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Icariine stimulates proliferation and differentiation of human osteoblasts by increasing production of bone morphogenetic protein 2

YIN, Xiao-xue; CHEN, Zhong-qiang; LIU, Zhong-jun; MA, Qing-jun; DANG, Geng-ting

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Abstract

Osteoporosis is defined conceptually as a skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture. It can occur at any age and in any racial or ethnic group, though more common in post-menopausal women, and especially in Asia or Caucasia areas. The most serious consequence of osteoporosis is fracture, which is associated with increase in mortality, substantial morbidity and social costs.1 Early intervention is now possible with the help of some effective medications, which may reduce the risk of first and recurrent fractures. However, these medications remain costly and are not affordable for the majority of Chinese. Therefore it is in urgent need to develop efficient and lower-cost methods.

Epimedium pubescens is recorded in the Chinese pharmacopoeia (2000) as a traditional Chinese medicine “Yinyanghuo”, which has been used as tonics, aphrodisiacs and antirheumatics. Icariine is a flavonoid isolated from this herb and is the main active compound of it. Recently, Epimedium pubescens was found to have a therapeutic effect on osteoporosis rat models induced by ovariectomy.2-4 It increases mineral content, promotes bone formation and increases lumbar bone mineral density (BMD). In addition, Epimedium pubescens was also found to induce osteoclast apoptosis and inhibit bone resorption in vivo.5 Further studies demonstrated that Icariine was able to stimulate MC3T3-E1 cell proliferation and increase alkaline phosphatase (ALP) activity, promote type I collagen expression and stimulate Smad4 mRNA level,6 and that Icariine could produce the most significant promoting effect on the proliferation of osteoblast-like UMR 106 cells.7

All these suggest that Epimedium pubescens extracts is a potential drug against osteoporosis, and flavonoids such as Icariine might be the active constituents stimulating osteoblasts. However, the studies concerning the mechanism by which Icariine stimulates osteoblasts proliferation and differentiation are far from being exhaustive. On the other hand, the existing researches were mainly based on cellular models of murine cells or human osteosarcoma cells, but few were with normal human osteoblast cells, which raised the problem of extrapolation to human beings. Undoubtedly, human cells, taken from adult bone, cultured and well-differentiated, are most suitable for studies on the cellular effects of Icariine.

In this study, we assessed the direct effect of Icariine on the viability, proliferation and differentiation of primary human osteoblasts derived from inducing human marrow mesenchymal stem cells (hMSCs) directionally in vitro, and discussed the possible mechanism of Icariine effect. Bone morphogenetic protein 2 (BMP-2) is a well-known bone cell-differentiating factor and bone-formation stimulator, and its mRNA level is very important to the formation of bone, hence we assayed the mRNA level of BMP-2 when osteoblasts were cultured in the presence of Icariine.

METHODS

Reagents

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from the HyClone Co., USA; trypsin and Trizol reagent were purchased from the Gibco Co., USA; and L-ascorbic acid and β-glycerophosphate from Sigma Chemical Co., USA. bicinchoninic (BCA) protein assay reagent was obtained from Prierce, USA. Icariine was purchased from National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) and the purity was 99%. rhBMP-2 was purchased from the China Academy of Military Medical Science. BMP-2 and β-actin primers were compounded by Shanghai Shenggong Co., China.

Human osteoblast cell culture

hMSCs were harvested from a 45-year-old donor undergoing orthopedic surgery with bone grafts from the iliac crest. The donor had no history or apparent involvement of any disease affecting bone metabolism as found by laboratory tests including serum calcium, inorganic phosphorus.

Informed consent was obtained from the donor, and the study protocol was approved by the Institutional Review Board of Peking University.

Human osteoblast cells were obtained by inducting hMSCs directionally in the way described by Yin et al.8 Briefly, hMSCs were isolated from the donor's posterior iliac crest marrow aspirates and cultured in an osteo-inductive conditioned medium, which was composed of complete DMEM-High Glucose (10% FBS, 100 U /ml Penicillin G, 100 μg/ml Streptomycin Sulfate) supplemented with 10-8 mol/L Dexamethasone, 50 μg/ml L-ascorbic acid and 10 mmol/L β-glycerophosphate. After 20 days in culture at 37°C with 5% CO2, the adherent confluent hMSCs were trypsinized and passaged. Cells were tested for their osteogenesis characteristics at PD 8. The results demonstrated the cell expressed Alkaline phosphates, Collagen type I, Osteonectin and Osteopontin, which were the specific proteins of osteoblasts. These evidences suggest that hMSCs have been induced into osteoblasts directionally at PD 8.8-10 PD8 and PD9 human osteoblasts were enlarged and seeded for the experiments. Icariine and rhBMP-2 were prepared as stock solution and diluted with the conditioned medium to the desired concentrations. Control wells received no Icariine but equivalent amounts of the solvent only.

Influence of cell proliferation

The influence of cell proliferation was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Human osteoblast cells were plated at a density of 2×104 cells per well in 96 flat-bottomed well plates. After incubated for 24 hours, the culture medium was replaced by medium containing various test agents. The cells were incubated at 37°C for successively 2, 4 or 6 days. The medium was replaced with one containing fresh test agents every 3 days. At 4 hours before the end of incubation, the cells were washed twice with 10 mmol/L phosphate-buffered saline (PBS, PH 7.2), and then incubated with 0.5 mg/ml MTT for the last 4 hours. The medium was then decanted, the formazan salts were dissolved with 200 μl DMSO, and the absorbance was determined at 570 nm using an enzyme linked immunosorbent assay (ELISA) reader (BIO-TEK Instruments, USA ).

Quantitative assay of ALP activity

ALP activity was assayed by total automatic biochemistry instrument (Hitachi, Japan), with p-nitrophenyl-phosphate as the substrate. Human osteoblast cells were seeded into 48-well plates at a density of 5×104 cells per well and cultured for 24 hours. Then the test agents were added to the wells and incubation was continued for another 3 days. Each well was then washed three times with physiological saline solution, and the cells were obtained by scraping the culture dish and these are suspended in 1 ml physiological saline solution. The cells were then completely lysed by sonication for 10 minutes with a sonifer cell disruptor (Cosmo Bio, Japan). The sonicates were centrifuged for 10 minutes at 20 142 g, and the supernatants were used as samples for the ALP activity assay. The protein concentrations were determined with the BCA protein assay reagent. ALP activity was expressed by the ratio of reading of the instrument and the corresponding protein concentration (U/μg).

Alizarin red S and Von Kossa staining for mineralization

Calcified nodules on the cells were demonstrated by Alizarin red S and von Kossa staining. Human osteoblast cells were seeded into 48-well plates at a density of 2×105 cells per well and cultured for 24 hours, and then the test agents were added to the well. The medium was replaced every 3 days. Fourteen days after incubation ended, cell cultures were washed twice in PBS, fixed with formalin/methanol/H2O (1:1:1.5) 0.5 ml/well for 15 minutes at room temperature, and then washed 3 times with pure water. For Alizarin red S staining, the cells were stained for 1 minute with 200 μl Alizarin red S, and the cell monolayer was next washed 4-5 times with water and air-dried. The stained calcified nodules that appeared bright red in color were identified and counted with light microscopy. Each result is expressed as the mean of 6 wells±standard deviation (SD). For Von Kossa staining, the cells were placed in a 5% silver nitrate solution for 15 minutes and kept in ultraviolet for 1 hour, then washed 3 times with PBS, and were deoxidized by Na2S2O3. The stained calcified nodules appeared brown.

RT-PCR analysis

Expression of BMP-2 mRNA was examined using RT-PCR, with the amplification of β-actin as control. Human osteoblast cells were seeded in 75-cm2 Nunc culture bottles at a density of 1×106 cells per bottle and cultured for 24 hours, then 20 μg/ml of Icariine was added. The control group was incubated without any drug. The medium was replaced every 3 days. After incubation for 7 days, total RNA of cells was extracted using Trizol reagent. cDNA was synthesized using 5 μg total RNA, random primer 1 μl, dNTPs 2 μl, and 200 U M-MLV Reverse Transcriptase (Promega, USA ) at 37°C for 1 hour. The BMP-2 gene in mRNA was detected by RT-PCR. The specific primers of BMP-2 were:11 Forward: 5′-GTCCTGAGCGAGTTCGAGTT-3′; Reverse: 5′-TG AAGCTCTGCTGAGGTGAT-3′. The specific primers of β-actin were:12 Forward: 5′-CAGGAGATGGCCAC TGCCGCA-3′; Reverse: 5′-TCCTTCTGCATCCTGT CAGCA-3′.

Amplification was carried out for 30 cycles, each of which was at 94°C for 15 seconds, 59°C for 15 seconds, and 72°C for 30 seconds in a 25 μl reaction mixture containing 3 μl cDNA, 25 pmol of each primer, 0.25 mmol dNTPs, and 2 U of Taq DNA polymerase (Promega, USA). The products of PCR were analyzed with 1% agarose gel electrophoresis and visualized with ethidium bromide staining. The expected sizes for BMP-2 and β-actin PCR products were 308 bp and 275 bp, respectively. Each RT-PCR product was compared to those obtained by amplifying β-actin cDNA from the same sample, band intensities were evaluated by densitometry.

Statistical analysis

Data are expressed as mean±standard deviation (SD), and t-test was performed for statistical analysis using the SPSS package (10.0 for windows). A P<0.05 was considered statistically significant.

RESULTS

MTT assay

Human osteoblast cells induced from hMSCs appear spindle and polygonal. (Fig 1) When the cells were exposed to various concentrations of Icariine and rhBMP-2 and incubated for a further 2, 4 or 6 days, cell viability in the presence of Icariine was significantly higher on day 6 than those in the absence of Icarrine. The maximum stimulatory effect on cell proliferation was achieved on day 6 when Icariine was at a concentration of 20 μg/ml, though increase was not seen on day 2 or 4 with the same concentration. rhBMP-2 did not show any stimulatory effect at the concentrations used (Figs. 2 and 3).

F1-6
Fig 1.:
PD8 human osteoblast cells induced from hMSCs appear spindle and polygonal. (Inverted phase contrast microscope, original magnification ×100)
F2-6
Fig 2.:
Effect of Icariine and rhBMP-2 on human osteoblast cells proliferation. The cells were incubated with Icariine or rhBMP-2 for 6 days. * P<0.05, compared with the control without Icarrine.
F3-6
Fig 3.:
Time-dependent effects of Icariine and rhBMP-2 on human osteoblast cells proliferation. The cells were incubated with 20 μg/ml Icariine or 50 ng/ml rhBMP-2 for 2, 4, 6 days. * P<0.05, compared with the control without Icarrine.

Quantitative assay of ALP activity

To evaluate the effect of Icariine on the differentiation of human osteoblasts, we examined the activity of ALP, a well-known marker of osteoblastic differentiation. The activity of ALP of the cells in the presence of Icariine at 10 μg/ml or 20 μg/ml for 3 days increased significantly. Furthermore, the maximum stimulatory effect was achieved at the concentration of 20 μg/ml. The activity of ALP of the cells stimulated with rhBMP-2 increased in a dose-dependent manner. rhBMP-2 at 25 ng/ml and 50 ng/ml enhanced the activity of ALP by about 1.4 fold and 1.9 fold, respectively (Fig. 4).

F4-6
Fig 4.:
Effect of Icariine and rhBMP-2 on ALP activity in human osteoblast cells. The cells were incubated with Icariine or rhBMP-2 for 3 days. * P<0.05, compared with the control without Icarrine or rhBMP-2.

Alizarin red S and Von Kossa staining for mineralization

The calcified nodules appeared bright red in color by Alizarin red S staining (Fig. 5) or brown by Von Kossa staining (Fig. 6). rhBMP-2 and Icariine at 10 μg/ml and 20 μg/ml could stimulate the formation of calcified nodules significantly. Furthermore, Icariine at 20 μg/ml stimulated an approximately 2.2 fold increase in calcified nodules when compared to the control, and the stimulative activity was more potent than that of rhBMP-2. The amounts of the nodular structures were similar when stimulated by rhBMP-2 of 25 ng /ml or 50 ng/ml. Furthermore, Icariine at 40 μg/ml slightly decreased the amount of nodules (Fig. 7).

F5-6
Fig 5.:
Alizarin red S staining for mineralization. The calcified nodules appeared bright red in color (original magnification ×100). A: The cells were incubated with Icariine at 20 μg/ml; B: The cells were incubated with Icariine at 10 μg/ml; C: The cells were incubated with Icariine at 0 μg/ml; D: The cells were incubated with rhBMP-2 at 50 ng/ml; E: The cells were incubated with rhBMP-2 at 25 ng/ml; F:The cells were incubated with rhBMP-2 at 0 ng/ml.
F6-6
Fig. 6.:
Von Kossa staining for mineralization. The calcified nodules appeared brown in color. A: original magnification ×10. B: original magnification×200. C: original magnification×400.
F7-6
Fig. 7.:
Human osteoblast cells after exposure to the Icarrine and rhBMP-2 for 14 days in 48-well culture plates and staining with Alizarin Red-S. Calcified nodules were counted under a light microscope. Each value is the mean±SD of the 6 dishes. Key: * P<0.05, compared with the control value without Icarrine.

RT-PCR analysis

mRNA expression of BMP-2 gene was detected using RT-PCR in the cultures in both the presence and the absence of Icariine for 7 days. The β-actin mRNA level was analyzed in the same samples as a reference gene. Compared to the β-actin mRNA level, the relative levels of the BMP-2 mRNA were markedly higher in the cultures with Icariine at 20 μg/ml than in the control cultures, as could be seen on the ethidium bromide-stained agarose gels (Fig. 8).

F8-6
Fig. 8.:
Effect of Icariine on BMP-2 mRNA synthesis in human osteoblast cells. Lane 1: DL 2000 DNA ladder markers; Lane 2: RT-PCR products of osteoblast cells treated with Icarrine for 7 days. Lane 3: RT-PCR products of osteoblast cells of control. The cells were incubated with Icariine at 20 μg/ml for 7 days.

DISCUSSION

Osteoporosis is widely recognized as a major public health problem, which can be debilitating and result in chronic pain, fractures, inability in normal daily activities, depression, social withdrawal, loss of independence, and deformities. The pathology of osteoporosis is complex, involving multiple factors. For linear growth of bone, it is constantly being remodeled (built and reabsorbed and rebuilt again). This process is critical to the formation of healthy bone tissue and allows the body to repair microfractures within the bone exerted by the daily stress. In remodeling, bone is absorbed in the remodeled site by osteoclasts, and the vacant cavity is then occupied by osteoblasts, which fill the space with new, stronger bone. This is a continuous, on-going process. Multiple factors can cause the loss of bone mass including increased bone turnover, which results in an imbalance of osteoclasts and osteoblasts at the resorption site. Depletion or too few osteoblasts can also lead to osteoporosis because the remodeled site is not properly filled with osteoblast cells during the bone remodeling process. Therefore, agents with an anabolic action on the bone may be effective in increasing the activity of osteoblasts and treating osteoporosis.

Chinese herb is relatively safe and cheap, and it is worthwhile that we explore and find some agents which can stimulate the proliferation and differentiation of osteoblasts. Recently, Epimedium pubescens received much attention since more and more studies in animals and cell culture systems have suggested that Icariine, an active compound of Epimedium pubescens, played an important role in the prevention of osteoporosis.13-16 Hence, the current study explored the effect of Icariine on human osteoblast.

The present study utilized primary human osteoblast cultures to systematically analyze the effect of Icariine on osteoblastic cells in vitro. The primary human osteoblast was obtained by inducting hMSCs directionally.8-10 There are several advantages in using primary human cell cultures, including the capacity to take into account the age and biological status of the patient from whom they derive. Compared with animal or human origin cells, such as osteosarcoma cell lines, primary human osteoblast cells have obvious advantages in revealing the effect of drugs because their susceptibility to drugs may be parallel with that as in vivo. Our previous work proved that when cultured in media with 10-8 mol/L Dexamethasone, 50 μg/ml L-ascorbic acid and 10 mmol/L β-glycerophosphate, at least 85% hMSCs could be induced into osteoblasts directly. The induced cell expressed osteoblast specific markers, such as ALP, Collagen type I, Osteonectin and Osteopontin. And when implanted the induced cell into the muscle of nude mice, ectopic osteogenesis could be observed.8-10

When cultured in the presence of Icariine for 6 days, the viability of human osteoblasts experienced a significant increase that was not seen on day 2 and day 4. Xue et al6 found that Icariine decreased the population doubling time of MC3T3-E1 cell and speculated that it could stimulate cell proliferation. And the present result is consistent with that notion.

Osteoblasts are bone-forming cells, the sequential expression of type I collagen, ALP, osteocalcin and the deposition of calcium are known as markers of osteoblastic differentiation.17 Human osteoblasts cultured for 3 days in the presence of 10, 20 μg/ml of Icariine exhibited a significant increase in ALP activity, and the formation of mineralized nodule increased significantly after the cells cultured for 14 days in the presence of 10, 20 μg/ml Icariine. 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 indicated that Icariine stimulated osteoblast differentiation at various levels from the osteoprogenitor stage to the terminal differentiation stage.

BMPs, which belong to the transforming growth factor beta (TGF-β) superfamily, were originally identified as compounds that induce bone and cartilage formation in ectopic extraskeletal sites in vivo. Extensive studies have demonstrated that BMPs, including BMP-2, are potent bone cell-differentiating factors as well as bone-formation stimulators.18,19 Several members of this family (BMPs2-15) have been identified, and their corresponding genes were cloned from human complementary DNA libraries.20 Through recombinant gene technology, BMPs are available in sufficient amounts for basic research and clinical trials. Recent studies have shown that rhBMP-2 and rhBMP-7 induced structurally sound orthotropic bone in various experimental systems.21,22 Therefore, we choose rhBMP-2 as the positive control of Icariine. The present study showed that rhBMP-2 could not stimulate the proliferation of human osteoblasts, but could induce the differentiation of human osteoblasts by increasing ALP activity and mineralized nodule formation at the given concentrations. This finding is consistent with that of Lecanda F et al,23 who reported that in the presence of BMP-2, proliferation of hMSCs and human osteoblasts was decreased, but the mRNA levels of most of the bone matrix proteins were elevated. Furthermore, one of the most intriguing findings in the present study is the finding that BMP-2 mRNA in the cultured osteoblasts increased in response to Icariine in the culture medium.

In summary, we demonstrated that Icariine stimulated the proliferation and differentiation of human osteoblasts and that the stimulatory effects are probably mediated, in part, by the upregulation of BMP-2.

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Keywords:

Icariine; osteoblasts; cell proliferation; cell differentiation; bone morphogenetic protein

© 2007 Chinese Medical Association