Oral administration of phenolic antidiarrheic ingredients prevents ovariectomy-induced bone loss
Introduction
Bone-forming osteoblasts and bone-resorbing osteoclasts are known to delicately regulate the bone formation and maintenance in bone tissues [1], [2]. Imbalance between osteoblasts and osteoclasts leads to the pathogenesis as well as etiology of certain metabolic bone diseases including osteoporosis, Paget's disease and osteopetrosis [3], [4]. The balancing mechanism atleast in part involves the endocrine control by estrogen and parathyroid hormone as well as the paracrine (autocrine) control by insulin-like growth factor and bone morphogenetic protein, respectively. The initiation of remodeling events and the subsequent proliferation and differentiation steps of osteoblasts must be tightly regulated, and the balance of osteoblast proliferation, differentiation and apoptosis could therefore determine the size of the osteoblast population at any given time. In particular, osteoblastic apoptosis plays a critical role during embryonic limb development, skeletal maturation, bone fracture healing and bone regeneration, in addition to a role in adult bone turnover [5], [6].
Several independent lines of evidence indicate the possible usefulness of particular natural products containing polyphenol molecules for such beneficial treatments. For example, a protective action is shown against osteopenia and/or osteoporosis in experimental animals with catechin [7], rutin [8], green tea polyphenol [9], olive oil polyphenol [10] and apple polyphenol [11]. However, little attention has been paid to pharmacological properties of compounds with a single phenol moiety on both osteoblastogenesis and osteoclastogenesis to date. In the present study, therefore, we have focused on major ingredients with a single phenol moiety of a traditional antidiarrheic drug used for more than 100 years in Japan with regard to possible pharmacological properties beneficial for the prophylaxis as well as therapy of different bone diseases using both in vitro and in vivo experimental analyses. These include 2-methoxyphenol (2MP), 2-methoxy-4-methylphenol (2M4MP) and 2-methoxy-4-ethyphenol (2M4EP) (Fig. 1), which are all phenolic constituents present in the effective ingredient, wood creosote, of the traditional antidiarrheic drug.
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Materials
2MP, 2M4MP and 2M4EP were obtained from Tokyo Kasei (Tokyo, Japan). Dulbecco's modified Eagle's medium (DMEM), alpha minimum essential medium (αMEM) and MEM were from Gibco BRL (Gaithersburgm, MD, USA). A cell counting kit was from Dojindo (Osaka, Japan). Dihydrodichlorofluorescein diacetate (DCFDA) was provided by Molecular probes (Eugene, OR, USA). Recombinant mouse macrophage-colony stimulating factor (M-CSF) and recombinant mouse receptor activator of nuclear factor-κB (NF-κB) ligand
Preventive effect on OVX-induced bone loss
To examine the possible pharmacological actions on OVX-induced bone loss, we conducted oral administration of three phenolic antidiarrheic ingredients to ovariectomized mice for subsequent investigation of the bone mineral density, in addition to different histomorphometric parameters. Ovariectomy-induced a significant reduction of bone mineral density in both total tibia (Fig. 2, left panel) and total femur (Fig. 2, middle panel), in addition to that in distal femur (Fig. 2, right panel), when
Discussion
The essential importance of the present findings is that the intermittent oral administration of the particular phenolic ingredients of a traditional antidiarrheic drug, such as 2M4MP and 2M4EP, significantly prevented the decrease in bone mineral density and the alternations of different osteoclastic parameters in histomorphometric analyses in ovariectomized mice in vivo. As the antidiarrheic drug has been usually used at a dose ranging from 400 to 900 mg/day for more than 100 years in Japan,
Acknowledgements
The authors are greatly indebted to Ms. Tsuchihashi Y. and Ms. Yamada J. for their excellent technical assistance. This work was supported in part by Grants-in-Aids for Scientific Research to E.H., T.T. and Y.Y. from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Dr. Nobuaki Moriguchi and Mr. Nobuyuki Matsushima are both employees of Taiko Pharmaceutical Co. Ltd., Japan. All other authors have no conflicts of interest.
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These two authors equally contributed to this work.