CN115969867A - Application of avicularin in preparation of medicine/health-care product for preventing and treating osteoporosis - Google Patents
Application of avicularin in preparation of medicine/health-care product for preventing and treating osteoporosis Download PDFInfo
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- CN115969867A CN115969867A CN202210823731.0A CN202210823731A CN115969867A CN 115969867 A CN115969867 A CN 115969867A CN 202210823731 A CN202210823731 A CN 202210823731A CN 115969867 A CN115969867 A CN 115969867A
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- 230000035899 viability Effects 0.000 description 1
- 231100000747 viability assay Toxicity 0.000 description 1
- 238000003026 viability measurement method Methods 0.000 description 1
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Abstract
The invention discloses application of avicularin in preparation of a medicine/health-care product for preventing and treating osteoporosis, the avicularin is a plant flavonoid compound and a quercetin derivative with biological activity, is derived from various plants, comprises lespedeza, fructus rubi and guava, and has the effects of resisting oxidation, allergy, inflammation, tumor and liver protection activity. In the present invention, we found that avicularin, which down-regulates the expression of osteoclast-associated genes and proteins, inhibits RANKL-induced osteoclastogenesis through in vitro studies; the related proteins are C-FOS, CTSK, NFATc1 and MMP-9; the related genes are CTSK, MMP9 and TRAP; through in vivo studies, we found that avicularin can alleviate bone loss in castrated mice; therefore, the avicularin is considered to be an effective ingredient in a potential medicament or health-care product for preventing and treating osteoporosis.
Description
Technical Field
The invention belongs to the field of medicines and health-care products for preventing and treating osteoporosis, and particularly discloses application of avicularin in preparing medicines/health-care products for preventing and treating osteoporosis.
Background
The mechanism of osteoporosis formation is mediated by both osteoclasts and osteoblasts, and an imbalance between their coordinated processes results in abnormal bone resorption and bone formation disorders, resulting in decreased bone stability and ultimately osteoporosis. This imbalance is caused by various factors such as hormone deficiency, aging, and drug side effects, which are all responsible for the abnormal activity of osteoclasts. Currently, drugs for treating osteoporosis are mainly focused on inhibiting the etiology or process of pathogenesis, especially osteoporosis in menopausal women, and mainly on selective estrogen receptor modulators, bone resorption inhibitors or bone formation promoters. However, with the long-term use of these drugs, serious side effects such as osteonecrosis of the jaw and cardiovascular diseases may occur, and furthermore, hormone replacement therapy may also result in an increased risk of ovarian and breast cancer. Therefore, it is of great importance to find new effective and safe treatments for osteoporosis.
Osteoclasts are multinucleated giant cells derived from monocyte/macrophage hematopoietic precursor cells. Has the functions of resorption and degradation of bone matrix. M-CSF and NF- κ B receptor activator ligand (RANKL) are two key molecules that promote osteoclastogenesis, and RANKL, by binding to its receptor RANK, activates mature osteoclasts and regulates monocyte-precursor osteoclast formation in the presence of M-CSF. When RANKL binds RANK, the expression of TNF receptor-associated factor 6 is up-regulated, resulting in activation of downstream signaling molecules such as MAPK and NF- κ B. These signaling pathways are responsible for activating key transcription factors, such as c-Fos and the nuclear factor that activates T cells, cytoplasm 1 (NFATc 1), leading to increased osteoclast-specific gene expression, inducing osteoclast differentiation and mature osteoclast formation.
Natural products are now used as an important, safe and convenient resource for the treatment of cancer, inflammation, oxidative activity, immunosuppression and allergy. Avicularin (AL, quercetin-3-alpha-l-arabinofuroside) is a plant flavonoid compound and a quercetin derivative with biological activity, is derived from various plants including lespedeza, fructus rubi and guava, and has the effects of resisting oxidation, allergy, inflammation, tumor and liver protection. A recent study has shown that avicularin can reverse the multidrug resistance of human gastric cancer by increasing the expression levels of B-cell lymphoma 2 (Bcl-2) associated X protein (Bax) and BOK. Vo et al suggest that Avicularin exerts anti-inflammatory effects by inhibiting extracellular signal-related kinase signaling pathways in lipopolysaccharide-stimulated RAW264.7 macrophages. Another study showed that Avicillarin might inhibit lipid accumulation in mouse adipocytes 3T3-L1 cells. These evidence suggests that Avicularin has a specific regulatory role in cell growth and inflammatory responses, possibly with an inhibitory effect on cell growth. However, it is not clear whether Avicularin can inhibit osteoclast activation and further inhibit the occurrence of osteoporosis.
Disclosure of Invention
In order to solve the problems, the application of the avicularin in preparing the medicine/health-care product for preventing and treating the osteoporosis is disclosed. We demonstrated therapeutic effects of avicularin using Ovariectomy (OVX) -induced mouse osteoporosis model and in vitro bone marrow derived macrophage (BMM) or RAW264.7 cell-induced osteoclast differentiation model.
The technical scheme of the invention is as follows:
application of polygonin in preparing medicine/health product for preventing and treating osteoporosis is provided. In some embodiments, avicularin can alleviate the progression of osteoporosis in OVX mice, and based on these findings, we consider avicularin as a potentially safe and effective treatment option for osteoporosis.
Further, the application of the avicularin in preparing medicines/health products for preventing and treating osteoporosis is that the avicularin inhibits the RANKL-induced osteoclast differentiation. In some embodiments, we found that osteoclast differentiation was induced in vitro with 50ng/mL M-CSF and 50ng/mL RANKL, and Avicillarin intervention was performed on BMMs cells at concentrations of 0, 10, 100, 300. Mu.M, respectively. The results show that Avicularin inhibits the formation of multinucleated osteoclasts in a dose-dependent manner compared to RANKL-induced group.
Further, the application of the polygonin in preparing the medicine/health-care product for preventing and treating osteoporosis inhibits the bone resorption function of osteoclast and the formation of F-actin ring. In some examples, we found that RANKL group-induced BMMs produced a large number of bone resorption pits, with the bone resorption area and number of resorption points decreasing in a dose-dependent manner after treatment with different concentrations of Avicularin; the RANKL-induced F-actin was observed to have a typical intact circular structure by immunofluorescence. Following Avicillarin treatment, the size, number, and number of F-actin rings decreased dose-dependently.
Furthermore, the application of the avicularin in preparing the medicine/health-care product for preventing and treating osteoporosis is realized, and the avicularin down-regulates the expression of osteoclast-related genes and proteins; the related proteins are C-FOS, CTSK, NFATc1 and MMP-9; the related genes are CTSK, MMP9 and TRAP. In some embodiments, we found that the Westernblotting results indicate that Avicillarin treatment significantly down-regulated the expression of C-FOS, CTSK, NFATc1 and MMP-9; after RANKL induction, the expressions of MMP9, TRAP and CTSK are all increased, and after Avicillarin treatment, the mRNA level is obviously reduced in a concentration-dependent mode relative to the RANKL group.
Furthermore, the application of the avicularin in preparing the medicine/health-care product for preventing and treating osteoporosis is that the avicularin inhibits the formation of the osteoclast induced by rankl by inhibiting the activation of an NF-kB signal path in the process of inhibiting the generation of the osteoclast.
Further, the application of the polygonin in preparing the medicine/health product for preventing and treating osteoporosis is that the polygonin inhibits phosphorylation and degradation of I kappa B alpha and p 65. The expression level of phosphorus-P65 in RANKL group is ascending and reaches peak at 10min, and after Avicillarin dry prognosis, the phosphorylation expression level is lower than that of RANKL group in the same period, and Avicillarin also has an inhibition effect on the phosphorylation level of I kappa B alpha and can inhibit the degradation of I kappa B alpha.
Further, the application of the avicularin in preparing the medicine/health-care product for preventing and treating osteoporosis can relieve bone loss of castrated mice. In some embodiments, avicularin intervention can protect mice from OVX-induced bone loss, improving BV/TV, BMD, and tb.sp in a dose-dependent manner.
Further, the application of the polygonin in preparing the medicine/health care product for preventing and treating osteoporosis is that the dosage of the polygonin is 1.25mg/kg/d to 5mg/kg/d.
Further, a drug/health product for preventing and treating osteoporosis contains avicularin.
Further, the polygonin is an injection or an oral preparation.
The invention has the following beneficial effects that the invention discloses the application of the avicularin in the preparation of the medicine/health care product for preventing and treating osteoporosis for the first time, and the therapeutic effect of the avicularin on the osteoporosis is researched by using an Ovariectomy (OVX) -induced mouse osteoporosis model and in vitro bone marrow-derived macrophages (BMMs) and RAW264.7 cell-induced osteoclast differentiation, and the mechanism of the avicularin for inhibiting the osteoclast differentiation is further defined; the research data can help to develop the medicine and health care product containing the avicularin for treating or preventing the osteoporosis, so as to produce a new medicine and health care product for treating the osteoporosis more effectively and safely, and lay a foundation for further researching a new medicine composition with synergistic effect.
Drawings
FIG. 1 is a CCK-8 viability assay; (A) CCK-8 assay to assess BMMs cell viability, (B) CCK-8 to assess RAW264.7 cell viability;
figure 2 is that Avicularin can inhibit RANKL-induced osteoclast formation in vitro: (A) Representative images of in vitro TRAP staining, (B) quantification of the area of each group of TRAP osteoclasts; note that n =3 per group; scale bar =750 μm; * Represents P <0.05, represents P < 0.01 (in contrast to the RANKL group only);
FIG. 3 is a graph of Avicillarin inhibiting osteoclastic bone resorption activity in vitro; (a) a typical bone resorption image; (B) Bone resorption plate resorption experimental analysis showed total hydroxyapatite resorption area in 50ng/mLMCSF, 50ng/mLRANKL and 0, 10, 100 or 300 μ MAL treated BMMs; note that n =3 per group; scale bar =750 μm; * Represents P <0.05, represents P < 0.01 (in contrast to the RANKL group only);
FIG. 4 is a graph of Avicillarin inhibiting actin ring formation in vitro; (A) Representative images of osteoclast-associated functional proteins MMP9, actin rings, and nuclear staining; (B) number of nuclei/number of osteoclasts; note that n =3 per group; scale bar =200 μm; * Represents P <0.05, represents P < 0.01 (in contrast to the RANKL group only);
FIG. 5 shows that Avicillarin inhibits the expression of osteoclast-associated protein; (A) Western blotting of cell lysates with antibodies against osteoclast-associated proteins MMP9, CTSK, c-fos and NFATc 1; (B-E) osteoclast-associated protein concentration quantification; note that n =3 per group; * Represents P <0.05, represents P < 0.01 (in contrast to the RANKL group only);
FIG. 6 shows that Avicillarin inhibits osteoclast-associated gene expression; (A-C) quantitative analysis of mRNA expression of MMP9, CTSK, TRAP; note that n =3 per group; * Represents P <0.05, represents P < 0.01 (in contrast to the RANKL group only);
FIG. 7 is a graph of Avicillarin inhibiting RANKL-induced osteoclast differentiation by inhibiting the NF- κ B signaling pathway; (A) Lysates of raw264.7 cells were treated with antibodies against P-P65, P-I.kappa.B-alpha, RANKL + AL at 0, 10, 20, 30 and 60 min for Western blotting; (B-D) histograms show the relative protein levels of P-P65, P-I.kappa.B-alpha, I.kappa.B-alpha in RANKL and RANKL + AL treated raw264.7 cells at 0, 10, 20, 30 and 60 min; note that n =3 per group; ns represents no statistical significance, P <0.05, P < 0.01 (as compared to RANKL group only);
FIG. 8 shows that Avicillarin has a protective effect on OVX-induced osteoporosis model mouse bone loss; (A) Typical 3D-CT images of femurs of mice in a sham operation group, a model group and an AL treatment group; (B) Body weight change line plots for each group of mice at modeling and at sacrifice; (C-E) comparative analysis of bone structural parameters including bone density (BMD, unit: g/cm 3), bone volume/tissue volume (BV/TV, unit:%), trabecular bone resolution (Tb.sp, unit: mm); (F) Quantitatively analyzing the content of CTX-1 in the serum of mice in a sham operation group, a model group and an AL treatment group; note that n =3 per group; ns represents no statistical significance, P <0.05, P < 0.01 (compared to model group);
FIG. 9 is H & E staining of femoral sections of mice in sham, model and AL treatment groups; (3 cases per group);
FIG. 10 is a graph showing the quantitative analysis of the surface area and number of H & E positive cells in femoral sections of the sham, model and AL treated groups; note that n =3 per group; * P < 0.01 (compared to model group);
FIG. 11 is TRAP activity staining of femoral sections of mice in the sham, model and AL treatment groups; (3 cases per group);
fig. 12 is a graph for quantitatively analyzing the surface area and the number of trap-positive cells in femoral sections of the sham operation group, the model group, and the AL treatment group; note that n =3 per group; * Represents P < 0.01 (compared to model group);
FIG. 13 is a graph of Avicillarin reduces the number of MMP9 and NFATc1 positive cells in bone tissue of OVX-induced osteoporosis model mice, thereby alleviating bone loss in OVX mice; (A) IHC staining of osteoclast-associated protein MMP9 in femoral sections of mice in sham, model and AL treatment groups; (B) IHC staining of osteoclast-associated protein NFATc1 in femoral sections of mice in sham, model and AL treatment groups; (C-D) quantitatively analyzing the number of MMP9 and NFATc1 positive cells in femoral sections of mice of the sham operation group, the model group, and the AL treatment group; note that n =3 per group; ns represents no statistical significance,. Indicates P < 0.01 (compared to model group);
FIG. 14 is a H & E stained section of liver and kidney tissues of mice treated with the drug, and no significant pathological changes were observed.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 reagents or instruments used in the examples of the present invention are not indicated by manufacturers, and are all conventional reagent products commercially available.
Materials and methods:
in vitro culture and differentiation of cells
RAW264.7 cells (FuHengBioLogy, shanghai, china) were passaged by incubation with DMEM (GEHealthcare, pittsburgh, USA) medium containing 10% FBS (Gibco, rockville, USA) and 100u/mL penicillin-streptomycin-amphotericin B (NCMBiotech, suzhou, china) prior to plating and drug intervention, with the medium changed every other day. Bone Marrow Macrophages (BMM) were isolated from femurs and tibias of female C57BL/6J mice. It was cultured for 24 hours in DMEM medium (GEHealthcare, pittsburgh, USA) containing 10% FBS (Gibco, rockville, USA) and 50 ng/mLM-CSF. Non-adherent BMM was then induced with 50ng/mLM-CSF (R & DSsystems, minneapolis, MN, USA) and 50ng/mLRANKL (R & DSsystems, minneapolis, MN, USA). The concentration of DMSO in the working solution was less than 0.1%. BMM media was changed every two days.
Cell viability assay
RAW264.7 cells or BMMs cells were seeded in 96-well plates at a density of 1X 104/well for 24 hours. Then treated with 0, 10, 30, 100, 300 or 600 μmavicular in for 1, 2 or 3 days. We then incubated each well with 10. Mu.l of CCK-8 reagent (Beyotime, shanghai, china) for 1-2 hours and measured the Optical Density (OD) at 450nm using a microplate reader (BioTek, USA).
In vitro TRAcP staining
BMM was seeded in 24-well plates (1X 10) 5 Per well), incubated with 50ng/mLM-CSF and 50ng/ml lrankl, and treated with 0, 10, 30, 100, or 300 μmavicular in for 3 days. The cells were then washed twice with PBS for 15 minutes each, fixed with paraformaldehyde, and stained using the TRAcP staining kit (Sigm) according to the manufacturer's instructionsa, USA) and osteoclasts (cells with more than three nuclei) were quantified using ImageJ software (NIH, bethesda, maryland, USA).
Hydroxyapatite absorption assay
Culture of 1X 10 per well in 24-well hydroxyapatite-coated plates (Corning Life sciences, st. Lowell, mass., USA) 5 BMM and 5 days of addition of 50ng/mLRANKL and 50ng/mLM-CSF to the coated plate. Then, the well plate was washed with PBS to remove cells, and the region of hydroxyapatite reabsorption was observed by an optical microscope OlympusCKX53 (Tokyo, japan) and quantified with ImageJ software (NIH, bethesda, maryland, USA).
Immunofluorescence staining
Differentiated osteoclasts were washed three times with PBS, fixed with 4% paraformaldehyde for 30 minutes, and then permeabilized with triton x-100 for 10 minutes. Cells were stained with primary antibodies against MMP9 (1, ab38898, abcam) and NFATc1 (1, ab25916, abcam) overnight at 4 ℃, followed by washing and placing in the dark with a secondary antibody AlexaFluor555 (1. After 10 minutes of staining with DAPI, cells were imaged using the EVOSM5000 cell imaging system (ThermoFisherScientific, bothell, WA, USA). The osteoclast area and the number of nuclei per osteoclast were quantified using ImageJ software.
Quantitative RT-PCR analysis
BMM cells at 1X 10 5 The density per well was inoculated into 6-well plates and cultured in medium containing 50ng/ml RANKL and 0, 10 or 100. Mu.M Avicularin until osteoclasts were formed. Total RNA was then extracted from osteoclasts using an RNA Rapid extraction kit (ESScience, shanghai, china). The extracted total RNA was reverse-transcribed into cDNA, and then real-time quantitative PCR was performed using SYBRGreenPCRMastermix (applied biosystems Vilnius, lithounia). The PCR cycle parameters were as follows: 10 minutes at 94 ℃, 15 seconds at 95 ℃, 60 seconds at 60 ℃ and 40 cycles. Primers used for PCR (sangon biotech, shanghai, china) were as follows:
CTSK
forward primer = SEQ ID NO:1=5'-GAAGAAGACTCACCAGAAGCAG-3'
Reverse primer = SEQ ID NO:2=5'-TCCAGGTTATGGGCAGAGATT-3';
TRAP
forward primer = SEQ ID NO:3=5'-CACTCCCACCCTGAGATTTGT-3',
reverse primer = SEQ ID NO:4=5'-CATCGTCTGCACGGTTCTG';
MMP9
forward primer = SEQ ID NO:5=5'-CTGGACAGCCAGACACTAAAG-3'
Reverse primer = SEQ ID NO:6=5'-CTCGCGGCAAGTCTTCAGAG-3';
GAPDH
forward primer = SEQ ID NO:7=5'-AGGTCGGTGTGAACGGATTTG-3',
reverse primer = SEQ ID NO:8=5'-TGTAGACCATGTAGTTGAGGTCA-3'.
Each sample was repeated 3 times to ensure accuracy.
Western blot analysis
Total protein was isolated from RAW264.7 cells induced with RANKL (50 ng/mL) in 6-well plates. After a corresponding time of 100. Mu.M Avicillarin treatment, the protein samples were separated in an electrophoresis apparatus and then transferred to a PVDF membrane (Merck Millipore, MA, USA). Next, the membrane was blocked and incubated with the corresponding primary antibody overnight at 4 ℃. The antibodies used were as follows: MMP9 (1. After washing with TBST (CWBiotech, beijing, china) for 15 minutes, the membrane was incubated with the secondary antibody for 2 hours. Finally, the protein was measured with a chemiluminescent HRP substrate (millipore corporation, MA, USA).
Laboratory animals and drug therapy
The animal experiments were conducted according to the protocol approved by the committee of animal care institutions of traditional Chinese medicine, zhang hong Kong City (approval document:). We purchased C57BL/6J mice (JOINN laboratories, suzhou, china) (lot number: 202114550) 6 weeks old and randomized into sham, OVX + low dose Avicillarin, OVX + high dose Avicillarin groups (5 mice per group). We prepared 6mM stock solutions of Avicillarin (Sigma-Aldrich, sydney, australia) in dimethyl sulfoxide (DMSO; thermoFisher scientific, scoresby, australia) and diluted to the corresponding concentrations using PBS (ThermoFisher scientific, scoresby, australia). OVX and OVX + low/high dose Avicularin mice were subjected to bilateral ovariectomy induced osteoporosis under anesthesia, and sham surgery in which only ovaries were externalized without ovariectomy was performed on sham surgery group mice. Starting from the third week after the model is established, drug intervention is carried out, mice in the OVX + low-dose Avicillarin group are injected with Avicillarin 1.25mg/kg in the abdominal cavity every 2 days, mice in the OVX + high-dose Avicillarin group are injected with Avicillarin 5mg/kg in the abdominal cavity every 2 days, sham operation groups and OVX groups are given with the same amount of physiological saline, the mice are killed after 8 weeks, and blood is taken from femurs, livers, kidneys and orbital bones on both sides and is centrifuged, and then supernatant is taken for further research.
micro-CT scanning and analysis
Femurs (n = 5/group) of 4 groups of mice were analyzed using SkyScan1176 high resolution micro computed tomography scanner (SkyScan, knottich, belgium) to obtain μ CT images thereof. The scanning parameters were 9 μm, 80kV (voltage) and 100mA (current) per layer. Then, the relevant 3D images were processed and introduced into CTAn software (Brukermicro-CT, kontich, belgium) to obtain data such as bone density (BMD), bone volume/tissue volume (BV/TV), trabecular spacing (Tb.Sp), etc.
Histological and immunohistochemical analysis
After micro-CT analysis, the femurs were stored at 4 ℃ for histological and immunohistochemical analysis. The femurs of 4 groups of mice were first decalcified with 15% ethylenediaminetetraacetic acid (EDTA, sigma), paraffin-embedded, microtomed and 5 μm thick. Sections were then stained for hematoxylin eosin (H & E), tartrate-resistant acid phosphatase (TRAcP) and Immunohistochemistry (IHC). The primary antibodies are MMP9 (ab 38898, abcam) and NFATc1 (ab 25916, abcam). The stained sections were photographed using an OlympusCX43 optical microscope (tokyo, japan).
Statistical analysis
Statistical analysis was performed in this experiment using SPSS25 software. Data are shown as mean ± Standard Deviation (SD), where SD represents the difference between three independent experimental values. The comparison between the two groups was performed by t-test, and the comparison between the two or more groups was performed by one-way analysis of variance (ANOVA). P <0.05 indicates that the data is statistically significant.
Example 1
Avicillarin inhibits RANKL-induced osteoclastogenesis
CCK-8 analysis showed that treatment of Raw264.7 with 10, 30, 100, 300. Mu. MAvicular in did not reduce its viability within 1d, 2d, 3d (FIG. 1). Osteoclast differentiation was induced in vitro with 50ng/mL M-CSF and 50ng/mL RANKL, and Avicillarin intervention was performed on BMMs cells at concentrations of 0, 10, 100, 300. Mu.M, respectively. The results showed that Avicularin inhibited the formation of multinucleated osteoclasts in a dose-dependent manner compared to the RANKL-induced group (figure 2).
Example 2
Avicillarin affects osteoclast bone resorption and F-actin ring formation
To verify whether Avicularin could inhibit osteoclastic bone resorption, we analyzed its effect on osteoclastic resorption activity using a hydroxyapatite resorption assay. The RANKL group induced BMMs produced a large number of bone resorption pits, and the number of bone resorption areas and resorption points decreased in a dose-dependent manner after treatment with different concentrations of Avicularin (fig. 3). The bone resorption areas of the RANKL group were 10, 100 and 300 μmol/lavicular stem prognosis, respectively, and the results showed that Avicularin inhibited the in vitro resorptive activity of osteoclasts in a dose-dependent manner.
The polarity of F-actin is crucial in the process of osteoclastogenesis and bone resorption. Therefore, we explored the effect of Avicillarin on F-actin loop formation. After co-staining with an antibody against phalloidin and osteoclast-specific protein MMP9, it was observed by immunofluorescence that RANKL-induced F-actin had a typical intact cyclic structure. Following Avicillarin treatment, the size, number, and number of F-actin rings decreased dose-dependently. Together, these results indicate that Avicularin can inhibit the formation of F-actin rings and osteoclast function (fig. 4).
Example 3
Avicillarin inhibits osteoclast-associated protein expression
Osteoclast differentiation and activation is associated with RANKL. Western blot was used to assess whether puerarin inhibits RANKL stimulated osteoclast-associated protein expression. RAW264.7 cells were treated with a medium containing or not containing RANKL and cultured with Avicillarin at a concentration of 100. Mu. Mol/L. Westernblotting results showed that Avicillarin treatment significantly down-regulated the expression of C-FOS, CTSK, NFATc1 and MMP-9 (P < 0.05) (FIG. 5).
Example 4
Avicillarin inhibits expression of osteoclast-associated genes
RANKL stimulates osteoclast formation and bone resorption functions accompanied by the up-regulation of osteoclast-specific genes, such as CTSK, MMP9, TRAP. qRT-PCR was used to assess the effect of Avicillarin on osteoclast-associated genes. BMMs cells were induced with RANKL containing medium and intervened by adding different concentrations of Avicillarin (10, 100. Mu. Mol/L). The results show that after RANKL induction, the expression of MMP9, TRAP, and CTSK were all elevated, and after Avicularin treatment, mRNA levels were significantly decreased in a concentration-dependent manner relative to the RANKL group (fig. 6), which further consolidated the anti-osteoclast function of Avicularin.
Example 5
Avicillarin inhibits activation of NF-kB signaling pathway during osteoclastogenesis
The upregulation of osteoclast genes is controlled by the activation of various RANKL response signaling pathways, among which the NF- κ B pathway plays an important role in osteoclast activation. To investigate the effect of Avicillarin on NF-. Kappa.B signaling after RANKL stimulation, we performed Western blot analysis (FIG. 5A), and the results showed that the expression level of phospho-P65 in RANKL group was up-regulated and peaked at 10min, while after Avicillarin dry prognosis, the expression level of phosphorylation was lower than that in RANKL group in the same time period, and Avicillarin also inhibited the phosphorylation level of I.kappa.B.alpha.and inhibited its degradation (FIG. 7).
Example 6
Avicillarin reduces mouse Ovariectomy (OVX) -mediated bone loss
Based on the effect of Avicularin on osteoclasts in vitro, we examined the anti-osteoporosis effect of Avicularin in OVX mice, an OVX model is commonly used to model postmenopausal osteoporosis. 21 days after bilateral OVX or sham surgery, mice were treated with intraperitoneal injections of saline or Avicillarin, and OVX mice were administered with different concentrations of Avicillarin (1.25 mg/kg/d or5 mg/kg/d) for 8 weeks. There was no difference in body weight between the control group and the administered group within 8 weeks of the experiment. Transverse microscopic CT images of the mouse femur were obtained at the end of treatment, and quantitative analysis of the OVX mouse bone morphometric parameters was consistent with its osteoporotic phenotype, i.e. a significant reduction in bone volume (BV/TV), bone Mineral Density (BMD), an increase in trabecular spacing (tb.sp), and Avicularin intervention protected mice from OVX-induced bone loss, improving BV/TV, BMD and tb.sp in a dose-dependent manner (fig. 8).
H & E staining results showed (fig. 9) a significant decrease in Bone Surface (BS) in OVX group compared to sham group, and a concentration-dependent increase in Bone Surface (BS) after Avicularin intervention (fig. 10). TRAcP staining of bone tissue sections showed a significant increase in osteoclast number/bone surface (n.oc/BS) and osteoclast surface/bone surface (oc.s/BS) in the OVX group compared to the sham group (fig. 11), and a significant decrease in osteoclast number/bone surface (n.oc/BS) and osteoclast surface/bone surface (oc.s/BS) in the Avicularin-treated OVX group mice, in a concentration-dependent manner (fig. 12).
Results of immunofluorescence staining analysis of osteoclast functional protein MMP9 and related transcription factor NFATc1 show: the number of MMP 9-and NFATc 1-positive cells was significantly increased in the OVX group compared to the sham group, and the number of MMP 9-and NFATc 1-positive cells was significantly decreased in the Avicularin intervention group compared to the OVX group (fig. 13).
The mouse serum bone resorption index CTX-1 is detected by an ELISA method and compared among groups, and the result shows that the content of the Avicillarin stem-control group serum CTX-1 is obviously reduced compared with that of a model group, and the difference among the groups has statistical significance.
From the general examples 1 to 6
Osteoclast is the only bone-resorbing cell of hematopoietic origin, osteoclast-mediated bone resorption is indispensable in the bone remodeling process, and excessive osteoclast activity leads to imbalance in bone homeostasis. Bone resorption involves a number of steps including bone adhesion, cytoskeletal reorganization, formation of fold boundaries, and the like. Osteoclast activation is initiated by matrix recognition, the osteoclast's cytoskeleton having the ability to polarize the osteoclast into the osteocyte interface, forming a ruffled boundary that forms a gasket-like structure with the isolated resorptive microenvironment, called the actin loop. In the cytoplasm, osteoclasts synthesize and secrete lytic enzymes to acidify the lacuna created by exocytosis, and then, osteoclasts transport osteoclast-associated functional proteins such as CTSK, MMP9, etc. into acidic absorption microenvironment via vesicles, and internalize degradation products by endocytosis. It can be seen that osteoclast number, area and secretion of functional proteins polarized onto osteocytes play an important role in bone resorption. In this study, we found that Avicularin concentration-dependently inhibited RANKL-induced osteoclast formation, with a significant decrease in osteoclast number and area seen by TRAP staining with increasing concentration of the intervention drug Avicularin. By immunofluorescence staining, the formation of an osteoclast actin ring can be obviously inhibited by Avicillarin, and meanwhile, the fluorescence signal of a functional protein MMP9 is weakened due to drug intervention. The WB and PCR results are consistent, the expression of related functional proteins is reduced in Avicillarin concentration dependency, and the bone plate absorption test also more directly shows that Avicillarin inhibits the osteoclastic activity. Taken together, avicularin inhibits osteoclast formation and activation in vitro to inhibit bone resorption activity.
Activation of the NF-. Kappa.B pathway is critical to osteoclast formation and activation, and Avicillarin has been reported to inhibit cell cycle progression and promote apoptosis by inhibiting NF-. Kappa.B activity. Also results indicate that Avicillarin decreased protein expression of p-p65 and decreased the p-p65/p65 ratio in bradykinin-induced MG-63 cells. In this study, the Westernblot results show that Avicularin can inhibit phosphorylation and degradation of I κ B α and p 65. In the time-dependent observation of Avicularin on the NF- κ B pathway, p65, I κ B α peaked 10min after RANKL intervention, whereas phosphorylation of p65, I κ B α was significantly down-regulated under the action of Avicularin drugs. NFATc1 and C-fos are downstream factors of NF-kB pathway and are related to osteoclast activation, and the research result shows that Avicillarin remarkably inhibits the protein expression of NFATc1 and C-fos in the osteoclast differentiation process, so that Avicillarin inhibits the formation and activation of osteoclast by inhibiting NF-kB signal pathway.
In an in vivo model, this study demonstrated the inhibitory effect of Avicularin on bone resorption activity in vivo by an ovariectomy-induced mouse osteoporosis model. In this study, liver and kidney tissue sections were free of lesions seen in drug toxicity 8 weeks after Avicillarin intervention in the treated mice (FIG. 14), indicating that Avicillarin can achieve safe and effective drug concentrations in vivo. Meanwhile, the bone density of the distal femur of the model group mice was significantly lower than that of the blank group, indicating that OVX surgery was successful, morphometric analysis of bone parameters showed significant improvements in bone mass and trabecular structure, indicating that administration of aviculrin protected mice from estrogen-deficient bone loss after OVX, whereas aviculrin reduced the number of TRAP-positive cells and inhibited RANKL-induced differentiation of TRAP-positive cells in histological observation of the distal femur. These results indicate that the anti-osteoporosis effect of Avicularin is mediated by inhibiting osteoclast differentiation.
Taken together, our in vitro studies indicate that Avicillarin can inhibit osteoclast activation by inhibiting the NF-. Kappa.B signaling pathway. In vivo experiments show that Avicillarin can alleviate the development of OVX mouse osteoporosis. Based on these findings, we believe Avicularin is a potentially safe and effective treatment of osteoporosis.
The above-mentioned embodiments only represent a limited number of preferred embodiments of the present invention, and the description is specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (10)
1. Application of polygonin in preparing medicine/health product for preventing and treating osteoporosis is provided.
2. Use of avicularin according to claim 1, wherein the avicularin inhibits RANKL-induced osteoclast differentiation in the preparation of a medicament/health product for preventing and treating osteoporosis.
3. Use of avicularin according to claim 1 for the preparation of a medicament/health product for preventing and treating osteoporosis, wherein the avicularin inhibits the bone resorption function of osteoclasts and the formation of F-actin rings.
4. Use of avicularin according to claim 1 in the preparation of a drug/health product for preventing and treating osteoporosis, wherein the avicularin down-regulates the expression of osteoclast-associated genes and proteins; the related proteins are C-FOS, CTSK, NFATc1 and MMP-9; the related genes are CTSK, MMP9 and TRAP.
5. Use of avicularin according to claim 1, wherein the avicularin inhibits rankl-induced osteoclastogenesis by inhibiting the activation of NF- κ B signaling pathway during osteoclastogenesis.
6. The use of avicularin according to claim 1 in preparing a drug/health product for preventing and treating osteoporosis, wherein the avicularin inhibits phosphorylation and degradation of I κ B α and p 65.
7. The use of avicularin in the preparation of a drug/health product for preventing and treating osteoporosis according to claim 1, wherein the avicularin relieves bone loss of castrated mice.
8. The use of polygonin in the preparation of drugs/health products for preventing and treating osteoporosis in accordance with claim 7, wherein the dose of said polygonin is 1.25mg/kg/d to 5mg/kg/d.
9. A medicine/health product for preventing and treating osteoporosis is characterized by containing avicularin.
10. The drug/health product for preventing and treating osteoporosis of claim 9, wherein the avicularin is an injection or an oral preparation.
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