Previous research indicates that kaempferol (Kae) promotes osteogenesis, but its underlying mechanism of action remains unclear. The present study hypothesized that the osteogenic effects of Kae were mediated through mammalian target of rapamycin (mTOR). To validate this hypothesis, bone marrow mesenchymal stem cells (BMSCs) from ovariectomized (OVX) rats were differentiated into osteoblasts. The bone mineral density and bone microarchitecture of the OVX rats was measured
Osteoporosis is described as a ‘silent disease’, characterized by gradual bone loss that occurs in the absence of other symptoms over a period of years (
Kaempferol (Kae;
Mammalian target of rapamycin (mTOR) is a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases (
Therefore, mTOR was hypothesized to be a novel target for the development of new and effective osteoporosis therapies. The aim of the present study was to investigate whether Kae was able to enhance the osteogenic differentiation and function of bone marrow mesenchymal stem cells (BMSCs) via mTOR activation.
Kae (purity>98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Kae was dissolved in DMSO and diluted to 0.01% in PBS. Rapamycin (Rapa), a specific inhibitor of mTOR, was purchased from Selleck Chemicals. The Alizarin Red S (ARS) staining buffer and alkaline phosphatase (ALP) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute. Anti-runt-related transcription factor 2 (Runx2; cat. no. ab23981) and anti-Osterix (cat. no. ab22552) was purchased from Abcam. Anti-eukaryotic translation initiation factor 4E-binding protein 1 (4E/BP1; cat. no. 94525), anti-phosphorylated (p)-4E/BP 1 (cat. no. 2855) and anti-ribosomal protein S6 kinase B1 (S6K1; cat. no. 9202), anti-p-S6K1 (cat. no. 9204) was obtained from Cell Signaling Technologies, Inc. Horseradish peroxidase-labeled anti-immunoglobulin G secondary antibody (goat anti-mouse lgG; cat. no. SA00001-1; and goat anti-rabbit lgG; cat. no. SA00001-2) was obtained from ProteinTech Group, Inc. Anti-β-actin antibody (cat. no. KL002) was provided by Nanjing Jiancheng Bioengineering Institute.
A total of 30 adult (age, 6–8 weeks) female Sprague-Dawley (SD) rats weighing 180–220 g were obtained from the Nanchang University Laboratory Animal Center (Nanchang, China) and maintained under a 12-h dark/light cycle at 22–25°C and 40–70% humidity. Animals were allowed access to food and water
A total of 30 rats were randomly divided into five groups (n=6 in each group) as follows: i) Sham group, in which the abdominal cavities of the rats were opened and fat tissue around the ovaries removed; ii) OVX group, in which the rats were ovariectomized; iii) Kae treatment group (OVX + Kae), in which the OVX rats were continuously given Kae (100 mg/kg/day) via gavage for 8 weeks; iv) Kae and Rapa administration group (OVX + Kae + Rapa), in which the rats were treated as in the Kae treatment group and in addition received intraperitoneal injections of Rapa (0.2 mg/kg/day) for 8 weeks; and v) Rapa group (OVX + Rapa), in which the OVX rats received intraperitoneal injections of Rapa (0.2 mg/kg/day) for 8 weeks. The dose and timing of Kae and Rapa administration were ascertained in preliminary experiments. Briefly, the OVX rats were given Kae at 25, 50 and 100 mg/kg/day. Bone mineral density (BMD) was detected after 8 weeks to select the working concentration. Kae at 100 mg/kg/day was the most effective concentration in improving BMD in OVX rats. Rapa was administered at 0.2 mg/kg/day according to the manufacturer's protocol (Selleck Chemicals). Subsequent experiments were performed following the 8 weeks of treatment.
Following treatment, the rats were sacrificed and their right femurs and tibias were dissected. The 2D total bone mineral content was used to calculate the BMD as previously described (
Based on the median values of total BMD, selected trabecular microarchitecture of the femoral metaphysis was evaluated using micro-computed tomography (micro-CT; Scanco Medical AG) with Scanco image processing language software (version 5.08b; Scanco Medical AG). Scans were performed from the proximal growth plate in the distal direction (18 µm/slice) as the distal femur has a high concentration of trabecular bone compared with the proximal and middle regions of the femur. A volume of interest (VOI) was selected, defined as the cross-sectional area spanning 100 slices from the proximal growth plate. The 2D scans were used to produce 3D reconstructions of the bone microarchitecture, which were used to measure bone morphometric parameters of the selected VOI, including the bone volume fraction [bone volume/tissue volume (BV/TV)], trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th) and structure model index (SMI). The operator conducting the CT analysis was blinded to the treatments associated with the specimens. All examinations were conducted according to the principles and procedures described in the most recent National Research Council publication of the Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines (
BMSCs were flushed from the femurs and tibias of normal SD and OVX rats with PBS in a biosafety cabinet, using a 5 ml syringe fitted with a needle (21G). Mononucleated cells were isolated by density gradient centrifugation at 400 × g for 20 min at room temperature in rat lymphocyte separation medium (TBD Science) at a concentration of 1.091 g/ml. Isolated cells were cultured in low-glucose DMEM (HyClone; GE Healthcare Life Sciences) containing 15% fetal bovine serum (HyClone; GE Healthcare Life Sciences). All cells were maintained in a 37°C incubator within an atmosphere containing 5% CO2. BMSCs were identified by CD44 and CD34 labeling (
The cytotoxicity of BMSCs was determined using an MTT assay. Briefly, rat BMSCs were plated into 96-well plates at a density of 1×105 cells/well. After 24 h of culture in a 5% CO2 incubator at 37°C, adherent cells were treated with a range of Kae concentrations (0.1, 1, 10 and 100 µM) for 24 h. The MTT reagent (10 µl; 10 mg/ml) was subsequently added to each well, and cells were incubated for a further 4–6 h in a 5% CO2 incubator at 37°C. The medium was removed and 100 µl DMSO was added to each well. Plates were shaken for 10 min, and the absorbance was measured at 492 nm using a Spectra Max Paradigm microplatereader (Molecular Devices, LLC). Cytotoxicity was calculated as follows: Cytotoxicity (%)=(1-absorbance of sample/absorbance of control) ×100.
Third-passage BMSCs were randomly divided into 5 groups: i) Control group, in which BMSCs derived from normal rats were treated with osteogenic induction medium (Cyagen Biosciences, Inc.) to induce osteoblast differentiation; ii) OVX group, in which BMSCs derived from OVX rats were treated in the same way as the control group; iii) OVX + Kae group, in which BMSCs derived from OVX rats were incubated with 0.1, 1, 10 or 100 µM Kae and treated in the same way as the control group; iv) OVX + Kae + Rapa group, in which BMSCs derived from OVX rats were incubated with Kae and 10 µM Rapa and then treated in the same way as the control group; v) OVX + Rapa group, where BMSCs derived from OVX rats were incubated with 10 µM Rapa and treated in the same way as the control group. The dose of Rapa was determined during preliminary experiments. Briefly, MTT was detected to evaluate the optimal concentration of Rapa. Rapa (at 0.1, 1, 10 or 100 µM) was incubated with BMSCs. Rapa was found to exhibit a concentration-dependent cytotoxic effect. Therefore, 10 µM was selected. BMSCs were harvested at day 15 in all groups.
ARS staining was used to assess the osteogenic differentiation of BMSCs. Briefly, rat BMSCs were plated into 24-well plates at a density of 2.5×105 cells/well. After induction of osteogenesis, cells were fixed with 95% alcohol for 15 min at room temperature and the BMSCs were stained with ARS for 5 min at room temperature. Mature osteoblasts that differentiated from rBMSCs displayed intense brown-red staining after 5 min of ARS staining (magnification, ×40). Cells were imaged at ×400 magnification by light microscopy, after the addition of 10% (w/v) cetylpyridinium chloride to precipitate calcium ions. The absorbance of each well was measured at 562 nm using a Spectra Max Paradigm microplatereader (Molecular Devices, LLC).
The effects of Kae on osteoblasts were assessed using an ALP assay kit. The absorbance/optical density (OD) of each sample was measured at 492 nm using a microplate reader. ALP activity in the osteogenic induction medium (Cyagen Biosciences, Inc.) was determined as follows: ALP activity (Jinshi unit/100 ml)=(T-B)/(S-B)×0.02 mg/ml ×100 ml × a, where T=OD of the test sample; B=OD of the blank; S=OD of the standard; and a=dilution factor of the sample. According to the conditions above, a Jinshi unit is defined as the ALP activity that releases of 1 mg of phenol every 15 min after mixing with 100 ml liquid matrix at 37°C.
Cells were lysed in RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 1 mM PMSF and 1 mM EDTA. Then, extracts were centrifuged at 24,750 × g at 4°C for 15 min to remove insoluble material. Total protein concentrations were determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. An equivalent quantity of total protein (40 µg) per well was diluted in sample buffer containing 100 mM dithiothreitol and heated to 98°C for 5 min. Lysates were separated using 10–15% SDS-PAGE (Bio-Rad Laboratories, Inc.) and subsequently transferred to PVDF membranes. The membranes were blocked in 5% non-fat dry milk for 2 h at room temperature and incubated overnight at 4°C using primary antibodies (1:500) against β-actin, Runx2, 4E/BP 1, p-4E/BP1, S6K1 and p-S6K1. Membranes were washed in TBS with Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000) at room temperature for 1 h. Membranes were washed three times for 20 min in TBS-T, and protein bands were visualized by enhanced chemiluminescence (Proteintech Group, Inc.). Band intensities were measured and quantitated using Quantity One software (version 4.6.6; Bio-Rad Laboratories, Inc.) and β-actin was used for normalization.
SPSS (version 20.0; IBM Corp.) was used for statistical analysis. Data are presented as the mean ± SEM from six independent experiments. The variance homogeneity test and one-way ANOVA were performed between groups. Newman-Keuls test was used following ANOVA. P<0.05 was considered to indicate a statistically significant difference.
The 3D trabecular bone microarchitecture, used for the assessment of distal femoral metaphysis, was calculated from micro-CT images (
The cytotoxicity of various concentrations of Kae was tested on BMSCs. As shown in
ARS staining was used to assess the induction of osteogenesis in BMSCs. As illustrated in
ALP activity was measured during osteogenesis. As shown in
Western blot analysis was used to assess the expression of the osteogenesis-related transcription factors Runx2 and Osterix during osteogenesis.
Western blot analysis was used to determine the levels of phosphorylated and total 4E/BP1 and S6K1, important downstream regulators of the mTOR pathway. As shown in
Estrogen has a significant role in bone metabolism (
Runx-2, also termed core binding factor α-1, activates and initiates the differentiation of BMSCs into osteoblasts and regulates osteoblast maturation (
mTOR is a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases (
Studies have demonstrated that mTOR signaling has numerous key regulatory functions in various diseases, including cancer (
In summary, the present study demonstrated that Kae has a positive effect on bone formation and osteogenesis in OVX rats, which is possibly exerted via mTOR-Runx2/Osterix signaling. Since Kae naturally occurs in a variety of foods, it is likely that it may exert beneficial effects as a food supplement by improving postmenopausal osteoporosis. Further large scale randomized
Not applicable.
The present study was supported by the Traditional Chinese Medicine Project of the Health and Family Planning Commission of Jiangxi Province, China (grant no. 2017A274) and the Science and Technology Program of Health and Family Planning Commission of Jiangxi Province, China (grant no. 20185232).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
JL conceived and designed the present study and revised the manuscript for important intellectual content. JZ was involved in all experiments and was a major contributor in writing the manuscript. JW modified the study design and designed the structure of the article. YL performed the experiments, analyzed the data and wrote the manuscript. JM and XL completed the data analysis. BX and ZY performed the cell culture, ARS staining, ALP activity assay, western blotting. All authors read and approved the final manuscript.
Experiments were performed according to the Guide for the Care and Use of Laboratory (National Institutes of Health), and were approved by the Ethics Committee of Nanchang University (no. 2017-0122).
Not applicable.
The authors declare that they have no competing interests.
kaempferol
bone mesenchymal stem cells
mammalian target of rapamycin
ovariectomized
rapamycin
Chemical structure of kaempferol (Pubchem CID: 5280863).
Effects of Kae on bone microarchitecture in rats. (A) Representative trabecular bone microarchitecture of the femoral metaphysis for each group, obtained from micro-CT images. Quantification of various parameters of bone microarchitecture for each group: (B) BV/TV, (C) SMI, (D) Tb.N, (E) Tb.Th, and (F) Tb.Sp. (G) BMD parameters measured using micro-CT in each group. The data are expressed as the mean ± SEM (n=6). *P<0.05 vs. control group; ▲P<0.05 vs. OVX group; #P<0.05 vs. OVX + Kae group. Kae, kaempferol; micro-CT, micro-computed tomography; BV/TV, bone volume/tissue volume; Tb.N, trabecular number; Tb.Sp, trabecular separation, Tb.Th, trabecular thickness; SMI, structure model index; BMD, bone mineral density; Rapa, rapamycin; OVX, ovariectomized.
Effects of Kae on cell cytotoxicity. BMSCs were treated with different concentrations of Kae and cell cytotoxicity was assessed via an MTT assay. The data are expressed as the mean ± SEM of six individual experiments. *P<0.05 vs. 0.1 µM and #P<0.05 vs. 100 µM. Kae, kaempferol; BMSCs, bone mesenchymal stem cells.
Effects of Kae on BMSC commitment to an osteogenic fate. ARS staining was performed to observe the osteogenic effects of Kae on rat BMSCs (0–100 µM). ARS-stained images (magnification, ×400) show the effect of the treatment with (A) 0, (B) 0.1, (C) 1, (D) 10 and (E) 100 µM Kae. (F) Bar graph illustrating the quantitative analysis. Values are presented as the mean ± SEM of six individual experiments. *P<0.05 vs. 0 µM. Kae, kaempferol; rBMSCs, rat bone mesenchymal stem cells; ARS, Alizarin Red S.
Kae promotes the differentiation of BMSCs into osteoblasts. ARS staining was performed to observe the osteogenic differentiation of cultured BMSCs. (A) ARS-stained images (magnification, ×400) and respective (B) quantitative analysis. The data are expressed as the mean ± SEM (n=6 for each group). *P<0.05 vs. control group; ▲P<0.05 vs. OVX group; #P<0.05 vs. OVX + Kae group. Kae, kaempferol; BMSCs, bone mesenchymal stem cells; ARS, Alizarin Red S; Rapa, rapamycin; OVX, ovariectomized.
Kae increases the level of ALP in BMSCs. The data are expressed as the mean ± SEM (n=6 for each group). *P<0.05 vs. control group; ▲P<0.05 vs. OVX group; #P<0.05 vs. OVX + Kae group. Kae, kaempferol; BMSCs, bone mesenchymal stem cells; ALP, alkaline phosphatase; Rapa, rapamycin; OVX, ovariectomized.
Kae upregulates the expression of Runx2 and Osterix. Western blot analysis was used for the assessment of Runx2 and Osterix expression, and β-actin was used as the internal control. Pretreatment with Kae induced increased expression of Runx2 and Osterix in BMSCs, which was antagonized by Rapa. The data are expressed as the mean ± SEM (n=6 for each group). *P<0.05 vs. control group; ▲P<0.05 vs. OVX group; #P<0.05 vs. OVX + Kae group. Kae, kaempferol; BMSCs, bone mesenchymal stem cells; Runx 2, Runt-related transcription factor; Rapa, rapamycin; OVX, ovariectomized.
Kae decreases the phosphorylation of 4E/BP1. Relative levels of 4E/BP1 and p-4E/BP1 were detected by western blotting. Treatment with Kae decreased the levels of p-4E/BP1, which was antagonized by Rapa. The data are expressed as the mean ± SEM (n=6 for each group). *P<0.05 vs. respective sham group; ▲P<0.05 vs. respective OVX group; #P<0.05 vs. respective OVX + Kae group. Kae, kaempferol; Rapa, rapamycin; BMSCs, bone mesenchymal stem cells; 4E/BP1, eukaryotic translation initiation factor 4E-binding protein 1; p, phosphorylated; OVX, ovariectomized.
Kae increases the phosphorylation of S6K1. Protein levels of S6K1 and p-S6K1 were detected by western blotting. Treatment with Kae increased the levels of p-S6K1, an effect that was antagonized by Rapa. The data are expressed as the mean ± SEM (n=6 for each group). *P<0.05 vs. respective sham group; ▲P<0.05 vs. respective OVX group; #P<0.05 vs. respective OVX + Kae group. Kae, kaempferol; Rapa, rapamycin; BMSCs, bone mesenchymal stem cells; S6K1, ribosomal protein S6 kinase B1; p, phosphorylated; OVX, ovariectomized.