Hederagenin protects mice against ovariectomy-induced bone loss by inhibiting RANKL-induced osteoclastogenesis and bone resorption

Abstract

Aims

Postmenopausal osteoporosis and other osteolytic bone diseases are often caused by the elevation in osteoclastogenesis and/or increased osteoclastic bone resorption, leading to excessive bone loss. Hederagenin (Hed) is a pentacyclic triterpenoid saponin extracted from various natural medicinal plants and exhibits numerous biological activities and may offer benefits against bone-related conditions. We evaluated the effects of Hed on osteoclast formation and bone resorption in vitro and the in vivo therapeutic benefits in the mouse model of ovariectomy (OVX)-induced bone loss.

Main methods

In vitro, osteoclast formation were determined by TRAcp staining; bone resorption were examined using Hydroxyapatite resorption assay and Podosomal actin belt formation assay; Related molecular mechanisms were determined by western blot assay. Construction of OVX mice by bilateral oophorectomy to simulate bone loss in vivo.

Key findings

In vitro cellular assays showed that Hed inhibited RANKL-induced osteoclast formation and osteoclast bone (hydroxyapatite) resorption as well as marker gene expression from BMM culture. Mechanistically, Hed attenuated RANKL-induced intracellular reactive oxygen species (ROS) production, and MAPK signaling pathway (ERK and p38) activation which curbed the downstream induction of c-Fos and NFATc1. Consistent with the in vitro findings, Hed administration effectively protected OVX mice from bone loss by reducing osteoclast number and activity on bone surface.

Significance

Our data provided promising evidence for the potential use of Hederagenin in the treatment of osteoclast-mediated osteolytic bone diseases such as postmenopausal osteoporosis.

Introduction

Bone remodeling is a dynamic balance process whereby the amount of bone removed by the bone resorbing osteoclasts is equal to the amount of new bone deposited by the bone forming osteoblasts [1]. Osteoclasts are the only cells in the body capable of resorbing bone and their activities are tightly coupled to the activity of the bone forming osteoblasts to maintain the bone homeostasis [2]. Elevated osteoclast formation and/or over-activation of osteoclast activity is the major underlying cause of postmenopausal osteoporosis.

Therapeutic treatment options for managing osteoporosis include both pharmacological-based therapy and non-pharmacological-based approaches. Current FDA-approved pharmacological interventions are classified as either pro-anabolic agents or anti-resorptives such as bisphosphonates, calcitonin, denosumab (RANKL inhibitor), and raloxifene (selective estrogen receptor modulator; SERMs) [3]. However, the clinical efficacy of these agents has not been very satisfactory varying in patients and with concerns for long-term safety [4]. For example, bisphosphonates which are commonly used as first-line treatment options can lead to low-trauma atypical fractures, osteonecrosis of the jaw, and atrial fibrillation [5]. Therefore, new therapeutic agents with improved risk-benefit profiles for the prophylactic or therapeutic treatment of osteoporosis and other osteolytic bone disease are pressingly needed.

Therefore, osteoclasts are the prime targets for the identification and/or development of novel anti-osteoporotic agents. Osteoclasts are multinucleated giant cells derived from bone marrow hematopoietic precursors in response to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) [6]. M-CSF is essential in priming monocytic precursors towards the osteoclast lineage by inducing the expression of RANK on the cell surface [7]. The binding of RANKL, expressed on osteoblast cell surface or in its secreted form, to its receptor RANK initiates a series of signaling events that stimulate precursor cell fusion, bone resorption and actin cytoskeleton reorganization [8]. Among the many signaling pathways activated, the NF-κB and MAPK/AP-1 pathways and secondary messengers such as ROS are crucial [9]. NFATc1 is the most crucial transcription factor that regulates the expression of numerous osteoclast-specific genes [10].

There are increasing interests in alternative medicine and natural products with a growing number of reports in the literature for naturally-occurring chemical compounds derived from traditional Chinese medicinal plants that possess protective effects against osteoporosis and other bone loss conditions [11]. Hederagenin (Hed) is a pentacyclic triterpenoid saponin isolated from Hedera helix (common ivy) plant. Hed possesses an impressive list of biological effects including anti-tumor, anti-inflammatory, anti-oxidant, anti-diabetic, and anti-lipid peroxidation [12]. Mechanistically, Hed has been documented to inhibit NF-κB and MAPK activation and therefore may offer beneficial effects against excessive RANKL-induced osteoclastogenesis. Therefore in this study, we investigated the in vitro effects of Hed on BMM-derived osteoclast formation and function, and key RANKL-induced signaling cascades. We further explored the potential therapeutic benefits of Hed in an a murine model of ovariectomy (OVX)-induced bone loss.