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Icariin, the main active compound of the traditional Chinese medicine,
Osteoporosis is a common disease characterized by a reduction of bone mass resulting from the negative balance between bone formation and bone destruction (
Recently, icariin was found to be therapeutically effective in ovariectomized rats. It increases trabecular bone mineral density (BMD) and bone strength and prevents the suppression of serum Ca levels (
Bone is an active tissue that undergoes constant remodeling in which old bone is degraded by osteoclasts (bone-resorbing cells), and subsequently replaced by new bone formed by osteoblasts (bone-forming cells), via a process known as remodeling (
Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and trypsin-EDTA were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from the Sigma Chemical Co. (St. Louis, MO, USA). TRIzol reagent was purchased from Inc. (Grand Island, NY, USA). Monoclonal rabbit anti-human Smad4, Cbfa1/Runx2, and β-actin HRP secondary goat anti-rabbit antibodies were purchased from Abcam (Cambridge, MA, USA). Primers were synthesized by Sangon Biotech (Shanghai, China). Icariin (HPLC ≥98%) was produced by Nanjing Zelang Medical Technological Co., Ltd. (Nanjing, China). Stock solutions of icariin were prepared by dissolving the icariin powder in DMSO to a concentration of 10−3 M, and stored at −20°C. The working concentrations of icariin were obtained by diluting the stock solution with the culture medium. The final concentration of DMSO in the medium was <0.5%.
The hFOB 1.19 human osteoblastic cell line obtained from the Insitute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) was cultured in DMEM, supplemented with 10% (v/v) FBS, penicillin (100 U/ml) and streptomycin (100
The hFOB 1.19 cell viability was assessed by MTT colorimetric assay. The cells were seeded in 96-well plates at a density of 1.0x105 cells/well, cultured for 24 h and starved for 24 h in serum-free DMEM medium. The cells were treated with icariin at various final concentrations (10−15, 10−12, 10−9 and 10−6 M) and the vehicle control cells were treated with 0.5% DMSO for 48 h. In some experiments, the cells were treated with 10−9 M of icariin for different periods of time. The medium was discarded and replaced with 10
Calcified nodules of the hFOB 1.19 cells were demonstrated by Alizarin red S staining. The cells were seeded in 48-well plates at a density of 2x105 cells/well and cultured for 24 h, and then treated with or without icariin. The medium was replaced every 3 days. After 14 days, the cell cultures were washed 3 times with PBS, fixed with formalin:methanol: H2O (1:1:1.5) 0.5 ml/well for 30 min at room temperature, and then washed 3 times with double distilled water. The cells were stained with 0.1% Alizarin red S at 37°C for 30 min, and washed 5 times with double distilled water and air-dried. The stained calcified nodules that appeared bright red in color were identified by light microscopy.
The cells were seeded in 48-well plates at a density of 2x105 cells/well and cultured for 24 h, and then treated with or without icariin for 48 h. Cells were harvested after treatment and lysed with 100
After icariin treatment for 48 h, total RNA from the cells was isolated with TRIzol reagent (Invitrogen). Oligo(dT)-primed RNA (5
The treated cells were harvested and lysed with Nonidet P-40 lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40) supplemented with protease inhibitors (2
Data were analyzed using the SPSS package for Windows (version 13.0). Quantitative data were expressed as the means ± standard deviation (SD). Statistical analysis of the data was performed with the Student’s t-test and ANOVA. P-value <0.05 was considered to indicate a statistically significant difference.
Icariin (10−12, 10−9 and 10−6 M) significantly increased cell viability by approximately 111.67±4.72, 136.50±6.47 and 123.17±4.49% in hFOB 1.19 cells compared to the control cells (100±0.00%, P<0.01) (
The ALP activity in the hFOB 1.19 cells was increased by 1.09-, 1.21- and 1.13-fold when the cells were treated with icariin at the final concentrations of 10−12, 10−9 and 10−6 M, respectively, significantly higher than that of the control cells (P<0.05) (
Icariin induced a 1.24-, 1.38- and 1.32-fold increase in BMP-2 mRNA expression (P<0.01) (
To further explore the mechanism by which icariin regulates bone formation, we analyzed the protein expression levels of Smad4 and Cbfa1/Runx2 after icariin treatment using western blot analysis (
In the present study, we systematically evaluated the osteoprotective effects and mechanism of actions of icariin in the hFOB 1.19 human osteoblastic cell line. Our results clearly demonstrate that icariin enhances the cell viability and increases the amount of calcified nodules, as well as increasing the expression ratio of OPG/RANKL in hFOB 1.19 cells. In addition, our results show that icariin upregulates the expression of ALP, BMP-2, Smad4, Cbfa1/Runx2, OPG and RANKL, suggesting that it promotes osteoblastic bone formation by the BMP-2/Smad4 signal transduction pathway.
Osteoporosis, a progressive disorder of aging bone, is a worldwide health problem with a high prevalence. Bone is a dynamic tissue whereby old bone is removed by osteoclasts and new bone is formed continuously by osteoblasts. Bone generation, maintenance and healing are complicated processes in which osteoblasts, osteoclasts, and osteocytes are known to play important roles (
The study of Chinese herbs is worthwhile as this may lead to the discovery of certain agents which can stimulate the proliferation and differentiation of osteoblasts. Recently,
The results of the present study confirm that icariin stimulates the proliferation of hFOB 1.19 cells in a dose- and time-dependent manner. Osteoblasts are derived from mesenchymal stem cells. The sequential expression of type I collagen, ALP and the deposition of calcium are known as molecular markers. Human osteoblasts cultured for 48 h in the presence of 10−12, 10−9 and 10−6 M of icariin exhibited a significant increase in ALP activity, and the formation of mineralized nodules increased significantly after the cells were cultured for 14 days in the presence of 10−12, 10−9 and 10−6 M icariin. As the appearance of ALP activity is an early phenotypic marker for mature osteoblasts, and mineralized nodule formation is a phenotypic marker for a later stage of osteoblast differentiation, our results suggested that icariin stimulated bone formation. Further study is required to clarify the possible additional effects of icariin on renal Ca transport that contribute to the conservation of bone mass in animal models of osteoporosis.
BMPs play important roles in the regulation of bone induction, repair and maintenance (
Osteoblasts are recruited to the resorption area and osteoclasts are activated to resorb old bone in the bone remodeling process. OPG and RANKL are critical in determining osteoclastogenesis and bone homeostasis (
Icariin, an active ingredient identified in
This study was supported by the National Natural Science Foundation of China (Grant no. 81102609), the Natural Science Foundation of Fujian Province (Grant no. 2011J05076) and the Youth Foundation of Fujian Provincial Health Bureau (Grant no. 2011-2-31).
Chemical structure of icariin.
Effect of icariin on cell viability in hFOB 1.19 cells. The hFOB 1.19 cell viability increased at each concentration, with significant statistical differences observed between the cells treated with icariin and the control cells. (A) Cells were treated with the indicated concentrations of icariin for 48 h. (B) Cells were treated with 10−9 M of icariin for the indicated time periods. Cell viability was determined by the MTT assay. Data are averages ± SD (error bars) from at least 3 independent experiments. **P<0.01, statistically significant vs. control. C, control cells.
Effect of icariin on alkaline phosphatase (ALP) activities in hFOB 1.19 cells. The hFOB 1.19 cells were treated with or without icariin for 48 h. Cell lysates were used for ALP activity measurement. Results were obtained from 3 independent experiments, and data are averages ± SD (error bars), *P<0.05; **P<0.01 vs. control. C, control cells.
Effect of icariin on calcified nodules in hFOB 1.19 cells. The hFOB 1.19 cells were treated with or without icariin for 14 days. Alizarin red S staining for mineralization. The calcified nodules appeared bright red in color.
Effect of icariin on BMP-2, Smad4, OPG, RANKL mRNA expression and the OPG/RANKL ratio in hFOB 1.19 cells. The hFOB 1.19 cells were treated with or without icariin for 48 h. Total RNA was isolated and real-time PCR was performed to determine the mRNA expression levels of (A) BMP-2, (B) Smad4, (C) OPG, (D) RANKL and (E) OPG/RANKL, which were normalized to those of GAPDH. Results were obtained from 3 independent experiments, and data are averages ± SD (error bars), *P<0.05; **P<0.01 vs. control. C, control cells.
Effect of icariin on Smad4 and Cbfa1/Runx2 protein expression in hFOB 1.19 cells. The hFOB 1.19 cells were treated with or without icariin for 48 h. Total protein was isolated and (A) western blot analysis was performed to determine the protein expression levels of (B) Smad4 and (C) Cbfa1/Runx2, which were normalized to those of β-actin. Results were obtained from 3 independent experiments, and data are averages ± SD (error bars), *P<0.05; **P<0.01 vs. control. C, control cells.