CN111991388A

CN111991388A - Application of genkwanin in preparation of products for preventing and treating osteoporosis

Info

Publication number: CN111991388A
Application number: CN202010743573.9A
Authority: CN
Inventors: 苏笠; 黄标通; 李英华; 富欣; 张晨曦
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2020-11-27

Abstract

The invention relates to the technical field of medicines, and discloses application of genkwanin in preparation of medicines for preventing and treating osteoporosis, application of genkwanin in preparation of health-care products for preventing and treating osteoporosis, and an in-vitro action mechanism of genkwanin. The invention proves that the genkwanin can prevent the bone loss of mice caused by ovariectomy, and the previous evidence shows that the genkwanin has an anti-inflammatory effect, the genkwanin is also reported to remarkably enhance the killing activity of natural killer cells and the cytotoxicity activity of T lymphocytes, so that the genkwanin has an immunoregulation effect, the antibacterial, anti-plasmodium, free radical scavenging and chemoprevention activities of the genkwanin are also reported, the genkwanin is directly used as a medicine, the potential value of the genkwanin in the medicine for treating the osteoporosis caused by female menopause is proved, and effective treatment guidance is provided for the osteoporosis caused by female menopause.

Description

Application of genkwanin in preparation of products for preventing and treating osteoporosis

Technical Field

The invention relates to the technical field of medicines, in particular to application of genkwanin in preparation of a product for preventing and treating osteoporosis.

Background

Bone is composed of an organic protein matrix formed by crystallization of calcium and phosphate and inorganic hydroxyapatite, repaired by osteoblast mediated bone formation and osteoclast mediated bone resorption formation, however, an imbalance between formation and resorption results in many diseases including rheumatoid arthritis, periodontitis and aseptic loosening of orthopedic implants.

Postmenopausal osteoporosis (PMO) is a common bone disease and is characterized by bone over-absorption, so that the bone balance of old women is damaged, at present, a plurality of anti-osteoporosis medicines have positive effects on bone health and are often accompanied with serious side effects, such as jawbone osteonecrosis caused by bisphosphonate and liver cancer risk increased by calcitonin, and therefore, the application of genkwanin in preparing products for preventing and treating osteoporosis is provided to solve the problems.

Disclosure of Invention

The invention aims to prove the action mechanism of genkwanin in vitro, the protection effect on bone loss caused by ovariectomy of mice and the potential value of genkwanin in osteoporosis drugs caused by female menopause by utilizing technologies such as cell culture, real-time fluorescence quantitative PCR, cell survival experiments, TRAP staining, bone plate absorption experiments, western blotting experiments, mouse osteoporosis models caused by ovariectomy, H & E and TRAP staining, Micro-CT scanning and the like.

In the first aspect of the invention, the application of genkwanin in preparing medicines for preventing and treating osteoporosis is provided.

Further, the invention provides application of genkwanin in preparing health care products for preventing and treating osteoporosis.

Further, the invention provides an application of genkwanin in preparation of a product for preventing and treating osteoporosis, wherein the genkwanin is mainly used for inhibiting RANKL-induced osteoclast formation to prevent bone loss caused by ovariectomy of mice.

Further, the genkwanin has an inhibitory effect on osteoclast differentiation and function.

Furthermore, the external action mechanism of the genkwanin is mainly through regulating miR-101/MKP-1/MAPK pathway in macrophage activated by LPS.

The medicine for preventing and treating osteoporosis contains genkwanin as effective component.

A health product for preventing and treating osteoporosis contains genkwanin as effective component

The product with genkwanin as effective component can be made into various preparations including but not limited to tablet, capsule, granule, injection, lyophilized powder, etc.

The invention has the beneficial effects that: the invention utilizes the technologies of cell culture, real-time fluorescence quantitative PCR, cell survival experiments, TRAP staining, bone plate absorption experiments, protein immunoblotting experiments, mouse osteoporosis models caused by ovariectomy, H & E and TRAP staining, Micro-CT scanning and the like to discuss the protective effect of the genkwanin on bone loss caused by ovariectomy of mice, and results show that the genkwanin can prevent the bone loss of mice caused by ovariectomy, and previous evidence shows that the genkwanin has anti-inflammatory effect, and reports that the genkwanin can obviously enhance the killing activity of natural killer cells and the cytotoxicity activity of T lymphocytes, thus showing that the genkwanin has immunoregulation effect, further reporting the antibacterial, antimalarial protozoa, free radical scavenging and chemopreventive activity of the genkwanin, directly using the genkwanin as a medicine, proving the potential value of the genkwanin in the osteoporosis medicine caused by women with the genkwanin, provides effective treatment guidance for osteoporosis caused by female menopause.

Drawings

In FIG. 1, (A) the chemical structure of Genkwanin, (B) the effect of different concentrations of Genkwanin on osteoclast toxicity, (C) the effect of different concentrations of Genkwanin on osteoclast inhibition, (D) the effect of different concentrations of Genkwanin on the area of TRAP-positive multinucleated osteoclasts, (E) the effect of different concentrations of Genkwanin on the number of TRAP-positive multinucleated osteoclasts;

fig. 2 shows (a) a schematic diagram of coriander culture, (B) TRAP positive cell areas under different concentrations of coriander, (C) the number of TRAP positive cells under different concentrations of coriander, (D) a culture plate of coriander on hydroxyapatite at different concentrations, and (E) bone resorption areas under different concentrations of coriander;

FIG. 3 (A) (B) (C) expression of Genkwanin at various concentrations on C-Fos, TRAP and NFATc 1;

FIG. 4 shows (A) the signal pathway assay of Genkwanin, (B) the induction of RANKL by treating BMMs with RANKL at 25ng/ml for 1, 3 and 5 days, respectively, (C) the induction of RANKL with a decreasing trend of p-p38 expression after 20, 30 and 60 minutes, and (D) and (E) the expression of C-Fos and NFATc1 after the treatment of Genkwanin;

fig. 5 (a) is a comparison graph of genkwanin trabecular loss in an OVX model, (B) is a graph of tibial trabecular loss in three-dimensional reconstruction, (C) is a graph of BV/TV and tb.n reduction in the OVX model, (D) is a graph of BS/BV increase after ovariectomy, and (F) is a graph of tb.sp no change;

FIG. 6 shows (A) a decrease in the number of TRAP-positive multinucleated osteoclasts after Genkwanin treatment and (B) the protective effect of Genkwanin on OVX-induced bone loss.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first embodiment is as follows:

reagent and method

(I) reagent

Genkwanin (HPLC. gtoreq.98%) was purchased from Kyowas Dester (ESTIE) biotech Inc., alpha-MEM and serum (FBS) were purchased from Sammer fly (Thermo Fisher Scientific), RANKL and M-CSF were purchased from R & D Systems, TRAP staining kit was purchased from Techhiki Biotech GmbH, Inc., PCR primers were designed and synthesized by Nanjing King-Shirui Biotech Co., Ltd, and cytoskeletal stains, Phalloidin-iFluor 488 Reagent (ab176753) were purchased from Abcam (Cambridge, UK), Prime Script RT Master Mix (# RR036A) and TB Green Premix Ex Taq (RR420A) were purchased from Takara Bio Inc (Shiga Prefecture, Japan).

(II) Experimental method

1. Cell culture

Taking a C57BL/6 mouse with the age of 4-6 weeks, taking off the cervical vertebra, killing the mouse, and aseptically separating the tibia and the femur on two sides; washing the sterile chamber with serum-free alpha-MEM medium until the chamber becomes white, and blowing off the cell mass; osteoclasts were obtained after 4 days of culture by transferring 75 cm square culture flasks in a 37 ℃ incubator using α -MEM medium containing 10% serum (FBS), a 1% antibiotic mixture of penicillin and streptomycin, and 50ng/mL M-CSF.

2. Cell viability assay

In order to explore the toxicity of the genkwanin on the BMMs, a cell counting kit (CCK-8) is used for measuring the influence of different concentrations of the genkwanin on the survival rate of the BMMs; BMMs were seeded in 96-well plates at 8000 cells per well using complete alpha-MEM (containing 10% fetal bovine serum, 1% penicillin/streptomycin antibiotic cocktail, and M-CSF (50 ng/ml)); after 24 hours of culture, the medium was changed and daphnetin (0,0.313,0.625,1.25,2.5,5,10and20 μ M) was added at different concentrations to the medium; replacing the culture medium every two days; after 96 hours of cell incubation, the medium was aspirated and washed with Phosphate Buffered Saline (PBS); on this basis, 10. mu.L of CCK-8 kit solution was added to each well, incubated at 37 ℃ for 4 hours, and the absorbance at 450nm was measured using an ELX800 absorbance microplate reader (Bio-Tek, Winooski, Vt).

3. Osteoclastogenesis and TRAP staining

BMMs were plated in 96-well plates (5000 cells/well) using complete α -MEM medium (containing 10% fetal bovine serum, 1% penicillin/streptomycin) and M-CSF (50ng/ml) and RANKL (25ng/ml) with or without vari-ous concentrations of daphnetin; osteoclast is formed after culturing for 5 days, and the culture medium is replaced every 2 days; after washing with PBS, fixing with 4% Paraformaldehyde (PFA) for 20 minutes, adding staining solution, incubating at 37 ℃ for 1 hour, and counting under an optical microscope, the stained cells with more than 3 nuclei were TRAP positive cells.

In addition, we also investigated the time-dependent inhibitory effect of genkwanin on osteoclast differentiation, and BMMs stimulated with RANKL were added with genkwanin at Day1 (Day1-Day6), Day3 (Day3-Day6), Day5 (Day5-Day6), and Day6, respectively, and cells were TRAP stained according to the above experimental procedure, and then the size of osteoclasts was observed.

4. Actin ring immunofluorescence

BMMs were inoculated into 96-well plates containing different concentrations of Genkwanin, complete α -MEM medium (containing 10% fetal bovine serum, 1% penicillin/streptomycin) and M-CSF (50ng/ml) and RANKL (25 ng/ml); after 5 days of culture, the cultured cells were washed with PBS and fixed with 4% Paraformaldehyde (PFA) for 20 minutes; three washes with PBS, actin rings with phallodin, nuclei with DAPI, and observation of the experiment results with a fluorescence microscope.

5. Osteoclast bone resorption function identification

Seeding 4 days with M-CSF (50ng/ml) and RANKL (25ng/ml) in 6 well plates; separating cells, culturing on a hydroxyapatite-coated 96-well plate, and adding genkwanin with different concentrations into a culture medium; after 48 hours, the cells were removed and the wells were examined under a light mirror; the bone resorption area was observed with an optical microscope and the hydroxyapatite resorption area was measured with ImageJ software.

6. RNA extraction and Real-Time qPCR

Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific) as described after culturing osteoclasts in the absence or presence of different concentrations of Genkwanin with M-CSF at a concentration of 50ng/ml and RANKL at a concentration of 25ng/ml for 7 days; 1mg of the extracted cDNA reverse transcribed and synthesized from RNA was used as a template for the subsequent Real-Time qPCR reaction; performing Real-time PCR on a qTOWER Real-time PCR Thermal Cycler (Analytik Jena, Jena, Germany) in a reaction mixture containing TB Green Premix Ex taq, cDNA and forward and reverse primers; the reaction conditions are 95 ℃ and 3 min; circulating for 40 times at 95 ℃ for 10 s; 60 ℃, 20s, and72 ℃, 20 s; the last extension step was extended at 72 ℃ for 20 seconds.

RT-PCR primers are given in the following table

7. Immunoblot detection of protein expression

To explore the mechanism by which genkwanin affects osteoclast formation, BMMs were seeded in 6-well plates (5 × 105 cells/well) with complete α -MEM medium (without fetal bovine serum), then cells were cultured for 2 hours in medium with or without genkwanin (50 μ M), then RANKL stimulation (25ng/ml) was performed for 5,10, 20, 30 and 60 minutes in RANKL-induced signaling events; in the late-stage RANKL-induced signal event, BMMs cells were cultured with 25ng/ml RANKL with or without coriander for 1, 3 and 5 days; after treatment, the treated BMMs were washed with PBS and separated using Radioimmunoprecipitation (RIPA) lysis buffer (Millipore, Billerica, MA, USA) containing PMSF. Centrifuging the lysate at 12000rpm for 15min, and collecting the supernatant; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by trans-blotting onto a nitrocellulose membrane, blocked with 5% skim milk for 1 hour and then washed three times with TBST (Tris-buffered saline with); the membrane was incubated with the specific primary antibody (dilution 1:1000) overnight at 4 ℃ and then with the appropriate fluorescently labeled secondary antibody (IRDye; dilution 1:1000 at room temperature) for 1 hour; protein bands were visualized using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and relative protein expression was calculated using ImageJ software.

8. Mouse Ovariectomy (OVX) -induced bone loss model

Twenty 8-week-old female C57BL/6 mice, an average of 20g, purchased from the center of animals of the chinese academy of sciences, (shanghai, china), randomly divided into 4 groups (5 each n ═ n) named sham group, OVX +1mg/kg genkwanin (Low) and OVX +10mg/kg genkwanin (High), respectively, and all animals except the sham group were bilaterally ovaried out; after surgery, mice can be rested for one week, then OVX +1mg/kg genkwanin (Low) and OVX +10mg/kg genkwanin (High) are administered separately, mice in sham and OVX groups are injected intraperitoneally with PBS of the same volume, finally all animals are sacrificed, their tibia is excised and processed, and microtomography (Micro-CT) analysis and histological staining are performed.

9. Micro-CT scanning

The left tibia of each mouse was collected and fixed in 4% PFA for 2 days; then scanning the Micro-CT with high resolution, wherein the equidistant resolution is 9 μm, and the x-ray energy is set to 80KV and 100 μ A; three-dimensional (3D) images were analyzed by SkyScan nreco program and SkyScan CTAn software (Bruker, Billerica, MA, USA) processing; quantitative and qualitative analysis of bone parameters in an area of 0.5mm below the growth plate; it was characterized by measuring the bone volume BV/TV, trabecular number (tb.n), trabecular thickness (tb.th) and small tb.sp per tissue volume.

10. Histological identification

Tibia was fixed in 4% paraformaldehyde for 2 days, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks, and H & E and TRAP staining were served by hangzhou cistronic biotechnology limited.

Second, experimental results

Firstly, the chemical structure of genkwanin is shown in fig. 1A, and toxicity of different concentrations of genkwanin on osteoclasts is detected by a CCK-8 method. The results show that genkwanin is not toxic to BMMs at concentrations below 10. mu.M (FIG. 1 b). Several different concentrations of genkwanin (1.25,2.5 and 5 μ M) were then selected to evaluate the effect on RANKL-induced osteoclast formation. TRAP staining results showed that osteoclast inhibition by genkwanin was enhanced with increasing concentration of genkwanin (fig. 1C). Compared to the control group, the area of TRAP-positive multinucleated osteoclasts (>3 nuclei) was reduced (fig. 1D), and the number of TRAP-positive cells was significantly reduced (fig. 1E). Finally, the results show that genkwanin inhibits osteoclast differentiation in a dose-dependent manner at concentrations below 10 μ M.

To find out at which stage daphnetin mainly inhibits osteoclast differentiation, we added daphnetin to RANKL and MCSF treated BMMs cultures at three different time points (fig. 2A).

The results show that D1-D6 has a more pronounced effect on osteoclasts than D5-D6. Both TRAP-positive cell area and number were smaller than control (fig. 2B and 2C). These results suggest that genkwanin inhibits osteoclast formation in the early stage of osteoclast differentiation. Mature osteoclasts were cultured on different concentrations of daphnetin hydroxyapatite culture plates and it was observed whether daphnetin could inhibit osteoclast function (fig. 2D). We found that genkwanin reduced the percentage of hydroxyapatite coated plate surface area absorption pits relative to the control (fig. 2E). Thus, genkwanin inhibits the effect of osteoclasts on bone resorption in a dose-dependent manner.

Genkwanin inhibits gene expression to regulate osteoclast differentiation, maturation and function. We investigated the effect of genkwanin on osteoclast gene expression. We used RT-PCR to assess gene expression levels. BMMs were inoculated with MCSF (50ng/mL) and RANKL (25ng/mL) and various concentrations of genkwanin were added until osteoclast formation. Genkwanin significantly inhibited the expression of c-Fos, TRAP and NFATc1 genes in a dose-dependent manner (FIG. 3).

Genkwanin inhibits the RANKL-induced MAPK signaling pathway. Previous reports have shown that when RANKL binds RANK, the mitogen-activated protein kinase (MAPK) signaling pathway is activated. In addition, we have also investigated the inhibitory effect of genkwanin on osteoclastogenesis. We detected the signal pathway by immunoblotting techniques (fig. 4). Cells were cultured in medium with or without daphnetin (40 μm) for 2 hours, and RANKL stimulation (25ng/ml) for 5,10, 20, 30, 60 minutes RANKL induced early signaling events (fig. 4). As shown in FIG. 4C, the relative p-p38 expression decreased significantly at 20, 30, and 60 minutes. In the late signaling event induced by RANKL, BMMs1, 3, and 5 days were treated with RANKL at 25ng/ml, respectively (fig. 4B). c-Fos and NFATc1 expression was lower after Genkwanin treatment than in controls (FIGS. 4D and 4E).

Genkwanin can prevent bone loss of mice caused by ovariectomy. The inhibitory effect of genkwanin on bone loss in mice was studied. We have built an OVX model to simulate PMO. Analysis showed that the genkwanin-treated group had less loss of trabeculae compared to the OVX group (fig. 5A). This was further confirmed by three-dimensional reconstruction of the tibia (fig. 5B). Second, morphometric analysis showed a significant reduction in BV/TV, Tb.N (FIGS. 5C and 5E). And a significant increase in BS/BV following ovariectomy (fig. 5D). However, tb.sp did not change significantly compared to OVX group (fig. 5F).

Histological analysis of H & E and TRAP stained tibial sections demonstrated that genkwanin can prevent bone loss from ovariectomy in mice (fig. 6). In TRAP staining, TRAP-positive multinucleated osteoclasts decreased in number following treatment with genkwanin (fig. 6A). H & E staining further confirmed the protective effect of genkwanin on OVX-induced bone loss (fig. 6B). In conclusion, our results show that genkwanin can prevent the bone loss of mice caused by ovariectomy.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. Application of genkwanin in preparing medicine for preventing and treating osteoporosis is provided.

2. Application of genkwanin in preparing health product for preventing and treating osteoporosis is provided.

3. The application of genkwanin in preparing products for preventing and treating osteoporosis is characterized in that the genkwanin is mainly used for inhibiting RANKL-induced osteoclast formation to prevent bone loss caused by ovariectomy of mice.

4. The use of genkwanin according to claim 3 in the preparation of products for preventing and treating osteoporosis, wherein the genkwanin has an inhibitory effect on osteoclast differentiation and function.

5. The application of genkwanin in preparing products for preventing and treating osteoporosis as claimed in claim 3, wherein the external action mechanism of genkwanin mainly regulates miR-101/MKP-1/MAPK pathway in macrophage activated by LPS.

6. The medicine for preventing and treating osteoporosis is characterized in that the effective component is genkwanin.

7. The health product for preventing and treating osteoporosis is characterized in that the effective component is genkwanin.

8. The product with the genkwanin as the effective component can also be prepared into various preparations, and is characterized by comprising but not limited to tablets, capsules, granules, injection, freeze-dried powder and the like.