Emodin-8-O-β-d-Glucoside from Polygonum Amplexicaule D. Don var. Sinense Forb. Promotes Proliferation and Differentiation of Osteoblastic MC3T3-E1 Cells

Polygonum amplexicaule D. Don var. sinense Forb. (Polygonaceae) (PAF) is a famous traditional herb used to treat fractures, rheumatoid arthritis, muscle injury and pain. The present study was designed to investigate a PAF derived-chemical compound emodin-8-O-β-d-glucoside (EG) on the proliferation and differentiation of osteoblastic MC3T3-E1 cell in vitro. A compound was isolated from PAF extract by HPLC and identified as emodin-8-O-β-d-glucoside (EG) by spectroscopic methods. EG significantly promoted cell proliferation at 0.1–100 ng/mL, and increased the cell proportion in S-phase from 16.34% to 32.16%. Moreover, EG increased alkaline phosphatase (ALP) expression in MC3T3-E1 cells at the concentration from 0.1 to 100 ng/mL and inhibited PGE2 production induced by TNF-α in osteoblasts at the concentrations ranging from 10–100 ng/mL, suggesting that cell differentiation was induced in MC3T3-E1 osteoblasts. Taken together, these results indicated compound EG directly stimulated cell proliferation and differentiation of osteoblasts, therefore this study preliminarily explored the pharmacological mechanism of PAF to promote the healing of bone rheumatism and various fractures.


Introduction
In recent years, Traditional Chinese Medicines (TCMs) have become important sources of potential therapeutic agents because of their tremendous diversity. Various strategies have been used to investigate the phytochemical and pharmacological effects of TCMs based on their therapeutic potentials [1]. Polygonum amplexicaule D. Don var. sinense Forb. (Polygonaceae) (PAF) is a herbal drug used to treat fractures, rheumatism, osteoporosis, muscle injuries and pain. It has also been reported to be effective to treat atherosclerosis and its antibiotic and antivirus effects were also examined with positive results. However, which chemical compound in PAF plays the therapeutic role is not clear, and its pharmacological mechanism to promote the healing of fractures is still unknown.
The chemical compounds from herbal medicines have fewer side effects and are more suitable for long-term use compared with total extracts. In this study, the chemical compound emodin-8-O-β-Dglucoside (EG) was isolated from PAF. EG is also the component in traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc and functions to protect from focal cerebral injury induced by ischemia and reperfusion [2]. However, no results have been reported about its potential to promote the healing of bone injury.
The MC3T3-E1 osteoblastic cell line is a widely adopted model for the research of osteogenesis in vitro [3]. By use of MC3T3-E1 cell model, several medical herbs such as Icariin [4], Actaea racemosa [5], safflower seeds [6], Ulmus davidiana planch [7], Drynaria fortunei [8], Drynariae Rhizoma [9], Soybean isoflavones [10,11] and Homalomen occulta [12] have been proven to be functional to promote osteogenesis. The purpose of this study is to identify the chemical compound in PAF promoting the healing of bone fractures, which may be able to replace PAF in clinical use.

Indentification of PAF Compounds
As PAF extract promotes cell proliferation (data not shown), we isolated the components in PAF extract by HPLC ( Figure 1A), and a compound in the absorption peak with a retention time of 13.0 min was harvested for further spectroscopic analysis. By comparison of the spectroscopic parameters with published data [13], this compound was identified as emodin-8-O-β-D-glucoside (EG) ( Figure 1B).

The Effect of EG on Proliferation of MC3T3-E1 Cells
To elucidate whether EG plays a role in PAF induced cell proliferation, MC3T3-E1 cells were treated with 1 ng/mL EG for 7 days. Cell numbers were detected at day 1 to day 7 by CCK-8 assay.
The results showed that EG significantly promote cell proliferation from day 1 to day 7. The doseresponse curves indicated that 0.01 to 100 ng/mL of EG significantly stimulated the proliferation of MC3T3-E1 cells (p < 0.01). Thus, we can conclude that EG contributed to PAF induced cell proliferation. (A) EG induced time-dependent cell growth. MC3T3-E1 ells in 96-well plates were cultured with 1 ng/mL for 7 days. Cell numbers were measured by CCK-8 assay; (B) EG induced dose-dependent cell growth. Cells in 96-well plates were cultured with EG for 3 days. Cell numbers were measured by CCK-8 assay. Each point represents the mean ± S.D. of five determinations. * P < 0.05, ** P < 0.01 compared with controls by student t-test. "DB" means positive control diethylstilbestrol; "EG" means emodin-8-O-β-D-glucoside; "M" means medium.

The Effect of EG on Cell Cycle Progression
Cell proliferation is regulated by cell cycle progression. MC3T3-E1 cells were treated with various concentrations of EG, and cell cycle analysis was performed by PI staining and flow cytometry. The results showed that EG dose-dependently up-regulated the cell proportion in S-phase significantly ( Figures 3A and 3B). When cells were cultured with 0.1 to 100 ng/mL of EG, the cell proportion in Sphase increased from 16.34% to 32.16% ( Figure 3A and 3B). These results indicated that EG promoted cell proliferation by regulating cell cycle progression.

The Effect of EG on ALP Expression in MC3T3-E1 Cells
The healing of fractures is related to the differentiation of osteoblasts. In this study, the effect of EG on the expression of ALP, a marker for osteoblast differentiation, was detected in MC3T3-E1 cell. The results showed that the treatment of MC3T3-E1 cells with 0.1 to 100 ng/mL of EG significantly upregulated the expression of ALP (Figure 4), which was similar to that of the positive control diethylstilbestrol (DB). Therefore, the herb PAF has a markedly positive effect on the differentiation of MC3T3-E1 cells. Cells were cultured with indicated EG or DB for three days in the presence of 10 mM β-glycerophosphate (β-GP). ALP concentration was detected by ELISA. Results were expressed as means ± S.D. of five determinations. * P < 0.05, ** P < 0.01 compared with controls by student t-test. "DB" means positive control diethylstilbestrol; "EG" means emodin-8-O-β-D-glucoside.

The Effect of EG on PGE2 Production in MC3T3-E1 Cells
PGE 2 is an endogenous factor to stimulate osteolysis through the EP4 receptor [14]. We examined the effect of EG on PGE 2 production in MC3T3-E1 cells. As shown in Figure 5, the treatment of MC3T3-E1 cells with TNF-α increased the expression of PGE 2 , but EG (10-100 ng/mL) significantly inhibited the expression of PGE 2 induced by TNF-α. To investigate the pharmacological mechanism of PAF to promote the healing of fractures and rheumatism, we selected the well-recognized osteoblastic MC3T3-E1 cell line as in vitro model. As the healing of fractures and rheumatism is related to the proliferation and the differentiation of osteoblastic cell, we examined cell growth in osteoblastic MC3T3-E1 cells by CCK-8 assay in vitro. The results showed that the low dosage of EG stimulated cell growth. At the same time, the results of cell cycle assay demonstrated that EG effectively promoted cell cycle progression, the proportion of cells in S-phase of cells significantly increased. These results indicated that EG from PAF promoted cell proliferation by regulating cell cycle progression. Moreover, the effect of EG on cell growth in osteoblastic MC3T3-E1 cells was comparable with DB (as positive control).
ALP is the most widely recognized biochemical marker for osteoblastic activity [15]. Although its precise mechanism of action is poorly understood, this enzyme is believed to play an important role in bone metabolism [16]. Therefore, we examined the effects of EG from the root tubers of PAF on the ALP activity of osteoblastic MC3T3-E1 cells. The results showed that the compound EG from PAF increased ALP expression, and the effect of EG on ALP expression was similar to that of the positive control DB. PGE 2 is an endogenous factor to stimulate osteolysis through the EP4 receptor [14]. It is known that some bone-resorbing agents like TNF-α [17][18][19] act on osteoblasts and stimulate PGE 2 release from osteoblasts [20]. The released PGE 2 acts on stroma cells and enhances factors that support the differentiation from stem cells to osteoclasts to augment osteolysis. Furthermore, it is known that the presence of PGE 2 caused a significant decrease in bone ALP activity and a corresponding increase in bone acid phosphatase activity. In the present study, EG inhibited PGE 2 production induced by TNF-α in osteoblasts, suggesting that EG may inhibit the osteolysis by down-regulating the release of PGE 2 induced by TNF-α. This result partly elucidated the pharmacological mechanism of PAF to promote the healing of bone fractures and rheumatism.

Plant Material
The fresh root tubers of PAF were collected in Enshi County of Hubei Province, China, in October 2008 and authenticated by the College of Pharmacy, South Central University for Nationalities (SCUN). The voucher specimen (SCUN0810) was deposited in the Herbarium of the College of Pharmacy, SCUN, China.

Sample Preparation
Ten mg of emodin-8-O-β-D-glucoside (EG) and diethylstilbestrol (DB, as positive control) were weighed respectively, and then diluted with DMEM medium. The concentrations of DB and EG ranged from 0.01-100 ng/mL.

Cell Culture
MC3T3-E1 cells were cultured in DMEM supplemented with 10% FBS, 100 μg/mL penicillin and 100 U/mL streptomycin in a mixture of 95% air and 5% CO 2 in a humidified incubator at 37 °C.

Cell Proliferative CCK-8 Assay
The cells (2 × 10 4 cells/well) were seeded in 96-well plates and cultured for 24 h to obtain adherent monolayer cells. After washed twice with phosphate-buffered saline (PBS), the cells were treated with various concentrations of PAF extracts or EG for 1-7 days, and cell proliferation was evaluated by cell count kit-8 assay (CCK-8, Beyotime Inst Biotech., China) according to the manufacturer's instructions [21]. Briefly, 10 μL CCK-8 was added to each well (100 μL medium). After incubation for 2 h at 37 °C, the absorbance at 450 nm was measured by an enzyme-linked immunosorbent assay (ELISA) plate reader. The experiments were carried out in five replicates and the results were expressed as mean ± S.D.

Cell Cycle Analysis
The cells (2 × 10 5 cells/well) were seeded in 6-well plates and cultured for 24 h to obtain adherent monolayer cells. The adherent cells were cultured for another 24 h in serum-free medium for synchronization. After washing twice with PBS, the cells were cultured with various concentrations of PAF extracts or EG. Then the cells were harvested and fixed with 70% cold ethanol at 4 °C overnight. After washing with PBS and centrifuging at 2,000 rpm for 3 min, the cells were incubated with 100 μL RNase (1 mg/mL) for 30 min at 37 °C, and then 50 μL (1 mg/mL) propidium iodide (PI, Jingmei, Shenzhen, China) was added and the cells were incubated at 4 °C for 30 min in the dark. The cell cycle was detected by flow cytomerty [22,23] (BD Bioscience, San Jose, CA, USA). The data were analyzed by Modfit software (Verity Software House, Topsham, ME, USA).

Alkaline Phosphatase (ALP) Activity Assay
MC3T3-E1 cells were cultured to grow to 80-100% confluent. The medium was replaced with phenol red-free α-MEM containing 5% charcoal-dextran-treated FBS (CD-FBS; GIBCO). Cells were cultured with various concentrations of EG, supplemented with 10 mM β-glycerophosphate (β-GP) (G9422, Sigma Chemical Co., St. Louis, MO), which was added to initiate in vitro cells differentiation [24,25]. After 3 days, the medium was removed and the monolayer cells were gently washed twice with PBS. The cells were harvested to 1.5 mL tubes and centrifuged for 20 min at 3,000 rpm. Supernatant was discarded, and PBS (PH 7.2-7.4) was added to prepare a solution with 1 × 10 7 cells/mL. Freeze-thaw cycles were repeated three times to break the membrane of the cells to release intracellular components. Then the solution was centrifuged for 15 min at 12,000 rpm. The supernatant was harvested for the measurement of ALP concentration by use of an ELISA kit (R&D System Inc., Minneapolis, MN, USA). The experiments were carried out in five replicates and data were expressed as mean ± S.D.

Prostaglandin (PG) E2 Assay
The cells were cultured according to the method as mentioned above except that the β-GP was replaced with 10 −10 M TNF-α. The content of PGE 2 in the supernatant was measured with an ELISA kit (R&D System Inc., Minneapolis, MN, USA) according to the manufacturer's instructions [26]. Immediately after the color development, the absorbance is read at 450 nm. The experiment was carried out in five replicates and data were expressed as mean ± S.D.