CN110538174B

CN110538174B - Application of dracorhodin in preparing medicine

Info

Publication number: CN110538174B
Application number: CN201910917991.2A
Authority: CN
Inventors: 刘予豪; 何伟; 徐家科; 张庆文; 周驰; 马超
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-09-26
Filing date: 2019-09-26
Publication date: 2023-03-07
Anticipated expiration: 2039-09-26
Also published as: CN110538174A

Abstract

The invention relates to application of dracorhodin in preparation of a medicine, in particular to application of dracorhodin or dracorhodin perchlorate in preparation of a medicine for inhibiting osteoclast formation or inhibiting osteoclast bone absorption or preventing and treating osteolytic diseases, and belongs to the technical field of medicines. The application of the dracorhodin or dracorhodin perchlorate in preparing the medicine for inhibiting osteoclast formation or inhibiting osteoclast bone absorption or preventing and treating osteolytic diseases has the effect of inhibiting osteoclast formation or inhibiting osteoclast bone absorption or preventing and treating osteolytic diseases. The invention provides a new application of dracorhodin or dracorhodin perchlorate and also provides a new medicinal component for treating osteoclast formation or bone absorption function or osteolytic diseases.

Description

Application of dracorhodin in preparing medicine

Technical Field

The invention relates to application of dracorhodin in preparation of a medicine, in particular to application of dracorhodin or dracorhodin perchlorate in preparation of a medicine for inhibiting osteoclast formation or inhibiting osteoclast bone absorption or preventing and treating osteolytic diseases, and belongs to the technical field of medicines.

Background

Normal bone tissue in the human body is a dynamic and constantly changing tissue, and the normal bone metabolic balance is mainly maintained by the bone remodeling function of osteoblasts and the bone resorption function of osteoclasts. When the activity of osteoclast in vivo is enhanced, the bone homeostasis is broken, so that the bone mass is lost, and various osteolytic related diseases such as osteoporosis, osteonecrosis and the like are induced, mainly manifested by the microstructure damage of bone tissues, the reduction of bone density, the reduction of bone strength, the increase of brittleness and the like.

Osteoclasts are multinucleated cells differentiated from mononuclear macrophages and hematopoietic cells. Macrophages are dependent on two major cytokines during differentiation into osteoclasts: nuclear factor kappa beta ligand (RANKL) and macrophage colony stimulating factor (M-CSF). RANKL is an important cytokine for osteoclast differentiation, survival and bone resorption, and it exerts a biological effect by binding to receptor RANK. The combination of RANK and RANKL activates signal pathways such as mitogen-activated protein kinase (MAPK), NF-kB, calcium ions and the like, finally activates transcription factors such as c-Fos, T cell cytoplasm 1 (NFATc 1) and the like which are necessary for osteoclast differentiation, and accordingly triggers the expression of related genes for controlling osteoclast differentiation and functions, such as tartrate-resistant acid phosphatase (TRAcP), cathepsin K (CTSK), calcitonin receptor (Calcitonine receptor), metalloproteinase (MMP 9), integrin beta 3, NFATc1, c-Fos and the like. M-CSF is an important factor inducing the survival and proliferation of osteoclast precursors, and can also mediate the sensitivity of Bone Marrow Macrophages (BMMs) to RANKL. Thus, disruption or attenuation of RANKL-mediated molecular signaling pathways may treat diseases associated with osteoclasts, such as osteoporosis, osteonecrosis, paget's bone disease, osteomyelitis, bone cysts, myeloma, bone metastases, osteoblastomas, non-ossifying fibroids, giant cell tumors of the bone, and the like.

The inventor finds that the dragon's blood chalcone extract can inhibit NFATc1 activity, inhibit ROS activity and inhibit osteoclast differentiation through MAPK, calcium ion signal channels and the like, thereby preventing and treating osteolytic diseases such as osteoporosis, osteonecrosis and the like. However, dragon's blood also includes other kinds of compounds, such as the flavonoid dracorhodin, and further intensive research is needed to find out whether other kinds of compounds have the effects of inhibiting osteoclast differentiation, and preventing and treating osteoporosis and osteonecrosis.

Disclosure of Invention

Dracorhodin, one of the main components of dragon's blood, belongs to a flavonoid compound, and researches and reports that dracorhodin can promote wound healing, promote tumor cell apoptosis and the like, but no report on the aspect of inhibiting osteoclast and improving osteolytic diseases exists. The invention confirms that the dracorhodin perchlorate compound can inhibit NFATc1 activity induced by RANKL through cell experiments, and inhibit osteoclast formation and bone absorption functions, and the action mechanism relates to a calcium ion signal pathway, a MAPK signal pathway and a NF-kB signal pathway.

The invention aims to overcome the defects of the prior art, defines that the dracorhodin serving as a component of the dragon blood has the functions of inhibiting osteoclast formation or inhibiting osteoclast bone absorption or preventing and treating osteolytic diseases, and provides the application of the dracorhodin or dracorhodin perchlorate in preparing a medicament for inhibiting osteoclast formation or inhibiting osteoclast bone absorption or preventing and treating osteolytic diseases.

In order to achieve the purpose, the invention adopts the technical scheme that: use of dracorhodin or dracorhodin perchlorate in preparing medicine for inhibiting osteoclast formation or osteoclast bone absorption or preventing and treating osteolytic diseases.

Preferably, the medicament is used for inhibiting the expression of related genes Acp5, c-fos, ctsk, mmp9, ctr or Nfatc1 in the process of inducing osteoclast formation by RANKL.

Preferably, the medicament is used for inhibiting the expression of related proteins NFATc1, c-Fos, integrin beta 3, MMP9 or CTSK in the process of inducing osteoclastogenesis by RANKL.

Preferably, the medicament is for inhibiting the activity of NFATc.

Preferably, the medicament is used for inhibiting a calcium ion signaling pathway, a MAPK signaling pathway or a NF-kB signaling pathway in the process of inducing osteoclast differentiation by RANKL.

Preferably, the dosage form of the medicament is a solid tablet or powder.

The invention provides an application of a pharmaceutical composition in preparing a medicament for inhibiting osteoclast formation or osteoclast bone absorption or preventing and treating osteolytic diseases, wherein the active ingredient of the pharmaceutical composition comprises dracorhodin or dracorhodin perchlorate; the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; the dosage form of the pharmaceutical composition is solid tablets or powder.

Preferably, the osteolytic disease is osteoporosis, osteonecrosis, paget's disease of the bone, osteomyelitis, bone cyst, myeloma, bone metastasis, osteoblastoma, non-ossifying fibroids or giant cell tumor of the bone.

Compared with the prior art, the invention has the beneficial effects that:

(1) The dracorhodin or dracorhodin perchlorate can inhibit the formation of osteoclasts induced by RANKL and inhibit the bone resorption function of osteoclasts induced by RANKL, inhibit the expression of genes Acp5, c-Fos, ctsk, mmp9, ctr and Nfatc1 related to osteoclasts induced by RANKL, inhibit the expression of proteins NFATc1, c-Fos, integrin beta 3, MMP9 and CTSK related to osteoclasts induced by RANKL, inhibit MAPK and NF-kappa B signal pathways induced by RANKL and inhibit the activity of NFATc1 induced by RANKL, and therefore, the dracorhodin or dracorhodin perchlorate can be used for preparing medicines for inhibiting the formation of osteoclasts or inhibiting the bone resorption function or preventing and treating osteolytic diseases and has the effects of inhibiting the formation of osteoclasts or inhibiting the bone resorption of the osteoclasts or preventing and treating osteolytic diseases;

(2) The invention provides a new application of dracorhodin or dracorhodin perchlorate and also provides a new medicinal component for the treatment of osteoclast formation or bone absorption function or osteolytic diseases.

Drawings

Figure 1 is a graph of the results of d.p inhibition of RANKL-induced osteoclastogenesis; wherein, fig. 1A is a graph of the results of the number and size of osteoclast formation gradually inhibited by d.p at a gradient concentration; fig. 1B is a statistical graph of the number of mouse osteoclasts under the inhibition of d.p at different concentrations; FIG. 1C is a graph showing the results of the macrophage toxicity test of bone marrow in mice at different concentrations of D.P; fig. 1D is the chemical structural formula of d.p. Note: dracorhodin Perchlorate (d.p), dracorhodin Perchlorate; MNCs, multinucleated cells; absorbance, absorbance.

Figure 2 is a graph of the results of d.p inhibition of RANKL-induced osteoclastic bone resorption function; wherein, fig. 2A is a bone resorption experiment, arrows show that osteoclasts are in a bone resorption region of hydroxyapatite, and d.p inhibits the bone resorption range of osteoclasts; FIG. 2B is a statistical chart of a bone resorption experiment; figure 2C is fluorescent staining of actin rings on the surface of osteoclasts, the number and area of actin rings inhibited by d.p; FIG. 2D is a statistical plot of actin ring area. Note: bone Resorption; % area resorbed per OC, percent bone resorption area of individual osteoclasts; f-actin belt size per OC, single osteoclast actin ring size; fold Change, multiple.

Fig. 3 is a graph showing the results of d.p inhibition of RANKL-induced osteoclast-associated gene expression; the gradient concentration D.P inhibits the expression of Acp5, ctsk, mmp9 and other genes. Note: gene expression; fold Change, multiple.

Figure 4 is a graph of the results of d.p inhibition of RANKL-induced osteoclast-associated protein expression; wherein, fig. 4A is a representative protein band, d.p inhibits the expression of osteoclast differentiation associated proteins c-Fos, integrin β 3, MMP9, CTSK; FIG. 4B is a histogram showing the expression level of protein bands.

FIG. 5 is a graph of the results of the RANKL-induced MAPK, NF- κ B signaling pathway inhibited by D.P; wherein, FIG. 5A is a representative protein band, D.P inhibits the phosphorylation expression of JNK and p65 proteins in the differentiation process of osteoclast, and inhibits the degradation of I kappa B-alpha protein; FIG. 5B is a statistical graph of the expression level of protein bands.

Figure 6 is a graph of the results of d.p inhibition of RANKL-induced NFATc1 activity; wherein, fig. 6A shows the calcium channel opening condition, and the number and intensity of times that d.p inhibits the calcium channel opening; fig. 6B is qPCR result, d.p inhibits Ctr, nfatc1 gene expression; fig. 6C is a representative band of NFATc1 protein expression, d.p inhibits NFATc1 protein expression, and fig. 6D is a statistical graph of the expression amount of the protein band; fig. 6E shows the fluorescent staining result of NFATc1 protein, and d.p inhibits the expression level of NFATc1 in cytoplasm and nucleus, and inhibits the migration of NFATc1 into nucleus. Note: intensity; calcium ionization, calcium shaking.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1

The present example is a study of the effect of dracorhodin perchlorate (d.p) on RANKL-induced osteoclast formation, RANKL-induced osteoclast bone resorption function, RANKL-induced osteoclast gene expression, RANKL-induced osteoclast protein expression, RANKL-induced osteoclast-associated protein expression, RANKL-induced MAPK and NF- κ B signaling pathway, and RANKL-induced NFATc1 activity, by an osteoclast differentiation experiment, a cytotoxicity experiment, actin ring and cell nucleus staining, a bone resorption experiment, a calcium ion concussion experiment, an immunofluorescence staining experiment, a short time gradient Western blot experiment, a qPCR experiment, and a long time gradient Western blot experiment by the inventors of the present invention.

1. Materials and reagents

(1) Chinese chengdumanst biotechnology limited: dracorhodin perchlorate (d.p);

(2) the Australian gibco company: α -MEM medium, FBS (fetal bovine serum);

(3) santa Cruz Biotechnology, USA: a β -actin antibody, NFATc1 antibody, V-ATPase-d2 antibody, CTSK antibody, etc.;

(4) australian Promega corporation: MTS determination kit and PCR reaction kit

(5) University of australia, western australia: M-CSF (macrophage colony stimulating factor) agent, RANKL (nuclear factor kappa beta ligand) agent

2. Experimental methods

2.1 osteoclast differentiation assay

Bone Marrow Macrophages (BMMs) extracted from 6 week old C57BL/6J mice were cultured in T75 flasks containing complete medium (. Alpha. -MEM medium +10% FBS +1% P/S +50ng/ml M-CSF), and after the BMMs had differentiated to maturity, at 6X10 per well ³ The individual cell ratios were seeded into 96-well plates. Next day, 50ng/ul RANKL was added to each well to differentiate osteoclasts, and the experimental group was added with D.P at gradient concentrations of 0. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, and 30. Mu.M, while the control group was added with the same volume of D.PPBS. The differentiation solution is changed every two days until the osteoclast is differentiated and matured. After osteoclasts were differentiated and matured, they were fixed with 2.5% glutaraldehyde for 15 minutes, followed by TRAcP (tartrate-resistant acid phosphatase) staining, and the number of osteoclasts (number of nuclei. Gtoreq.3) was counted for each group under the microscope.

2.2 cytotoxicity assay

Differentiating mature BMMs at 6X10 per well ³ The individual cell ratios were plated in 96-well plates and the intervention started the following day: the experimental group was added with D.P at a gradient concentration of 0. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M per well, respectively, the control group was added with PBS of the same volume, and incubated in alpha-MEM medium containing 10% FBS and 1%P/S for 48 hours, then 20ul of MTS reagent was added per well, and after incubation for 2 hours in the dark, the absorbance at 490nm was measured using an ELISA reader (German BMG) to determine the MTS absorbance.

2.3 actin Ring and Nuclear staining

Differentiating mature BMMs at 5X10 per well ⁴ The density of each cell is planted into a 24-well plate, and next day, 50 ng/mu l of RANKL is added into each well of an experimental group to induce the BMMs to differentiate towards OC, wherein 30 mu M of D.P is added into a drug group, and PBS with the corresponding volume is added into a positive control group; the negative control group was treated as the positive control group, but induced differentiation was not performed. The culture medium or the differentiation solution is changed every two days until the OC of the positive control group is differentiated and matured on the sixth day. After the cells were differentiated and matured, each group of cells was washed once with PBS, then fixed with 4% paraformaldehyde solution (PH = 7.4) for 10 minutes, and washed twice with PBS; the membrane was broken for 5 minutes by adding 100. Mu.l of 0.1% Triton X-100 solution per well, followed by blocking for 10 minutes at room temperature using 3% BSA-PBS solution, followed by incubating Vinculin protein antibody for 2 hours at room temperature, washing the cells three times using PBS, and then staining for 70 minutes for actin loops under light-shielding conditions using a corresponding secondary protein antibody (FITC-labeled) and phalloidin simultaneously. After staining was complete, the cells were washed four additional times with PBS, followed by 10 min staining of nuclear DNA with Hoechst 33258 substrate. After the staining was completed, the bone slides were transferred to glass slides, randomly blocked with an anti-fluorescence quenching blocking solution, mounted with coverslips, and then confocal with NikonAnd (5) taking pictures by a microscope to observe the actin ring area and the cell nucleus number of each group of cells.

2.4 bone resorption test

Differentiating mature BMMs at 8X10 per well ⁴ The individual cells are planted in a collagen plate for osteoclast differentiation until osteoclasts begin to form, then the cells are transferred into hydroxyapatite plate for osteoclast induction, and dragon blood extract or PBS is added at the same time. After osteoclasts are differentiated and mature, one half of the wells in the same group of cells are stained with TRAcP, the other half of the cells are rinsed and dried, the number of osteoclasts and the hydroxyapatite absorption condition are respectively observed, and Image J software is used for quantitative analysis.

2.5 calcium ion shaking experiment

Differentiating mature BMMs at 3X10 per well ⁴ The density of each cell is planted into a 96-well plate, and on the next day, 50 ng/mu l of RANKL is added into each well of an experimental group to induce the BMMs to differentiate towards OC, wherein 30 mu M of D.P is added into a drug group, and PBS with the corresponding volume is added into a positive control group; the negative control group was treated as the positive control group, but induced differentiation was not performed. After 24 hours, the cells were washed twice with HBSS medium containing 2% FBS and 0.2mM carboxybenzenesulfonamide, then 100. Mu.l Fluo4-AM reagent was added per well, and incubated in a cell incubator for 45 minutes in the absence of light; continuously washing the cells once by using the HBSS culture medium, standing for 20 minutes in a dark place at normal temperature, capturing opening and closing signals of a calcium ion channel of a single cell within 3 minutes by using a fluorescence inverted microscope, and capturing once every two seconds; and the fluorescence intensity of each group of cells was quantitatively analyzed using NIS-Elements Viewer software.

2.6 immunofluorescence staining experiments

Differentiating mature BMMs at 5X10 per well ⁴ The density of individual cells was seeded into glass plates. The cells of each group were differentiated into OC by adding RANKL 50 ng/. Mu.l at

days

1, 3 and 5, respectively, and then 30. Mu.M D.P was added to the group of the concurrent drugs and PBS was added to the control group in a corresponding volume. The culture medium or the differentiation solution is replaced every two days until the OC in the positive control group is differentiated and matured on the sixth day. After the cells were differentiated and matured, each group of cells was washed once with PBS, and then fixed with 4% paraformaldehyde solution 10Minute, wash cells three more times with PBS; rupture of membranes for 5 minutes by adding 100. Mu.l of 0.1% Triton X-100 solution per well, followed by blocking for 30 minutes at room temperature using 3% BSA-PBS solution, followed by incubation of NFATc1 protein antibody for 2 hours at room temperature followed by 30 minutes of the corresponding protein secondary antibody (FITC label) in the absence of light; after one PBS wash, actin bands were stained with phalloidin for 20 min and nuclear DNA was stained with Hoechst 33258 substrate for 10 min. After the staining is finished, the cells are washed three times by PBS, then sealed by an anti-fluorescence quenching sealing solution, mounted by a cover glass, and photographed by a Nikon confocal microscope to observe the fluorescence intensity of each group of cells.

2.7 short-time gradient Western blot experiment

Differentiating mature BMMs at 2.5x10 per well ⁵ The density of each cell is planted in a 6-well plate, and the culture solution is replaced every two days until the cells grow to cover about 90% of the bottom area of the culture bottle. Starving the cells for 2 hours by using alpha-MEM medium without FBS, adding 30 mu M of D.P and a corresponding volume of PBS to the experimental group and the control group respectively, and culturing for 1 hour; subsequently, 50 ng/. Mu.l of RANKL was added to each experimental group and the corresponding control group at 0, 5, 10, 20, 30, 60 min, respectively. After the intervention was complete, the cells were lysed using RIPA lysate and the protein was extracted. Proteins were separated using SDS-PAGE electrophoresis, membrane-transferred using nitrocellulose membrane, followed by overnight incubation of protein antibodies at 4 ℃ for 2 hours with the corresponding secondary antibodies, followed by development using ECL developer, LAS4000 developer, and analysis of the images using Gel-pro software. The incubated target protein antibodies are anti-p-JNK, anti-I kappa B-alpha and p-p65, and the internal reference protein antibodies are anti-JNK, anti-beta-actin and p65 respectively.

2.8qPCR experiment

Differentiating mature BMMs at 1x10 per well ⁵ The proportion of individual cells was seeded in 6-well plates. Next day, 50ng/ul RANKL was added to each well to differentiate osteoclasts, gradient concentrations of D.P were added to the experimental group, the same volume of PBS was added to the control group, and the medium was changed every two days. After the sixth day (osteoclast differentiation and maturation), cells were lysed using Trizol lysate and RNA was extracted; use ofCarrying out reverse transcription on Oligo-dT, and then detecting by adopting a SYBR Green method and using a ViiATM7 Real-time PCR instrument, wherein the sequences of target gene primers are respectively as follows:

acp5 (forward primer: 5'-TGTGGCCATCTTTATGCT-3';

downstream primer 5'-GTCATTTCTTTGGGGCTT-3');

nfatc1 (upstream primer: 5'-CAACGCCCTGACCACCGATAG-3';

downstream primer 5'-GGCTGCCTTCCGTCTCATAGT-3');

ctr (upstream primer: 5'-TGGTTGAGGTTGTGCCCA-3';

5 'of downstream primer CTCGTGGGTTTGCCTCATC-3');

c-fos (forward primer: 5'-GCGAGCAACTGAGAAGAC-3';

downstream primer 5'-TTGAAACCCGAGAACATC-3');

ctsk (upstream primer: 5'-GGGAGAAAAACCTGAAGC-3';

downstream primer 5'-ATTCTGGGGACTCAGAGC-3');

mmp9 (upstream primer: 5'-CGTGTCTG GAGATTCGACTTGA-3';

downstream primer 5'-TTGGAAACTCACACGCCAGA-3');

hmbs (forward primer: 5'-AAGGGCTTTTCTGAGGCACC-3';

downstream primer 5'-AGTTGCCCATCTTTCATCACTG-3');

hprt1 (forward primer: 5'-TCAGTCAACGGGGGACATAAA-3';

downstream primer 5'-GGGGCTGTACTGCTTAACCAG-3');

and analyzing the expression quantity of each target gene by adopting a delta-delta Ct method.

2.9 Long-term gradient Western blot experiment

Differentiating mature BMMs at 1x10 per well ⁵ The individual cell ratios were seeded into 6-well plates. Next day, 50ng/ul RANKL per well was added to induce osteoclast differentiation, while 30uM d.p was added to the experimental group and the same volume of PBS was added to the control group, and the medium was changed every two days. After the sixth day (osteoclast differentiation and maturation), cells were lysed using RIPA lysate and protein was extracted. Use ofSDS-PAGE electrophoresis separates proteins, uses nitrocellulose membrane for membrane transfer, then incubates NFATc1, c-Fos, integrin beta 3, MMP9, CTSK and beta-actin protein antibodies overnight in an environment of 4 ℃, incubates corresponding secondary antibodies for 2 hours, then uses ECL developer, LAS4000 developer for development, and uses Gel-pro software to analyze images.

3. Statistical treatment

Statistical processing of experimental data of the invention: metering data adoption

Represents; SPSS 24.0 software is used for statistical analysis, non-pairing t test is adopted for comparison between two groups of independent samples, and corresponding single-factor or two-factor variance analysis is adopted for comparison between multiple groups of independent samples; defining p < 0.05 as statistically significant for the differences, denoted by "+"; data were plotted statistically using GraphPad Prism7 software.

4. Results

4.1 Inhibition of RANKL-induced osteoclastogenesis by d.p

In order to verify the inhibitory effect of dracorhodin, a component in the dragon blood, on osteoclast formation, D.P with gradient concentrations of 0 muM, 1 muM, 5 muM, 10 muM, 20 muM and 30 muM is respectively used for interfering the induced differentiation of BMMs, and the result is shown in figure 1. As can be seen from fig. 1A and 1B, the gradient concentration of d.p gradually inhibited the amount and size of osteoclast formation, and the d.p started to inhibit osteoclast formation at a low concentration (5 μ M), and the inhibitory effect at a concentration of 30 μ M was already significant; as can be seen from fig. 1C, the results of the drug toxicity test showed that the d.p drugs at concentrations of 0 μ M, 1 μ M, 5 μ M, 10 μ M, 20 μ M, and 30 μ M did not inhibit the activity of Bone Marrow Macrophages (BMMs) and were not toxic to Bone Marrow Macrophages (BMMs).

4.2 Inhibition of RANKL-induced osteoclast bone resorption by D.P

In order to verify the inhibition effect of dracorhodin, a component in the dragon blood, on the bone resorption function of osteoclasts, the invention intervenes the osteoclasts which are differentiated by using D.P with the concentration of 30 mu M, and the result is shown in figure 2. The results of fig. 2 show that the bone resorption area of the bone plate of the experimental group is significantly smaller than that of the control group; in addition, d.p inhibits the formation of the actin loop and focal adhesion protein on the surface of osteoclasts.

4.3 Inhibition of RANKL-induced osteoclast-associated gene expression by D.P

In the study, D.P with different concentration gradients (0 mu M, 20 mu M and 30 mu M) is selected to interfere with the RANKL-induced osteoclast formation, and the result is shown in figure 3. The qPCR result shows that D.P has inhibition effect on the gene expression of Acp5, c-fos related to osteoclast formation, ctsk related to bone resorption function and Mmp9, and is dose-dependent.

4.4 Inhibition of RANKL-induced osteoclast-associated protein expression by D.P

The research of the invention adopts 30 mu M D.P to intervene the formation of RANKL induced osteoclast, and the long-time gradient Western blot experiment result is shown in figure 4. The result shows that the D.P can obviously inhibit the expression of c-Fos, integrin beta 3, MMP9 and CTSK proteins related to osteoclast differentiation.

4.5 Inhibition of RANKL-induced MAPK, NF-kB signaling pathway by D.P

The experimental result of Western blot for interfering the formation of RANKL-induced osteoclast by using 30 mu M D.P in the research of the invention is shown in figure 5. The result shows that the D.P can obviously inhibit the phosphorylation expression of JNK and p65 proteins and inhibit the degradation of I kappa B-alpha protein, thereby indicating that the D.P can inhibit the RANKL-induced MAPK and NF-kappa B signal pathways.

4.6 Inhibition of RANKL-induced NFATc1 Activity by D.P

The calcium ion oscillation experiment result of the invention is shown in fig. 6, and the result shows that fig. 6A shows that d.p can inhibit the opening and closing of calcium ion channels; FIGS. 6B-D show the contemporaneous suppression of Ctr, nfatc1 gene expression and NFATc1 protein expression. In addition, immunofluorescent staining results showed that d.p can inhibit NFATc1 migration into the nucleus.

In conclusion, the dracorhodin or dracorhodin perchlorate can inhibit the formation of osteoclasts induced by RANKL, inhibit the bone resorption function of osteoclasts induced by RANKL, inhibit the gene expression related to osteoclasts induced by RANKL, inhibit the protein expression related to osteoclasts induced by RANKL, inhibit MAPK and NF-kB signal pathways induced by RANKL and inhibit the activity of NFATc1 induced by RANKL.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of dracorhodin or dracorhodin perchlorate as an active ingredient for the preparation of a medicament for the treatment of osteoporosis, osteonecrosis or Paget's disease of the bone, osteomyelitis, bone cysts, myeloma, bone metastases, osteoblasts, non-ossifying fibroids or giant cell tumors of the bone, characterized in that said treatment is by inhibition of osteoclastogenesis or inhibition of osteoclastogenesis bone resorption or prevention of osteolysis;

the structural formula of the dracorhodin is as follows:

the dracorhodin perchlorate has the following structural formula:

2. the use according to claim 1, wherein the medicament is for inhibiting expression of related genes Acp5, c-fos, ctsk, mp9, ctr or Nfatc1 in RANKL-induced osteoclastogenesis.

3. Use according to claim 1, wherein the medicament is for inhibiting the expression of related proteins NFATc1, c-Fos, integrin β 3, MMP9 or CTSK during RANKL induced osteoclastogenesis.

4. The use according to claim 1, wherein the medicament is for inhibiting NFATc activity.

5. The use according to claim 1, wherein the medicament is for inhibiting the calcium signaling pathway, the MAPK signaling pathway or the NF- κ B signaling pathway during RANKL-induced osteoclast differentiation.

6. The use according to claim 1, wherein the medicament is in the form of a solid tablet or powder.

7. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment of osteoporosis, osteonecrosis or Paget's bone disease, osteomyelitis, bone cysts, myeloma, bone metastases, osteoblastomas, non-ossifying fibroids or giant cell tumors of bone, wherein the active ingredient in the pharmaceutical composition is dracorhodin or dracorhodin perchlorate; the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; the dosage form of the pharmaceutical composition is solid tablets or powder; the treatment is by inhibiting osteoclastogenesis or inhibiting osteoclastic bone resorption or preventing osteolysis;

the structural formula of the dracorhodin is as follows:

the structural formula of the dracorhodin perchlorate is as follows: