Application of amygdalin A in the preparation of products for preventing and treating osteoporosis

Abstract

The present invention belongs to the field of medicine, and specifically relates to an application of amygdalin A in the preparation of a product for preventing and treating osteoporosis. The present invention first uses tartrate-resistant acid phosphatase staining, cell activity analysis, protein immunoblotting, etc. at the cellular level to explore the effects and molecular mechanisms of amygdalin A on osteoclastogenesis, and then uses biomembrane interference technology and SPR to explore the interaction between AA and RANKL and RANK and the effects of amygdalin A on the binding of RANKL to its receptor, and finally in the ovariectomized mouse osteoporosis model, hematoxylin-eosin staining, TRAP staining, real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay are used to verify the improvement of amygdalin A on bone loss in ovariectomized mice. The present invention first proves the biological activity of amygdalin A against osteoclastogenesis, and provides a basis for the development of amygdalin A as a potential osteoporosis drug.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to an application of amygdalin A in preparing a product for preventing and treating osteoporosis. Background Art

[0002] Osteoclasts are multinucleated cells formed by the fusion of multiple mononuclei in the hematopoietic stem cell lineage. They have a transparent pancake-shaped outline and clear boundaries. Their main characteristics are the absorption and degradation of mineralized bone matrix. The differentiation of osteoclast progenitor cells to mature osteoclasts depends on the presence of receptor activator of nuclear factor (RANKL) and receptor activator of nuclear factor (RANK). Among them, RANK is involved in the occurrence of many diseases and activates RANK receptors on the surface of osteoclasts. RANK is a multifunctional cytokine that involves many genes in the NF-κB signal transduction system. In addition, it is one of the important molecular targets for regulating inflammatory responses and immune responses. When RANKL and RANK act together, some regulatory transcription factors and enzymes increase, which can promote osteoclast differentiation, proliferation and survival. Therefore, reducing the activity and maturity of osteoclasts has become an important treatment for osteoporosis.

[0003] At present, bone resorption inhibitors are the main treatment for osteoporosis. Conventional drugs include bisphosphonates, estrogen replacement therapy and selective estrogen receptor modulators. Among them, bisphosphonates are currently the first choice treatment for osteoporosis. Their mechanism of action is to increase the accumulation of collagen in the bone matrix, improve bone quality, reduce the risk of fractures, inhibit osteoclast activity, hinder bone turnover, prolong bone mineralization, and reduce trabecular damage. Estrogen receptor modulators are therapeutic drugs developed based on estrogen. Estrogen binds to the receptor and regulates estrogen-related genes with ultra-high affinity, regulates the activation and differentiation of osteoclasts, and enhances osteoblast-mediated bone resorption.

[0004] Parathyroid hormone analogs, active vitamin D and fluoride are common bone formation promoters, among which parathyroid hormone is recognized as the most promising bone calcium and phosphorus promoter, and is gradually used in the prevention and treatment of primary osteoporosis. Its main mechanism of action is: promoting osteoblast proliferation and activation, inhibiting osteoclast apoptosis, stimulating osteoclast bone uptake, promoting bone calcium and phosphorus release and intestinal calcium and phosphorus reabsorption.

[0005] However, although many traditional osteoporosis drugs are widely used in clinical practice and have significant therapeutic effects, their various disadvantages have become increasingly obvious and have attracted people's attention as their usage increases. With the continuous deepening of scientific research, targeted therapy has become the development trend of future drugs. New drugs related to the RANK signaling pathway, such as denosumab; new drugs related to the Wnt signaling pathway, such as sclerostin antibodies, are a new type of bone formation promoter. In osteoblasts, sclerostin antibodies specifically bind to sclerostin, indirectly induce β-catenin to enter the cell nucleus, regulate gene expression, and promote the formation of osteoblasts.

[0006] When the body is affected by trauma or disease, a large number of osteoclasts with different functions appear in the body. These cells participate in regulating bone metabolism by secreting cytokines, adhesion molecules and other pathways, thereby promoting fracture occurrence. Therefore, controlling the unbalanced differentiation of osteoclasts in bone remodeling is particularly important for the treatment of osteoporosis. Osteoclasts have unique morphological and phenotypic characteristics, such as multiple nuclei, TRAP and calcitonin receptor expression. The differentiation and activation of osteoclasts are regulated by a variety of cytokines, among which RANKL is the most important one. It directly binds to the homologous receptor RANK and initiates dynamic intracellular signal transduction including NF-κB and mitogen-activated proteins (MAPKs). This synergistic effect induces the expression and activation of osteoclast prokaryotic factor c1 (NFATc1), c-Fos, TRAP, c-Src and cathepsin K, promoting the activation of osteoclasts. Osteoclasts have become a key target for the development of drugs for the treatment and prevention of osteoporosis.

[0007] At present, there are few reports on the study of natural compounds inhibiting osteoclastogenesis by interfering with RANKL-RANK interaction. Therefore, it is particularly important to study the natural compounds derived from plants to block RANKL-RANK interaction to improve osteoporosis. Summary of the invention

[0008] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide an application of amygdalin A in the preparation of products for preventing and treating osteoporosis.

[0009] Another object of the present invention is to provide the use of the above-mentioned amygdalin A in the preparation of products for inhibiting the generation or activity of osteoclasts.

[0010] Another object of the present invention is to provide a drug for preventing and treating osteoporosis, comprising the above-mentioned amygdalin A.

[0011] The fourth object of the present invention is to provide a drug for inhibiting the generation or activity of osteoclasts, comprising the above-mentioned amygdalin A. The object of the present invention is achieved by the following technical solutions:

[0012] A use of amygdalin A in the preparation of a product for preventing and treating osteoporosis, wherein the structural formula of the amygdalin A is shown in Formula I:

[0013] ;

[0014] The osteoporosis may be postmenopausal osteoporosis;

[0015] The use of the amygdalin A in the preparation of products for inhibiting osteoclast formation or activity;

[0016] The amygdalin A inhibits the phosphorylation of MAPKs, AKT or NF-κB pathways;

[0017] The amygdalin A inhibits the expression of transcription factors FATc1 and c-Fos that regulate osteoclastogenesis;

[0018] The amygdalin A inhibits the expression of osteoclast marker proteins, and the marker proteins are TRAP, c-Src, and cathepsin K or NFATc1;

[0019] The use of the amygdalin A in preparing a product for inhibiting the binding of RANKL and RANK;

[0020] The use of the amygdalin A in preparing products for improving bone loss or promoting bone formation;

[0021] The amygdalin alcohol can reduce the ARANKL content in serum;

[0022] The amygdalin A can increase the content of PINP and OPG in serum;

[0023] A drug for preventing and treating osteoporosis, comprising the above-mentioned amygdalin A;

[0024] A drug for inhibiting osteoclast formation or activity, comprising the above-mentioned amygdalin A;

[0025] The drug preparations include a variety of clinical drug dosage forms, such as tablets, injections, liposome nanoparticles, controlled release agents, etc.

[0026] The medicament may contain one or more pharmaceutically acceptable carriers, excipients or diluents;

[0027] The drug preferably further comprises pharmaceutical excipients or other compatible drugs;

[0028] The pharmaceutical excipients are conventional pharmaceutical excipients, such as solvents, disintegrants, flavoring agents, preservatives, colorants or adhesives, etc.

[0029] The other compatible drugs refer to the effective dose of amygdalin A as the drug raw material, and then combined with other natural drugs or chemical drugs;

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] (1) The present invention used tartrate-resistant acid phosphatase (TRAP) staining, cell viability assay (MTT), and protein immunoblotting (WB) to explore the effect of AA on osteoclastogenesis and its molecular mechanism. The results showed that AA significantly inhibited RANKL-induced osteoclastogenesis in vitro without cytotoxic side effects. Specifically, AA significantly inhibited the expression of RANKL-induced osteoclast-related specific proteins TRAP, c-Src, and cathepsin K, and inhibited the expression of key transcription factors NFATc1 and c-Fos. AA significantly inhibited the phosphorylation of NF-κB (p65, IκBα, IKKα / β), MAPKs (ERK1 / 2, p38, JNK), and AKT pathways downstream of RANK.

[0032] (2) The present invention uses biomembrane interferometry (BLI) and SPR to explore the interaction between AA and RANKL and RANK and the effect of AA on the binding of RANKL to its receptor. The results show that at the molecular level, AA directly binds to RANKL and RANK with strong affinity, and its K D The values ​​are 1.517×10 -6 M, 1.368×10 -6 M, SPR shows that the higher the AA concentration, the stronger the binding; AA interferes with the interaction between RANKL and RANK. In the presence of AA, the affinity between RANKL and RANK decreases 5.6 times. The higher the AA concentration, the more obvious the interference.

[0033] (3) In the osteoporosis model of ovariectomized mice, hematoxylin-eosin (H&E) staining, TRAP staining, real-time fluorescence quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to verify the improvement effect of AA on bone loss in ovariectomized mice. The results showed that AA significantly increased the trabecular bone area of ​​ovariectomized mice, reduced the activity of TRAP-positive osteoclasts, reduced bone loss in mice, and had no toxic side effects on mouse organs; AA significantly reduced the expression of osteoclast-specific marker proteins and genes TRAP, c-Src, cathepsin K, NFATc1 and c-Fos in bone tissue, reduced the content of RANKL in serum, and increased the content of osteogenic markers osteocalcin (OCN), type I procollagen amino-terminal propeptide (PINP) and osteoprotegerin (OPG).

[0034] In summary, AA directly binds to RANKL and RANK and inhibits their interaction, thereby inhibiting the activation of NF-κB, MAPKs and AKT pathways, downregulating the expression levels of transcription factors NFATc1 and c-Fos, leading to downregulation of osteoclast-specific marker genes and proteins, inhibiting osteoclast formation, and improving bone loss in ovariectomized mice. This invention demonstrates for the first time the biological activity of AA in inhibiting osteoclast formation, providing a basis for the development of AA as a potential osteoporosis drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The results of AA inhibiting RANKL-induced osteoclastogenesis without cytotoxicity in Example 1 are shown in Figure 1, wherein: (A): chemical structural formulas of amaranol A (AA) and amaranol B (AB); (B): TRAP staining method was used to detect the effects of different concentrations of AA and AB on 50 ng / mL RANKL-induced osteoclastogenesis, and representative images were collected under an optical microscope (magnification: 40 times); (C): quantification of TRAP-positive osteoclasts per well induced by 50 ng / mL RANKL (≥3 nuclei were counted as one osteoclast); (D): TRAP staining method was used to detect the effects of different concentrations of AA and AB on 10 ng / mL RANKL-induced osteoclastogenesis, and representative images were collected under an optical microscope (magnification: 40 times); (E): quantification of TRAP-positive osteoclasts per well induced by 10 ng / mL RANKL (≥3 nuclei were counted as one osteoclast); (F) MTT method was used to detect the effects of different concentrations of AA and AB on RAW Effect of 264.7 cell toxicity. Values ​​are expressed as mean ± SEM of three independent repeated experiments; ###P<0.001 compared with the control group; compared with the group treated with RANKL alone .

[0036] Figure 2The results of AA inhibiting the expression of osteoclastogenesis-related marker proteins and key transcription factors in Example 2, wherein, (A): Western blotting analysis of the effects of different concentrations of AA on the expression of TRAP, c-Src and cathepsin K proteins in RANKL-induced RAW 264.7 cells, showing representative images; (B-D): AlphaView software was used to analyze the grayscale values ​​of TRAP (B), c-Src (C) and cathepsin K (D) bands respectively; (E): Western blotting analysis of the expression of key transcription factors NFATc1 and c-Fos proteins in RANKL-induced RAW 264.7 cells, showing representative images; (F-G): AlphaView software was used to quantify the expression of NFATc1 (F) and c-Fos (G) proteins, and the values ​​were expressed as mean ± SEM of three independent repeated experiments, compared with the control group ###P < 0.001; compared with the group treated with RANKL alone .

[0037] Figure 3 The results of AA inhibiting the phosphorylation of MAPK and AKT pathways, where (A): RAW 264.7 cells were pretreated with serum-free DMEM medium for 20 min in the presence or absence of AA (10 μM), and stimulated with RANKL (10 ng / mL) at different time points. The cell lysates were subjected to protein immunoblotting analysis and identified with corresponding primary antibodies. Representative images are shown; (BE): The grayscale ratios of p-AKT, p-p38, p-JNK and p-ERK1 / 2 to the total amount of AKT, p38, JNK and ERK1 / 2 were calculated using AlphaView software. The values ​​are expressed as mean ± SEM of three independent repeated experiments. Compared with the RANKL treatment group at the same time point, .

[0038] Figure 4 The results of AA inhibiting the phosphorylation of the NF-κB pathway, where (A): RAW 264.7 cells were pretreated with serum-free DMEM medium for 20 min in the presence or absence of AA (10 μM), and stimulated with RANKL (10 ng / mL) at different time points. The cell lysates were taken for protein immunoblotting analysis and identified with the corresponding primary antibodies to show representative images; (BD): AlphaView software calculated the grayscale values ​​of p-IKKα / β, p-IκBα, and p-p65 and their total IKKα / β (IKKα+IKKβ), IκBα, and p65; the values ​​are expressed as mean ± SEM of three independent repeated experiments. Compared with the RANKL treatment group at the same time point .

[0039] Figure 5 The results of the interaction between AA and RANKL-RANK are shown in Figure 2. (A): BLI detected the interaction between AA (20 μM) and RANKL. The binding and dissociation times were both 300 s, and the affinity was 1.517×10 -6 M; (B): BLI detected the interaction between AA (20 μM) and RANK, with the binding and dissociation times both being 300 s and the affinity being 1.368×10 -6 M; (C): BLI detection of the interaction between RANKL and RANK in the presence or absence of AA (10 μM); (D): SPR determination of the interaction affinity between AA (5-0.625 μM, serial dilution) and RANKL, with a KD value of 2.788×10 -6 M; (E): SPR assay for the interaction affinity between AA (5-0.625 µM, serial dilution) and RANK, with a KD value of 1.765×10 -6 M; (F): SPR detection of the interaction between AA (0-20 μM) and RANKL-RANK.

[0040] Figure 6 The results of the effect of AA on the body weight and organs of ovariectomized mice are shown in the figure, where (A): Schematic diagram of the animal experiment; (B): Body weight of each group of mice during injection (g); (C): Liver weight (g); (D): Liver coefficient; (E): Liver H&E staining; (F): Kidney weight (g); (G): Kidney coefficient; (H): Kidney H&E staining, compared with the sham operation group, ##P < 0.01, compared with the OVX group, .

[0041] Figure 7 The figures show the results of AA inhibiting bone loss in ovariectomized mice, including: (A) H&E staining of mouse femoral sections, magnified 100 times, showing representative images; (B): quantification analysis of femoral trabecular area; (C): TRAP staining of mouse femoral sections, magnified 200 times, showing representative images; (D): TRAP activity analysis; (G): ELISA kit to detect the osteoclast marker RANKL in mouse serum; (E, F, H): the levels of osteoblast markers OCN (E), P1NP (F) and OPG (H); (I): Western blotting analysis of osteoclast-related marker protein expression in bone tissue; (JN): qRT-PCR analysis of the expression of osteoclast-related marker genes TRAP (J), c-Src (K), cathepsin K (L), NFATc1 (M) and c-Fos (N). DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0043] 1. The main experimental materials and reagents involved in the examples are shown in Table 1.

[0044] Table 1 Materials and reagents

[0045]

[0046] 2. Preparation of the main solutions involved in the examples

[0047] (1) Preparation of 10 mM AA solution

[0048] Accurately weigh an appropriate amount of AA powder into a 1.5 mL centrifuge tube (EP), add the corresponding volume of sterile dimethyl sulfoxide (DMSO) solution on a sterile operating table to make a 10 mM solution, fully dissolve and mix, and then divide it into 10 μL / vial and freeze it in a -20℃ refrigerator. (Avoid repeated freezing and thawing of the drug)

[0049] (2) Preparation of PBS buffer

[0050] Phosphate buffered saline (PBS) powder was placed in a 5L clean beaker, 2L of deionized water (up water) was added, and stirred with a magnetic stirrer until completely dissolved. After sterilization in a high-temperature sterilizer at 120℃ for 20 min, it was used in the cell sterility experiment.

[0051] (3) Preparation of 10 μg / mL RANKL solution

[0052] RANKL (R&D, USA) stored at -20°C was equilibrated to room temperature in advance. 0.01 g of bovine serum albumin (BSA) powder was weighed and added to 10 mL of PBS buffer to prepare a BSA working solution with a final concentration of 0.1%. The working solution was filtered through a 0.22 μm sterile filter on a sterile operating table. 1 mL of the filtered BSA working solution was added to 10 μg of RANKL protein, mixed thoroughly, and stored in aliquots at -20°C for later use.

[0053] (4) MTT preparation

[0054] Accurately weigh 0.2 g of MTT powder and place it in a 50 mL centrifuge tube. Add 40 mL PBS, stir thoroughly and ultrasonicate until the powder dissolves. Filter with a 0.22 μm filter and store in a dark place. Dissolve in advance and place in a 4°C refrigerator before use. (Avoid light during the preparation process)

[0055] (7) Preparation of WB reagent

[0056] Preparation of 10% APS solution: Accurately weigh 2 g of ammonium persulfate (APS) into a centrifuge tube, add 20 mL of up water to make the final concentration 10% (m / v), and store in a 4°C refrigerator for later use;

[0057] Preparation of 10% SDS solution: accurately weigh 2 g of sodium dodecyl sulfate (SDS) in a centrifuge tube in a fume hood, add 20 mL of up water to make the final concentration 10% (m / v), and store at room temperature for later use;

[0058] Preparation of 8% separation gel (4 pieces): 4.68 mL of up water, 2.5 mL of 1.5 M Tris-HCl (pH 8.8), 2.66 mL of 30% Acr-Bis, 0.1 mL of 10% SDS, 0.05 mL of 10% APS, 0.004 mL of TEMED;

[0059] Preparation of 4% stacking gel (4 pieces): 3.2 mL of up water, 1.25 mL of 1.5 M Tris-HCl (pH 8.8), 0.7 mL of 30% Acr-Bis, 0.05 mL of 10% SDS, 0.05 mL of 10% APS, and 0.005 mL of TEMED;

[0060] Preparation of 10× electrophoresis buffer (the amount required to make up to 1 L with water): Tris base 30.29 g, glycine 144.13 g, SDS 10 g;

[0061] Preparation of 10× transfer buffer (the amount required to make the volume to 1 L with water): Tris base 72.5 g, glycine 36.25 g, SDS 4.63 g;

[0062] Preparation of 10× TBST solution (water to 1 L): 88 g sodium chloride (NaCl), 24 g Tris base, 10 mL Tween-20, pH 7.6;

[0063] Preparation of blocking solution: weigh 2 g BSA or skim milk powder, inject into 40 mL PBS or 1×TBST to make 5% blocking solution, shake well to mix, and store in a 4℃ refrigerator for 3-4 days;

[0064] Preparation of primary antibody solution: The dilution medium of the primary antibody solution is 5% BSA dissolved in PBS, and 0.1% sodium azide is added. The dilution ratio of the antibody used for Western Blot (WB) experiments is 1:1000 (v / v);

[0065] Preparation of secondary antibody solution: Add the secondary antibody of the corresponding source at a ratio of 1:5000 to the blocking solution, mix well and store at 4°C, and recycle repeatedly. The validity period is 3 days;

[0066] (10) Preparation of 5× non-reducing loading solution: 21 mL of up water, 11.2 mL of 10% SDS, 7 mL of 0.5 M Tris-HCl (pH 6.8), 11.2 mL of glycerol, and 2.8 mL of 0.5% bromophenol blue. After preparation, store at -20°C. Add reducing loading solution at a ratio of 9:1 (v / v) of non-reducing loading solution and β-mercaptoethanol. Mix well and store at 4°C for later use.

[0067] 3. Statistical analysis of data in the examples:

[0068] The experimental data were processed by T-TEST and univariate level analysis, SPSS17.0 software and GraphPad Prism 5. The data of each index are expressed as mean ± standard error (mean ± SEM), P < 0.05 is considered to be statistically different; P < 0.01 indicates a significant statistical difference; P < 0.001 indicates an extremely significant statistical difference.

[0069] Example 1 AA inhibits RANKL-induced osteoclastogenesis

[0070] 1. Test methods

[0071] 1.1 Cell culture

[0072] (1) Cell culture conditions

[0073] The cell experiment used mouse mononuclear macrophage leukemia cells (RAW 264.7) and cultured them in a mixed medium containing 10% fetal bovine serum (FBS) and 1% double-antibody (P / S) DMEM. The incubator was a water-jacketed incubator with a culture temperature of 37°C, a CO2 content of 5%, and a humidity of 95%.

[0074] (2) Cell recovery

[0075] On a sterile operating table, aspirate the cell suspension and add it to a 50 mL centrifuge tube containing 10 mL of culture medium. Mix by pipetting and centrifuging at 1500 r for 3 min. Discard the supernatant, resuspend in 10 mL of culture medium and add to a 100 mm culture dish, and culture in a 37°C incubator.

[0076] (3) Cell passaging

[0077] When the cell growth density has reached 90% of the entire culture dish, aspirate the old culture medium, gently blow off the cells with new culture medium, place the cells in a 50 mL centrifuge tube and blow them apart, take 1 mL of the cell suspension in a new 100 mm culture dish containing 10 mL of culture medium, and place it in the incubator for continued culture.

[0078] (4) Cell cryopreservation

[0079] Aspirate the old culture medium, blow off the cells gently with new culture medium, count the cells, centrifuge at 1500 r for 3 min, prepare freezing solution (fetal bovine serum: sterile DMSO 9:1 (v / v)), remove the supernatant after centrifugation, resuspend the cells in freezing solution, and the cell concentration is 3×10 5 / mL-1×10 6 / mL. After fully resuspending, inject 1 mL of each tube into the cryopreservation tube and place it in the cell freezing box. The principle of cell freezing is "slow freezing and fast thawing". First put the box in a -80℃ refrigerator for 72 hours, and then transfer it to a -120℃ refrigerator for long-term storage.

[0080] 1.2 Cytotoxicity analysis

[0081] The MTT method was used to determine the effect of AA on the proliferation of RAW 264.7 cells. The specific method is as follows:

[0082] RAW264.7 cells were seeded in 96-well plates at a ratio of 15,000 / well and cultured in an incubator overnight. After stable attachment, they were treated with serum-free DMEM containing 1% P / S. Blank (no cells), control (with cells) and different concentrations of AA or AB treatment groups were set up respectively. The AA treatment concentrations were 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, and the AB treatment concentrations were 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. There were 6 parallels for each concentration, and 200 μL of AA solution or AB solution was added to each well. The same concentration of treatment solution was first prepared in a 60mm small dish and fully mixed, and then added to the 96-well plate using a spray gun. After treatment, it was cultured in an incubator for 48 h. After 48 hours, add 20 μL / well MTT in a dark place and incubate in a 37°C incubator for 4 hours. Carefully aspirate the liquid in the wells, add 200 μL / well DMSO, shake the plate until the purple crystals are completely dissolved, and measure the absorbance (OD) value at 492 nm with a high-end microplate reader.

[0083] 1.3 In vitro osteoclastogenesis analysis

[0084] RAW264.7 cells were seeded into a 96-well plate at a ratio of 2000 / well and cultured in an incubator overnight. After stable attachment, the cells were treated with DMEM medium containing 6% fetal bovine serum. A control group (with cells, no drugs and no RANKL induction), a RANKL-only treatment group (with cells, only RANKL induction) and a drug-treated group (with cells, RANKL induction after adding drugs) were set up respectively. The AA treatment concentrations were 5 μM, 10 μM, 15 μM, and 20 μM, and the AB treatment concentrations were 5 μM, 10 μM, 15 μM, and 20 μM. There were 4 parallels for each concentration. The treatment method was consistent with the above-mentioned MTT treatment. After treatment, the cells were immediately placed in the incubator for culture for 15 min. Then, RANKL (50 ng / mL or 10 ng / mL) was added to each well of the RANKL-only treatment group and the drug-treated group, and the cells were placed in the incubator for culture for 4-5 days (the number of osteoclasts generated was observed and TRAP staining was performed. One cell with > three nuclei was one osteoclast). TRAP staining was performed according to the TRAP kit.

[0085] 2. Test results

[0086] The structural formulas of AA and AB are as follows Figure 1 As shown in A, TRAP staining was used to directly observe the effects of AA and AB on osteoclastogenesis from an epigenetic biological perspective. In RAW 264.7 cells, after treatment with different concentrations of AA or AB (10, 20 μM), RANKL (50 ng / mL) stimulated RAW246.7 cells. After 5 days of culture, multinucleated transparent pancake-shaped cells appeared and were considered to be osteoclasts. The staining results showed that RANKL treatment could significantly increase the number of TRAP-positive cells (P< 0.001; Figure 1 B, C), while AA treatment significantly inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner (P < 0.05 or P < 0.001; Figure 1 In contrast, AB had no significant effect on RANKL-induced osteoclastogenesis ( Figure 1 B, C).

[0087] To further verify the effect of AA or AB on osteoclastogenesis, cells were stimulated with RANKL (10 ng / mL) under the conditions of different concentrations of AA or AB (5, 10, 15, 20 μM). TRAP staining results showed that RANKL could also significantly induce osteoclastogenesis. Consistent with the above results, AA treatment inhibited the number and size of osteoclastogenesis in a dose-dependent manner, among which 15 μM and 20 μM had the most significant inhibitory effects (P < 0.001; Figure 1D, E). However, when the concentration of RANKL was reduced, AB still had no significant inhibitory effect on the formation of osteoclasts ( Figure 1 D, E), we speculated whether the C-4′ phenolic hydroxyl group played a key role in its biological activity. Since 10 ng / mL RANKL can significantly induce osteoclastogenesis, this concentration was used for subsequent cell experiments.

[0088] To exclude that AA cytotoxicity inhibits osteoclastogenesis, cytotoxicity assays were performed in RAW264.7 cells. Figure 1 As shown in Figure F, it is clear that 5-20 μM AA and AB have no effect on the viability of RAW264.7 cells, indicating that the inhibitory effect of AA on osteoclastogenesis is not mediated by its cytotoxicity. The above results show that AA can significantly inhibit the formation of osteoclasts in vitro without cytotoxicity.

[0089] Example 2

[0090] 1. Test methods

[0091] 1.1 Cell protein extraction

[0092] (1) Marker protein

[0093] RAW 264.7 cells were inoculated into 60 mm dishes at a ratio of 300,000 / dish, 4 mL / dish, and placed in an incubator overnight. The next day, the original culture medium was removed and replaced with DMEM induction medium containing 6% FBS and 1% P / S. The AA treatment concentrations were 10 μM, 15 μM, and 20 μM, respectively. The corresponding volume of drugs was directly added to the small dish with the replaced culture medium. After treatment, it was placed in an incubator for 15 min, and then RANKL (10 ng / mL) was added. Except for the control, all other dishes were added, and then placed in an incubator for 24 h. A control group (with cells, no drugs, no RANKL induction) and a single RANKL treatment group (with cells, only RANKL induction) were set up. After 24 h, aspirate the culture medium, wash with pre-cooled PBS, prepare lysis buffer (high-efficiency RIPA cell lysis buffer: PMSF = 100:1 (v / v)), add lysis buffer (about 90-130 μL, depending on cell density) immediately after washing, lyse on ice for 30 min, scrape the protein into a 1.5 mL EP tube, centrifuge at 13500 g-15000 g in a centrifuge at 4°C for 13-15 min, transfer the supernatant lysate to a new 1.5 mL EP tube for WB blotting analysis.

[0094] (2) Signaling pathway

[0095] ① RAW264.7 cells were inoculated into 60 mm small dishes at a ratio of 2-2.5 million / dish, 4 mL / dish, and treated with serum-free DMEM medium overnight. The original medium was removed on the next day, and the cells were divided into a non-drug group and a drug-added group. The non-drug group was directly replaced with serum-free medium 3 mL / dish. The drug-added group was first mixed with AA in a centrifuge tube containing serum-free medium, and then added to the small dishes 3 mL / dish, respectively. The AA treatment concentration was 10 μM, and the cells were immediately placed in an incubator for 20 min after treatment.

[0096] ②20 min later, the two groups of cells were stimulated with RANKL at different time points (5 treatment time points, 0 min, 5 min, 15 min, 30 min, 60 min), specifically: RANKL (10 ng / mL) was added from high to low time points, one dish was treated with drugs at each time point, and one dish was not treated with drugs and RANKL was added at the same time. At 0 min, all the dishes were taken out and the culture medium was discarded. The excess culture medium was sucked away with a pump and placed on ice. The lysis solution was prepared in the same way as in (1) above, and 150 μL-200 μL / dish was added. After adding the lysis solution, it was shaken and placed on ice for 30 min. The subsequent steps were the same as in (1) above. Each treatment was repeated at least three times, and a control group (with cells, no drugs, and no RANKL induction) was set up.

[0097] 1.2 Protein quantification

[0098] The BCA quantitative kit was used for quantification. The standard protein was 1 mg / mL. In a 96-well dilution plate, 68 μL of standard protein was added to 68 μL of filtered PBS. After mixing, 68 μL was aspirated and added to the next well containing 68 μL PBS. The standard curve had five concentrations, namely 0.03125 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL. After mixing the 5 wells, 20 μL was aspirated with a gun and transferred to a 96-well ELISA plate. Three parallels were made for each concentration. 68 μL PBS in the sample well was added with 2 μL of protein sample (diluted 35 times) and mixed with a gun. After that, 20 μL was transferred to the ELISA plate. Three parallels were made for each sample. After the transfer, the color developing solution was prepared. The color developing solution was prepared (A solution: B solution = 50:1 (v / v)). 200 μL was added to each well. μL, add the colorimetric solution and place in a 37°C incubator for 30 min; after 30 min, measure the OD value using dual wavelength detection, 560 nm and 630 nm.

[0099] Protein concentration calculation: draw an XY scatter plot of the standard curve to obtain R2 and the standard equation, R2 ≥ 0.9990, substitute the absorbance value of the sample into the x of the equation, and the y value obtained is the concentration of the 35-fold dilution. Multiply it by 35 to get the concentration of the sample, and the sample volume is obtained based on the sample mass / sample concentration.

[0100] 1.3 Western blotting

[0101] (1) Sample preparation: Calculate the sample volume based on the sample mass. Add the corresponding sample volume into a 200 μL PCR tube. Add 5 μL of reducing loading solution to each tube. Centrifuge to mix well and boil the sample in a 95℃ sample cooker for 6-10 min. Sample preparation is complete. If electrophoresis is not performed immediately, the sample needs to be placed at 4℃.

[0102] (2) Preparation of gel: Select the corresponding separation gel according to the molecular weight of the antibody to be coated. First, place the gel plate on the gel rack, add the corresponding amount of components to a 50 mL centrifuge tube, shake thoroughly to mix, and then pour the gel. Seal the upper layer with up water. Allow the separation gel to solidify for more than half an hour. Use filter paper to absorb the up water and prepare concentrated gel. Add the concentrated gel on the upper layer of the separation gel and vertically insert a 0.75 mm comb. After gelling, remove the gel plate and soak it in up water for later use.

[0103] (3) Electrophoresis: Install electrodes, adjust the machine, inject electrophoresis buffer, and inject samples into the corresponding lanes. The first program is 50 V, 30 min; the second program is 120 V, 1.5 h.

[0104] (4) Transfer: Prepare 1× transfer solution and precool it. After the electrophoresis is completed, activate the polyvinylidene fluoride (PVDF) membrane with methanol and place it in the transfer solution. Soak the filter paper in the transfer solution. First stack three filter papers and then put a PVDF membrane in it. Use a special pry plate to take out the gel and stack the gel on the PVDF membrane in the direction of loading. It should be noted that there should be no bubbles between the gel and the membrane to avoid the protein from not being transferred to the position with bubbles. Stack three more filter papers on the top in the order of filter paper-membrane-filter paper. Fix them with the matching clips and place them in the transfer tank in the direction of black gel and white membrane. The current of one electrophoresis tank is 200 mA, constant current, and time is 1 h.

[0105] (5) Blocking: After transfer, place the PVDF membrane in the blocking solution and block it on a shaker for more than 1 hour.

[0106] (6) Primary antibody incubation: Take 5-10 mL of primary antibody solution (Tracp-5b (A16238), C-Src (sc-130124), CathepsinK (sc-48353), NFATc1 (sc-7294) or c-Fos (sc-271243), etc.) in a sealed small box, put in the sealed PVDF membrane, and incubate on a shaker at 4°C overnight.

[0107] (7) Secondary antibody incubation: The primary antibody was recycled multiple times. After recovery, it was quickly washed three times on a shaker with 1× TBST. The corresponding secondary antibody (HRP-anti-rabbit / mouse IgG) was added and incubated slowly at room temperature on a shaker for 1 h. The secondary antibody was recovered and washed three times with 1× TBST.

[0108] (8) Chemiluminescence: Prepare the exposure solution according to the instructions in a dark place, add the exposure solution to the exposure machine, remove the membrane, wipe off the excess liquid on the membrane, wipe off the exposure solution, start automatic exposure, save the data and analyze the data.

[0109] 2. Test results

[0110] 2.1 AA inhibits the expression of osteoclastogenesis-related marker proteins and key transcription factors

[0111] Osteoclast differentiation and bone resorption are associated with the expression of many specific genes and proteins, such as MMP-9, TRAP, c-Src, and cathespin K, most of which are target genes of NFATc1. RAW264.7 cells were induced with RANKL (10 ng / mL) as a positive control, and RANKL was induced after treatment with AA at the specified concentrations (10, 15, 20 μM). Figure 2 As shown in Figure 2, during osteoclastogenesis, AA significantly downregulated the expression levels of TRAP, c-Src, and cathepsinK ( Figure 2 In addition, the quantitative results showed that AA reduced the expression levels of related marker proteins in a concentration-dependent manner (P < 0.05 or P < 0.01 or P < 0.001; Figure 2 The results showed that AA could inhibit the expression of osteoclast-related marker proteins.

[0112] The production of osteoclasts is regulated by transcription factors. NFATc1 and c-Fos are two important transcription factors that regulate osteoclastogenesis. Upregulating the expression levels of NFATc1 and c-Fos can activate the expression of osteoclast-specific genes and proteins. This example further explores the effect of AA on key transcription factors for osteoclastogenesis. The results show that the expression levels of NFATc1 and c-Fos significantly increased after RANKL stimulation, while AA treatment significantly downregulated their expression levels in a dose-dependent manner ( Figure 2 EG), indicating that AA can inhibit the expression levels of NFATc1 and c-Fos, thereby inhibiting the expression of osteoclast-related marker proteins. The above results further illustrate that AA can inhibit the formation of osteoclasts.

[0113] 2.2 AA inhibits phosphorylation of MAPKs and AKT pathways

[0114] As mentioned above, MAPKs and AKT pathways are two important pathways for osteoclast formation, and their activation can upregulate the expression levels of NFATc1 and c-Fos, key transcription factors of osteoclasts. In order to clarify the molecular mechanism by which AA inhibits osteoclast formation, we further explored whether AA inhibits osteoclast formation by inhibiting the phosphorylation of MAPKs and AKT pathways. WB was used to examine the phosphorylation of MAPKs family and AKT in RANKL-induced RAW 264.7 cells. Figure 3 As shown in Figure 3, RANKL can significantly induce the phosphorylation of JNK, ERK and p38, while AA treatment significantly inhibited the phosphorylation of JNK, ERK and p38 induced by RANKL ( Figure 3 Similarly, AA treatment significantly inhibited the phosphorylation of AKT at 5 min and 15 min ( Figure 3 These results indicate that AA can inhibit the activation of MAPKs and AKT pathways.

[0115] 2.3 AA inhibits phosphorylation of NF-κB pathway

[0116] RANKL binding to the receptor RANK can activate the NF-κB pathway, which is one of the most important and earliest activated cascade reactions after RANKL stimulation of RAW 264.7 cells. In order to determine whether AA inhibits the activation of the NF-κB pathway in RAW 264.7 cells induced by RANKL, the same method was used in this example. After pre-treating the cells with or without AA (10 μM) for 20 min, the cells were stimulated with RANKL (10 ng / mL) at 0, 5, 15, 30, and 60 min, respectively.

[0117] The results showed that NF-κB signaling was significantly inhibited after AA treatment ( Figure 4 (A), phosphorylated p65, IκBα, and IKKα / β were significantly downregulated at 5 min (P < 0.05 or P < 0.01; Figure 4 The above results indicate that AA can significantly inhibit RANKL-induced NF-κB pathway activation.

[0118] Example 3 Intermolecular interaction analysis shows that AA inhibits RANKL-RANK interaction

[0119] 1. Test methods

[0120] 1.1 Biofilm Interferometry (BLI)

[0121] BLI technology can provide association and dissociation rate constants, as well as overall affinity KD values. BLI was used to investigate whether AA could bind to RANKL or RANK, and competition experiments were performed to test whether AA could interfere with the RANKL-RANK interaction. RANKL and RANK proteins were biotinylated with isothiocyanate-LC-biotin, and solutions containing biotinylated RANKL or RANK proteins were immobilized on the surface of a super streptavidin biosensor (ForteBioInc, MenloPark, CA, United States). The interaction of AA (20 μM) with proteins RANKL and RANK was monitored at 25°C for 300 s, and finally dissociated in PBS (pH 7.4) for 300 s, and data were collected in real time. In the competition experiment, AA (0 or 10 μM) was preincubated with immobilized RANKL for 10 min, and RANK containing AA (0 or 10 μM) was allowed to interact with immobilized RANKL for 300 s. Finally, 0 μM or 10 μM AA was used to measure the degree of dissociation after 300 s. The kinetic parameters and affinity constants were calculated using the Octet analysis software according to the “1:1 model”.

[0122] 1.2 Surface Plasmon Resonance (SPR)

[0123] SPR technology was used to further verify the affinity of AA to RANKL or RANK, as well as the interference with RANKL-RANK interaction. RANKL and RANK proteins were immobilized on the CM 5 chip, and the proteins were diluted with 10 mM sodium acetate solution at pH = 5 and pH = 4.5, respectively, and coupled by time coupling. In the affinity experiment, the running buffer used PBS-P solution containing 5% DMSO, and the affinity level of AA with RANK and RANKL was detected at 0, 0.625, 1.25, 2.5, and 5 μM, respectively. The competition experiment was carried out according to the "ABA-inject" method in the template, flanking solution: 0, 5, 10, 20 μM AA dilution, RANKL concentration was 0.2 μM, and the chip regeneration condition was Gly-HCl buffer (pH 2.5).

[0124] 2. Test results

[0125] The RANKL-RANK system plays an important role in osteoclast maturation and bone remodeling. The binding of RANKL to RANK can stimulate downstream NF-κB, MAPKs, and AKT pathway cascades. Therefore, the interaction between RANKL-RANK is a key target for the treatment of osteoporosis. Based on the inhibitory effect of AA on osteoclastogenesis in vitro, we further speculated whether AA inhibits osteoclast formation by interfering with RANKL-RANK binding.

[0126] In order to determine whether AA binds to RANKL and RANK, this example uses biomembrane interferometry (BLI) to detect their interaction and obtain the biomolecular interaction kinetic constant and affinity constant KD value. Figure 5 As can be seen from A and B, AA (20 μM) can directly bind to RANKL and RANK, and the binding signal value increases rapidly. However, it is not easy to dissociate from RANK and RANKL during the dissociation process, which belongs to "fast binding and slow dissociation". The KD values ​​are 1.517×10 -6 M( Figure 5 A), 1.368×10 -6 M( Figure 5 In order to verify that AA does bind to RANKL and RANK, further SPR experiments were performed and it was found that AA does bind to RANKL and RANK ( Figure 5 D, E), whose KD values ​​are 2.788×10 -6 M( Figure 5 D), 1.765×10 -6 M( Figure 5 E), which is consistent with the results of biomembrane interference technology, indicating that AA can directly bind to RANKL and RANK, and the RANKL-AA and RANK-AA complexes are very stable.

[0127] Since AA can directly bind to RANKL and RANK, we speculated that AA might interfere with the binding of RANKL-RANK. We designed a competitive experiment using BLI to explore whether AA could inhibit the binding of RANKL-RANK. Figure 5 As shown in C, the KD value of RANKL-RANK binding is 4.702×10 -8 M, and after adding AA (10 μM), its binding KD value was 2.645×10 -7 M, the affinity decreased by 5.6 times. To verify this result, SPR was also performed. Consistent with the BLI results, the inhibitory effect on RANKL-RANK interaction became more obvious as the AA concentration increased ( Figure 5The above results indicate that AA can interfere with the interaction between RANKL-RANK, thereby inhibiting the formation of osteoclasts.

[0128] Example 4

[0129] 1. Test methods

[0130] 1.1 Animal analysis

[0131] Construction of OVX mouse model: Female C57BL / 6 mice were purchased and adapted to the laboratory for 1 week. After 1 week, they were weighed and randomly divided into 4 groups according to their weight, namely sham operation group (Sham; n=10), model group (OVX; n=10), raloxifene treatment group (OVX+RLX; n=10) and AA treatment group (OVX+AA; n=10). After the allocation, the mice underwent ovariectomy. All animal experiments were carried out in accordance with the "Regulations on the Management of Experimental Animals". First, sodium pentobarbital (5 mg / kg) was injected intraperitoneally to anesthetize the mice. After successful anesthesia, the mice were fixed on the operating table. The bilateral ovaries of the Sham group mice were not removed, and they were sutured after cutting open; the bilateral ovaries of the OVX mice were removed and the fallopian tubes were ligated. After suturing, the mice were given a warm environment and sufficient food. After they were fully awake, they were sent back to the animal room for feeding. The mice recovered for about one week after surgery, and sodium penicillin was applied twice a day during this period to prevent infection.

[0132] Mice administration: After the weight of mice returned to normal levels after surgery, the weight of mice was recorded every other day. Injections were performed every two days by intraperitoneal injection according to body weight. The Sham group and OVX group were injected with medium (3% DMSO, 5% PEG400, 5% Tween 80, 87% PBS, the above percentages are volume percentages); the OVX+RLX group was injected with RLX (6.25 mg / kg); and the OVX+AA group was injected with AA solution (4 mg / kg). Four weeks after the injection, the mice were starved for 12 h and then blood glucose was measured. The mice were killed after blood was collected from the eyeballs. The weight of the liver and kidney of the mice was recorded and placed in formalin for subsequent H&E staining. The front legs were wrapped in tin foil and placed in liquid nitrogen and stored in a -80℃ refrigerator; the hind legs were placed in formalin and decalcified with EDTA decalcification solution (5 mL per tube) for 4 weeks three days later for paraffin section staining. After blood is collected, let it stand for 2 hours, centrifuge it at 3000 rpm at 4℃ for 20 min, take the supernatant into a new EP tube, centrifuge it again at the same speed for 10 min, transfer it to a new EP tube to obtain serum, seal it with sealing film and freeze it at -80℃.

[0133] 1.2 Western blot analysis

[0134] 1. Tissue protein extraction

[0135] (1) Grinding the front leg bone tissue

[0136] After the mouse injection, take the front legs of the mouse and store them at -80℃. When grinding, place the front legs on an ice box, shave the meat on the legs and put them in a mortar. Grind the front left leg and the front right leg together in a mortar, add liquid nitrogen, and grind until it is fine. After grinding, divide it into two parts, one for WB analysis and the other for real-time fluorescence quantitative (RT-qPCR) analysis, and put them into 1.5mL EP tubes and put them on ice. For RT-qPCR, add 1 mL of TransZol on a sterile operating table, shake evenly, seal with a sealing film, and freeze them back to -80℃ together.

[0137] (2) Bone tissue protein

[0138] 1) Weighing: Place a clean 1.5 mL EP tube in a small beaker filled with paper, set to zero (turn off the air conditioner), wipe the surface of the sample tube with paper towels, take out the clean tube from the beaker, and put the tube containing the sample in. The obtained value is the mass of the sample;

[0139] 2) Add lysis buffer: Add lysis buffer to sample at a ratio of 1:5 (1 mg plus 5 μL lysis buffer), and calculate the total amount required for preparation;

[0140] 3) Vortex: Vortex after adding the lysate. Precool the vortexer in advance. Vortex for about 10 seconds, let it stand for 5 minutes, and vortex again, for a total of 6 times (do not vortex too vigorously);

[0141] 4) Centrifugation: After vortexing, centrifuge at 14,000 g for 10 min, aspirate the supernatant and transfer it to a new centrifuge tube. Centrifuge again for 10 min and transfer it to a new 1.5 mL EP tube for quantitative analysis.

[0142] (ii) Protein quantification is the same as in Example 2.

[0143] (III) Western blotting analysis was the same as in Example 2.

[0144] 1.3 Real-time fluorescence quantitative PCR

[0145] 1. RNA extraction and concentration determination:

[0146] Bone tissue RNA was extracted using TransZol according to conventional methods. The obtained RNA was diluted 50 times with diethyl pyrocarbonate (DEPC) water (98 μL DEPC water plus 2 μL RNA). The RNA concentration was measured using an ultraviolet spectrophotometer and recorded (μg / μL).

[0147] 2. Reverse transcription (SYBR Green Assay)

[0148] DNA removal: Calculate the volume required for 1 g and the amount of RNase Free H2O according to the RNA concentration. Add the sample according to 10 μL of DNA removal reaction system (5×gDNA Eraser buffer 2 μL, gDNA Eraser 1 μL, the volume required for Total RNA 1 μg, and RNase Free H2O to make up to 10 μL). Mix well, centrifuge, and place in a PCR instrument. React at 42°C for 2 minutes. Stop when the temperature drops to 4°C.

[0149] RNA reverse transcription: The above reaction solution was taken out from the PCR instrument, and 20 μL of reverse transcription reaction system (5× PrimeScript Buffer 4 μL, Prime Script RT Enzyme Mix 1 μL, RT Primer Mix 1 μL, 10 μL of reaction solution after DNA removal, 20 μL of RNase Free H2O) was added and mixed, and the mixture was placed in the PCR instrument. The reaction was performed at 37°C for 15 min and at 85°C for 5 s. When the temperature dropped to 4°C, the stop point was clicked to obtain cDNA.

[0150] (3) RT-PCT (SYBR Green Assay)

[0151] This experiment involved 6 pairs of primers and 12 samples (4 treatment groups, 3 mice in each group). The specific primer sequences are shown in Table 2:

[0152] Table 2 Primers used in qRT-PCR

[0153]

[0154] Prepare system A: System A is the primer, add according to Table 3, first add reagent SYBR Mix, forward primer, reverse primer, and finally add ROX Reference.

[0155] Prepare system B: System B is cDNA. First add DEPC water and then add the corresponding cDNA according to the label on the tube.

[0156] 22.32 μL and 14.4 μL were added to the 96-well plate in the order of layout, centrifuged at 1000 rpm for 1 min after sealing the plate, vortexed for 2 min, and centrifuged at 2000 rpm for 2 min. Repeat twice, and add to the 384-well plate in the order of layout (10 μL / well). The three groups were placed in parallel and centrifuged for 1 min after sealing the plate. The 384-well plate was placed on a PCR instrument for PCR detection. According to the results of the PCR instrument detection, the endogenous gene GAPDH, 2 −ΔΔCt The expression level of the target gene was normalized with the GAPDH gene level.

[0157] Table 3 qRT-PCR reaction system

[0158]

[0159] 1.4 Organ coefficient determination

[0160] The liver and kidneys were weighed after separation. The organ index was calculated as follows: organ index = organ weight / body weight.

[0161] 1.5 Immunohistochemistry

[0162] (I) Hematoxylin-eosin (H&E) staining of liver and kidney

[0163] (1) After cutting the tissue, place it in a clamp (cut the liver horizontally at the thickest part and cut the kidney vertically), and rinse it with running water for 2 hours; (2) Dehydrate with ethanol: 60% (60 min) → 70% (overnight) → 80% (40 min) → 90% (10 min) → 95% (5 min) → 100% (5 min, twice), all the above percentages are volume percentages;

[0164] (3) Transparency: Soak the tissue in ethanol and xylene (transition) for 3 min, then soak in xylene (transparency) for 5 min and 3 min respectively;

[0165] (4) Wax immersion: Soak the tissue in melted paraffin wax for 60 min each time, for a total of 2 times (the new wax should be filtered three times);

[0166] (5) Embedding: First pour the wax into the embedding box, clamp the tissue from the centrifuge tube into the embedding box, use tweezers to adjust the position of the tissue, and when a film appears on the upper layer of the embedding box, stick a label paper on the opposite end of the tissue. When the tissue is no longer visible, put the embedding box in ice water for 1 hour, take out the wax block and put it at -20℃, and take it out again when slicing;

[0167] (6) Sectioning: Place on adhesive slides at 5 μm thickness, bake at 65°C for 4 h, and store at 4°C.

[0168] (7) Staining was performed according to the H&E instructions (Solabio);

[0169] (8) Photography: The liver and kidneys are imaged at 40x through the eyepiece.

[0170] 2. Bone tissue H&E and TRAP staining

[0171] (1) Decalcification: After soaking in formalin for 3 days, remove the femur of the mouse hind leg, shave the meat clean, and put it into a centrifuge tube containing EDTA decalcification solution (5 mL). Decalcify for 4 weeks and slice, and change the decalcification solution every two weeks.

[0172] (2) Preparation of bone dehydration working solution: Solution A (n-butanol: anhydrous ethanol = 6:4); Solution B (n-butanol: anhydrous ethanol = 8:2); Solution C (n-butanol: anhydrous ethanol = 10:0). The above percentages are all volume percentages.

[0173] (3) Take out the tissue that has been decalcified for more than 4 weeks and place it in a small box and rinse it with running water for 3 hours;

[0174] (4) Dehydration: 65% ethanol for 1 h → 75% ethanol overnight → 85% ethanol for 25 min → 95% ethanol for 5 min, liquid A for 2 h, liquid B for 3 h, liquid C for 4 h. The above percentages are all volume percentages.

[0175] (5) Wax immersion: The bone tissue was immersed in wax twice, 2 hours each time, for a total of 4 hours;

[0176] (6) Embedding: Place the tissue in an embedding box, adjust the position of the tissue, label it, place it in ice water for 1 h, and store it in a -20°C refrigerator;

[0177] (7) Paraffin section thickness is 7 μM;

[0178] (8) H&E staining of bone tissue (staining performed according to the kit), with images collected at 4x and 10x magnification, respectively.

[0179] (9) TRAP staining of bone tissue (staining was performed according to the kit - Nanjing Jiancheng), and 20x and 40x images were collected respectively.

[0180] 1.6 ELISA

[0181] During the detection, according to the instructions of the ELISA kit (PⅠNP Elisa kit, RANKL Elisa kit, OPG Elisa kit, etc.), the mouse serum was diluted with normal saline, and the contents of OCN, PⅠNP, RANKL and OPG in the serum were detected respectively.

[0182] 2. Test results

[0183] 2.1 Effects of AA on body weight and organs of ovariectomized mice

[0184] Based on the above experimental results, AA can interfere with RANKL-RANK interaction in vitro, reduce the activation of downstream NF-κB, MAPKs and AKT pathways, thereby inhibiting the expression of key transcription factors and marker proteins of osteoclasts, and inhibiting the formation of osteoclasts. So whether AA has the same effect in vivo and improves bone loss, this example uses 8-week-old C57BL / 6 female mice to establish an osteoporosis model to verify the effect of AA in vivo. The mice were divided into a sham operation group (Sham), a model group (OVX), a raloxifene hydrochloride (RLX) group, and an AA treatment group (AA). The mice were injected every other day for 4 weeks, and the weight of the mice was recorded ( Figure 6 A).

[0185] The body weight of mice during injection is shown in the figure ( Figure 6 B) In the 10 days before injection, the body weight of the sham group, model group and AA treatment group increased rapidly and was close to that of the sham group on days 8-10. After day 10, the body weight of the model group continued to increase more than that of the sham group, indicating that the model was successfully established. Compared with the model group, the body weight of the RLX treatment group was always lower than that of the model group, and there was a significant difference on days 10, 12 and 24 (P < 0.05). There was no significant difference in the body weight of the AA treatment group compared with the model group.

[0186] The liver is an important organ for the metabolism and biotransformation of human substances, and the kidney has the function of eliminating metabolism in the body and maintaining the balance of the body's internal environment. Therefore, the weight and organ coefficient of the liver and kidney are one of the important indicators for evaluating animal health. After the mice were ovariectomized, the weight of the liver and kidney increased slightly, and RLX had a protective effect on organ obesity induced by ovariectomy. Figure 6 As can be seen from Figures C and D, the liver weight and coefficient of mice in the RLX-treated group were significantly lower than those in the model group (P < 0.01 or P < 0.001; Figure 6 C, D), while there was no significant difference between the AA treatment group and the model group. Similarly, the kidney weight and coefficient of the RLX treatment group showed the same effect (P < 0.01; Figure 6 F, G) In the H&E staining results of liver and kidney, no significant pathological defects were found in the four treatment groups ( Figure 6 E, H), indicating that AA treatment had no toxic side effects on mice.

[0187] AA improves bone loss in ovariectomized mice

[0188] To explore whether AA can prevent bone loss in ovariectomized mice, this example measured the microstructure of trabecular bone in the femur and its related parameters. H&E staining was used to detect the microstructure of trabecular bone in the femur ( Figure 7 trabecular bone area ( Figure 7 The results showed that the trabecular bone area of ​​the model group mice was significantly reduced compared with that of the sham operation group (P < 0.001; Figure 7 Compared with the model group, the trabecular bone area in the RLX-treated group and the AA-treated group was significantly increased (P < 0.01 or P < 0.001; Figure 7 Figure 2 (A) shows that AA has a protective effect on bone loss in ovariectomized mice.

[0189] Bone remodeling is regulated by osteoclasts and osteoblasts. This example investigates whether AA can inhibit the activity of osteoclasts in vivo. TRAP staining was performed on mouse femurs. Figure 7 As shown in C, the purple part marks the activity of osteoclasts, and TRAP was quantified. Compared with the sham operation group, the TRAP activity was significantly increased in the model group (P < 0.01; Figure 7 D); compared with the model group, the TRAP activity in the RLX-treated group and the AA-treated group was significantly decreased (P < 0.05 or P < 0.01; Figure 7 D).

[0190] The levels of osteoblast and osteoclast markers in serum can also reflect the activity of osteoblasts and osteoclasts. The activity of osteoclasts was detected by detecting the RANKL content in mouse serum. The results showed that the RANKL content in the serum of the model group mice was significantly higher than that in the sham operation group (P < 0.01; Figure 7 G), while RLX and AA treatment significantly reduced serum RANKL content (P < 0.05; Figure 7 G), indicating that AA inhibited osteoclast activity; detection of osteoblast markers showed that compared with the sham operation group, the serum levels of osteocalcin (OCN), type I procollagen amino-terminal propeptide (PⅠNP) and osteoprotegerin (OPG) in the model group were significantly decreased (P < 0.01 or P < 0.001; Figure 7 E, F, H). On the contrary, compared with the model group, RLX significantly increased the content of OCN in serum (P < 0.05; Figure 7 E), while there was no statistical difference in the AA treatment group. Both the RLX and AA treatment groups could significantly increase the levels of serum PINP and OPG (P < 0.05 or P < 0.01; Figure 7 F, H), indicating that AA treatment has a certain osteogenic effect.

[0191] To further verify the effect of AA on osteoclast activity in vivo, immunoblotting analysis was performed on the mouse forelegs. The results showed that AA treatment could significantly reduce the expression levels of osteoclast-related marker proteins TRACP-5b, c-Src, NFATc1, and c-Fos in vivo ( Figure 7 I).

[0192] RT-qPCR analysis of bone tissues also showed that AA significantly inhibited the gene expressions of TRAP, c-Src, cathepsin K, NFATc1, and c-Fos (P < 0.05 or P < 0.01 or P < 0.01; Figure 7 The above results indicate that AA can inhibit the activity of osteoclasts in vivo and improve bone loss in ovariectomized mice.

[0193] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A use of amygdalin A in the preparation of a drug for preventing and treating osteoporosis, characterized in that The structural formula of the amygdalin A is shown in Formula I: 。 2. The use according to claim 1, characterized in that: The medicine contains one or more pharmaceutically acceptable carriers, excipients or diluents.

3. The use according to claim 1, characterized in that: The dosage form of the medicine is tablet, injection or liposome nanoparticle.

4. The use according to claim 1, characterized in that: The dosage form of the medicine is a controlled release agent.

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