Anti-osteoporotic activities of eurycoma longifolia : Effects on proliferation, differentiation and protein expression in osteoblasts and osteoclasts

Abstract

Eurycoma longifolia (EL) has acclaimed remarkable recognition due to its promising efficacy of stimulating bone formation in androgen-deficient male osteoporosis. Numerous in vivo studies have explored the bone-forming capacity of EL; however, the in vitro mechanism was not established yet. Therefore, the present study was aimed to investigate the in vitro cellular and molecular mechanisms of EL in regulating bone formation. The bone forming ability of EL was investigated by evaluating its effect on the proliferation, differentiation, and morphogenic modulation of osteoblasts (MC3T3-E1 cells). To comprehend the bone forming molecular mechanism of EL, the sequential expressions of various osteoblast-related protein biomarkers such as bone morphogenic protein-2 (BMP-2), alkaline phosphatase (ALP), Runx-2, osteocalcin, type I collagen, osteopontin, tissue growth factor (TGF)-β1 and androgen receptors were investigated. In order to understand the effect of ELon bone resorption, the proliferation and differentiation of osteoclasts (RAW 264.7cells) were studied by assessing tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclasts. The time-mannered expressions of osteoclast-related protein biomarkers such asmatrix metallopeptidase-9, cathepsin-K, TRAP, NFATc1, superoxide generation and superoxide dismutase (SOD) activity were also measured to comprehend the molecular mechanism of EL in osteoclastogenesis. The cell proliferation analysis revealed a promising efficiency of EL in promoting growth of MC3T3-E1 cells, particularly at the dose of 25 µg/mL. Significant increments in ALP activity and osteogenic differentiation (collagen formation and minerals deposition) were also observed in EL-treated MC3T3-E1 cells. The bone forming potential of EL was also evident from sequential analysis of osteoblast-related protein biomarkers whichindicated that the expression of these mediators was highly regulated in EL-treatedcell cultures compared to the control groups. Significant inhibitions in the differentiation and maturation of osteoclasts were observed in EL-treated RAW 264.7 cells. Taken together, EL down-regulated the RANKL-induced TRAP activity and expressions of MMP-9, cathepsin-K, TRAP, NFATc1 and generation of superoxide radicals in RAW 264.7 cells. In conclusion, the providential ability of EL inpromoting the proliferation and osteogenic differentiation of MC3T3-E1 cells and the remarkable down-regulation of osteoclastic differentiation and activation provides an in vitro basis for the prevention and treatment of male osteoporosis