Elsevier

Journal of Ethnopharmacology

Volume 96, Issue 3, 15 January 2005, Pages 489-495
Journal of Ethnopharmacology

Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 Cells

https://doi.org/10.1016/j.jep.2004.09.038Get rights and content

Abstract

Pharmacological factors are needed to prevent bone loss that occurs with increasing age. The chemical compounds that act on bone metabolism in herbal medicines, however, are poorly understood. Effects of traditional Korean medicine, Drynariae Rhizoma [Drynaria fortunei (kunze) J. Sm] extract (DR), on the osteoblastic proliferation and differentiation were investigated. The effect of DR, a natural phyto herb, on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. DR dose-dependently increased DNA synthesis (significant at 50–150 μg/ml). DR increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (50–150 μg/ml). Antiestrogen tamoxifen eleminated the stimulation of proliferation and ALP activity of MC3T3-E1, which were induced by DR. DR at concentrations ranged from 30–100 μg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that DR directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and DR is effective for bone anti-resorptive action in bone cells.

Introduction

The MC3T3-E1 pre-osteoblastic cell line is a well-accepted model of osteogenesis in vitro (Quarles et al., 1992). Growth and differentiation factors contained in the culture medium stimulate these cells to undergo a developmental sequence that includes proliferation of undifferentiated precursors of osteoblasts, which subsequently differentiate into post-mitotic osteoblasts capable of expressing the osteogenic phenotype. During the proliferative phase, these cells undergo DNA synthesis and cell division resulting in a rapid increase in cell number until the cultures become confluent. At this juncture, proliferation is downregulated and increased expression of the osteogenic phenotype is observed indicating the presence of mature osteoblasts. Osteoblasts produce alkaline phosphatase, process procollagen to collagen, and deposit extracellular matrix containing additional proteins (e.g. osteopontin, bone sialoprotein and osteocalsin) on the substrate, which is subsequently mineralized (Peterson, 1987).

A sharp decrease in ovarian estrogen production is the predominant cause of the rapid hormone-related bone loss during the first decade after menopause. Osteoporosis associated with estrogen deficiency after menopause is the most common cause of age-related bone loss (Gruber et al., 1984). Estrogen replacement therapy recommended only for women who are at high risk of osteoporosis and with out any contraindications, seems to be the most effective method to reduce the rate of postmenopausal bone loss, although some side-effects may be accompanied (Genant et al., 1989). Attempts have been made to use a combination of antiresorptive agents, such as estrogen, and bone formation-stimulating agents, such as growth hormone, to treat osteoporosis (Turner, 1991). However, the potential bone-forming agents available either may have serious side-effects, or may not improve bone quality, or may nor reduce the susceptibility to fracture. Thus, it would be most helpful to discover a natural dietary substance that minimizes bone loss in postmenopausal women. The phytoestrogens, therefore, are potentially important in the prevention of postmenopausal osteoporosis caused by estrogen deficiency. Since a large decrease in bone mass occurs in the postmenopause state, women are vulnerable to the osteoporosis known as postmenopausal osteoporosis (Ettinger et al., 1987). Several medications have also been reported to be effective for curing osteoporosis based upon the results obtained using these animal models from non steroidal sources, such as bisphosphonates (Kalu, 1991), calcitonin (Kalu, 1991), calcium products (Morris et al., 1991) and ipriflavone (Agnusdei et al., 1997) are clinically employed as effective medications.

Plants used in folk medicine have been accepted as one of the main sources of drug discovery and development. Because natural products of plant origin are still a major part of traditional medicinal systems, there is also a resurgence of interest in herbal medicines in western countries as an alternative source of drugs often for intractable diseases such as rheumatoid arthritis (RA) (Phillipson and Anderson, 1989). From ancient times in China, Japan and Korea, women who have had low back pain in climacteric and senescent periods have been treated with Hanbang medicines. However, no data are available as to the recovery of bone mass by any of these Hanbang medicines. In Korea, there is a rich treasury of ethnobotanical knowledge and over the past decade (Kim et al., 1999). During our field studies, we have coincided following Oriental and herbal remedy claimed to be used in the treatment of rheumatism, bone resorption and related inflammatory diseases.

Drynariae Rhizoma [Drynaria fortunei (kunze) J. Sm; Gol-Se-Bo in Korean and Gu-Sui-Bu in Chinese] extract (DR) is one of candidates known to be effective for the treatment of inflammation, hyperlipemia, arteriosclerosis, and gynecological diseases such as osteophoresis and bone resorption according to the ancient Chinese and Korean medicinal and herbal literature (Shi, 1983). To treat the osteoporosis, herbal formulation containing DR is being used in Oiental medicine (Hong, 2001, Chung, 2002). DR was also commonly used to manage disorders of orthopedics and had been claimed to have therapeutic effects on bone healing (Sun et al., 2002). Specifically, through tissue culture and isotope tracing, it was found that DR injection (50–200 μg/kg of animal) significantly promoted calcification of the cultivated chick embryo bone primordium, increased ALP activity in the cultivated tissue, and accelerated synthesis of proteoglycan (Ma et al., 1996). Later, Liu et al. (2001) has shown that DR has an antioxidant effect on rat osteoblasts from hydrogen peroxide-induced death and may promote bone recovery under similar pathologic conditions. DR should be intensively studied for its possible use in bone diseases (Chang et al., 2003, Lin et al., 2002).

Traditional medicines have been reevaluated by clinicians (Xiu, 1988), because these medicines have fewer side effects and because they are more suitable for long-term use as compared to chemically synthesized medicines (Ma et al., 1996). The need for safer and effective anti-inflammatory drug and the lack of enough scientific data to support the claims made in ancient literature prompted the present study. In order to evaluate the effectiveness of Hanbang medicines on osteoporosis, we have examined whether DR could prevent the progression of bone loss induced by ovariectomy in rats (Hong, 2001, Chung, 2002). It was also shown that DR extracts were shown to be potent inhibitors of the degradation of denaturated collagen by cathepsin K and of bone resorption in an in vitro model. Treatment and pretreatment of the DR to cat K strongly inhibited cat K activity and bone resorption activity, suggesting that cat K involves the osteoporosis pathway. The naturally occuring DR, which contains phenolic compounds, possesses estrogenic activity (Jeong et al., 2003).

In this study, we examined the effect of DR on the proliferation and differentiation of osteoblastic MC3T3-E1 cells (Li et al., 1995) in vitro. The results suggested that the DR is effective for bone resorptive action on bone cells.

Section snippets

DR preparation and chemicals

Drynariae Rhizoma extracts (specimen no. D-25-6) was massproduced as for clinical use, were kindly supplied by the Oriental Medical Hospital of Dongguk University College of Oriental Medicine (Kyungju, Korea). DR (60 g) extracted with 500 ml of boiling water for 3 h. After the extract was centrifuged at 7500 rpm for 30 min, the supernatant was lyophilized. For direct use, the extract solution was stored at 4 °C in aliquots. Depending upon the experimental group, DR was added (at 2% volume in culture

Effect of DR on the proliferation of MC3T3-E1 cells

Incorporation of tritiated thymidine [3H]dT has been used as a tool to assess the rate of DNA synthesis in cell cultures for many years (Peterson, 1987). Cells were cultured in plastic microplates, pulse labelled with [3H]dT, and subsequently harvested onto glass fibre filter discs. The filters were then proccessed by washing with PBS, TCA and ethanol, and finally the CPM was assessed in a scintillation counter. Under these conditions there was a positive linear relationship between CPM and

Discussion

To investigate the effect of DR on bone metabolism, we employed cell culture system. ALP is the most widely recognized biochemical marker for osteoblastic activity. Although it precise mechanism of action is poorly understood, this enzyme is believed to play a role in bone mineralization. Therefore, we examined the effect of DR (0–150 μg/ml) on the ALP activity of osteoblastic MS3T3-E1 cells. DR dose-dependently increased ALP activity and DNA synthesis. Thus, DR seems to stimulate the

Acknowledgment

This study was in part supported by Dongguk University Research Fund and National Research Laboratory Program (2002–2007 to C.H.K.) from Korean Ministry of Science and Ministry.

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