrin B increased, the fluorescence of NFATc1 was weakened compared with the control group, which was consistent with the results of the luciferase reporter gene experiment (such as Figure 5 D). In addition, after 3 and 5 days of treatment with schisandrin B (20 μM), the expression of NFATc1 was also reduced (e.g. Figure 5 E).

[0064] 2.5 Schisandrin B promotes the expression of antioxidant elements and reduces intracellular ROS

[0065] Natural antioxidants can inhibit osteoclast formation-related factors and offset the negative effects of ROS on bone metabolism-related molecular factors. In view of the antioxidant properties of Schisandrin B, the present invention tested its effect on the expression of antioxidant elements (NRF2-ARE). The results showed that the activity of antioxidant elements in the positive control group increased after the addition of RANKL, indicating that the ARE element responded to the stimulation of RANKL. After treatment with Schisandrin B, the activity of antioxidant elements increased significantly compared with the positive control group (such as Figure 6 A). In addition, H2DCFDA and MitoSOX fluorescent probes were used to stain ROS in cells and mitochondria to examine whether Schisandrin B can reduce ROS in cells. Figure 6 As shown in BD, under RANKL stimulation, the fluorescence staining of ROS in cells and mitochondria was enhanced, and ROS was significantly reduced after treatment with schisandrin B (p < 0.01), indicating that schisandrin B can remove reactive oxygen species in cells and mitochondria.

[0066] 2.6 Schisandrin B inhibits NOX1 and promotes the expression of related antioxidant enzymes

[0067] By detecting the gene expressions of intracellular Nox1 and anti-ROS related enzymes including quinone oxidoreductase 1 (Nqo1), heme oxygenase 1 (Hmox1) and superoxide dismutase 1 / 2 (Sod1 / 2), the possible pathway of Schisandrin B in scavenging ROS was explored. Figure 6 E, Compared with the positive control group, schisandrin B significantly inhibited the gene expression of Nox1 at a concentration of 20 μM, and the gene expressions of other antioxidant enzymes were increased. At the protein level, the inhibitory effect of schisandrin B on NOX1 and the promotion of related antioxidant enzymes were basically consistent with the gene level (such as Figure 6 FL).

[0068] 2.7 Schisandrin B can prevent trabecular bone loss and inhibit osteoclast activity in OVX model mice

[0069] To further verify the in vitro experimental results, the present invention constructed an ovariectomized (OVX) mouse model. After the administration of Schisandrin B, the mice were sacrificed, the lower limb bones were removed and fixed, and μCT scanning was performed on the tibia. The results are as follows: Figure 7 As shown in A, compared with the Sham group, the trabecular bone in the Ve group was significantly reduced (p < 0.001), indicating that the OVX mouse model was successfully established. Compared with the Ve group, the BV / TV, Tb.N and BS / TV values ​​of the tibial trabecular bone in the Schisandrin B-treated group were significantly increased, while Tb.Sp and Tb.Th did not change significantly (as shown in Figure 2). Figure 7 In addition, there was no significant difference in tibial cortical bone BMD among the groups (e.g. Figure 7G). In summary, schisandrin B can effectively prevent bone loss in OVX model mice. Based on the μCT results, the present invention further detected the CTX-1 and PINP levels in the serum samples of OVX mice. The results showed that compared with the Ve group, the CTX-1 content in the E2 group and the schisandrin B-treated group was significantly reduced, while the difference in PINP content between the groups was not statistically significant (e.g. Figure 7 LM). The results of TRAP staining of paraffin sections showed that the number of osteoclasts in mice treated with Schisandrin B was reduced (e.g. Figure 7 HK). The results of NFATc1 and NOX1 immunohistochemistry showed that the expression of NFATc1 and NOX1 in the Schisandrin B-treated group was decreased compared with that in the Ve group, indicating that Schisandrin B inhibits NFATc1 and NOX1 in vivo (e.g. Figure 7 In addition, the present invention performed HE staining on the liver and kidney of each group of mice to observe whether Schisandrin B has a damaging effect on the liver and kidney of mice. The results showed that there was no significant difference in the morphology of liver and kidney cells of each group of mice (such as Figure 7 N).

[0070] In summary, the present invention found that Schisandrin B can inhibit RANKL-induced osteoclast formation, hydroxyapatite plate bone resorption, F-actin ring formation and bone loss in OVX mice in vitro and in vivo. Further analysis of the molecular mechanism found that Schisandrin B inhibits RANKL-induced osteoclast formation by affecting MAPK (p38, ERK and JNK) and NF-κB signaling pathways. Based on this, the present invention believes that Schisandrin B may have great potential and clinical value for the prevention of osteoporotic diseases.

[0071] In the present invention, the effects of 8 main lignan components from the kidney-tonifying Chinese medicine Schisandrae Chinensis on osteoclast formation were preliminarily explored, and it was found that Schisandrin B and Schisandrin B can inhibit RANKL-induced osteoclastogenesis. The highest initial screening concentration of the present invention was 20 μM, which did not significantly inhibit osteoclastogenesis. Therefore, the present invention selected Schisandrin B to conduct a more detailed study on the inhibitory effect of RANKL-induced osteoclastogenesis. Schisandrin B was set to act on the RANKL-mediated osteoclastogenesis process at more concentrations, and it was found that Schisandrin B inhibited osteoclast formation in a dose-dependent and time-dependent manner, and had no significant inhibition on cell viability. It was found that Schisandrin B can interfere with the osteoclast differentiation process.

[0072] Normal bone is in a state of constant remodeling through osteoblast-mediated bone formation and osteoclast-mediated bone resorption, while osteoporosis is the result of an imbalance in the function of osteoblasts and osteoclasts, resulting in reduced bone formation or increased bone resorption, the latter of which is the main factor leading to osteoporosis in postmenopausal women, mainly due to accelerated bone loss caused by postmenopausal estrogen deficiency. Currently, drugs for the treatment of osteoporosis are mainly divided into anti-resorptive drugs and anabolic drugs for reducing bone resorption or increasing bone formation. Among them, anti-resorptive treatments, such as bisphosphonates, estrogens and selective estrogen receptor modulators, denosumab, target osteoclasts, while anabolic drugs, such as parathyroid hormone and parathyroid hormone-related protein, aim to improve bone formation. However, since the pathophysiology of osteoporosis varies from person to person, treatment may have serious side effects. Therefore, finding drugs with low toxicity and side effects and affordable and easily available to inhibit the number and activity of osteoclasts has become an important strategy for the treatment of osteoporosis and other osteolytic diseases. The inventors' results demonstrated for the first time that Schisandrin B can inhibit RANKL-induced osteoclast differentiation and bone resorption function.

[0073] In order to study the specific molecular mechanism of schisandrae alcohol B inhibiting osteoclast differentiation, the inventors studied the main signal pathways affecting osteoclast differentiation. MAPK and NF-κB pathways are two classic pathways for osteoclast differentiation. MAPK signaling pathways (including p38, ERK and JNK) are mainly involved in the regulation of osteoclast growth, differentiation, inflammatory response and other important physiological processes. The combination of RANKL and RANK will lead to an increase in the phosphorylation levels of JNK, p38 and ERK, and finally start the increase in the expression of osteoclast marker genes in the nucleus, promoting the formation of osteoclasts. Among these signal molecules, ERK is very important for the survival and differentiation of osteoclasts, P38 participates in the differentiation of osteoclasts but does not affect its bone absorption function, and blocking the activation of JNK can affect the formation of osteoclasts. Mice with JNK gene deletion will develop osteosclerosis due to incomplete bone absorption function. Therefore, inhibiting p38, ERK and JNK can effectively block the formation of osteoclasts induced by RANKL. In the present invention, it was found through experiments that schisandrae alcohol B inhibited the expression of TRAF6, reduced calcium ions and inhibited the transcriptional expression of NFATc1. The results of immunoblotting in the study showed that Schisandrin B significantly inhibited the phosphorylation of p65 protein and the degradation of IκBα protein in the NF-κB signaling pathway; Schisandrin B significantly reduced the phosphorylation levels of JNK, ERK and p38 proteins in the MAPK signaling pathway. The above results prove that Schisandrin B can inhibit the differentiation of osteoclasts by inhibiting the TRAF6-mediated NF-κB and MAPK pathways, affecting the transcription of calcium ions and NFATc1. c-Fos and NFATc1 are the main transcription factors of the downstream signaling pathway of RANKL-induced osteoclast differentiation, which affect the differentiation of osteoclasts by regulating the expression of osteoclast marker genes. The present invention found that after the intervention of Schisandrin B, the WB experiment found that c-Fos and NFATc1 were significantly inhibited in terms of protein expression levels. When further RT-PCR detection was performed, it was found that the expression of osteoclast marker genes such as Nfatc1, c-Fos and Acp5 in the drug group decreased in a dose-dependent manner. In summary, the above analysis results indicate that Schisandrin B may affect the expression levels of downstream important transcription factors c-Fos and NFATc1 proteins by blocking the MAPK and NF-κB signaling pathways, thereby inhibiting the expression of osteoclast marker genes and thus inhibiting osteoclast differentiation and bone resorption.

[0074] Most postmenopausal women with osteoporosis have bone loss associated with estrogen deficiency. Estrogen inhibits osteoclast differentiation and promotes osteoclast apoptosis by increasing the production of transforming growth factor-β; in the state of estrogen deficiency, RANKL expression is induced, leading to osteoclast formation. Therefore, based on the in vitro research results, the present invention constructs an OVX mouse model to simulate postmenopausal osteoporosis and explores the preventive effect of schisandrin B on this disease. The μCT results show that schisandrin B can effectively protect OVX mice from bone loss. Type I collagen cross-linked C-terminal peptide (type I collagen carboxy-terminal peptide, CTX-1) and type I procollagen amino-terminal peptide (type I procollagen amino-terminal peptide, PINP) are clinically relevant biomarkers of bone metabolism and important quantitative indicators reflecting bone resorption and bone formation. The serological test results of the present invention show that schisandrin B inhibits bone metabolic activity in mice by inhibiting CTX-1, rather than PINP. Histological results showed that Schisandrin B reduced the number of TRAP-positive cells in the tibia and reduced the expression of transcription factors NFATc1 and NOX1 proteins. Further, the present invention quantitatively analyzed the expression of osteoclast-specific genes Nfatc1 and Ctsk and antioxidant enzyme Nqo1 gene in the bone tissue of model mice. Schisandrin B effectively regulated their expression, and the inhibitory effects at the protein level and gene level were consistent. The above results suggest that Schisandrin B may reduce the number of osteoclasts by inhibiting the production of ROS by NOX protein and enhancing the expression of antioxidant enzyme NQO1 to eliminate ROS production, thereby inhibiting the expression of CTSK and the transcription of NFATc1, thereby reducing bone resorption and protecting the bone loss of OVX mice.

[0075] Conclusion: Schisandrin B has an inhibitory effect on osteoclast differentiation, bone resorption function and bone loss induced by OVX mice both in vivo and in vitro, and this effect may be related to the inhibition of RANKL-mediated MAPK and NF-κB signaling pathways. Therefore, the present invention may provide a new therapeutic idea for preventing osteoporosis.

Claims

1. The application of schisandrae alcohol B as the only active ingredient in the preparation of drugs for preventing and treating osteoporosis.

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