Abstract
Lycopus lucidus (LL) is a perennial herb that is traditionally used in Asia to treat edema, wound healing, and gynecological diseases such as irregular menstruation and menstrual pain. We hypothesized that LL would decrease the risk of developing osteoporosis, which is a condition related to gynecological diseases. In this study, we aimed to investigate the effect of a water extract of LL on osteoclastogenesis in vitro and osteoporosis in vivo. In vitro study, we used RAW 264.7 cells as osteoclast precursor cell. Osteoclast differentiation was induced by receptor activator nuclear factor-kappa B ligand (RANKL). We investigated the effect of LL on RANKL-induced osteoclastogenesis, tartrate-resistant acid phosphatase (TRAP) activity, and osteoclast-related genes. In vivo study, we used ovariectomized- (OVX-) induced osteoporosis rat model. OVX-induced Sprague-Dawley rats were randomly separated into sham, OVX, 17β-estradiol (100 μg/kg), wLL-L (15.2 mg/kg), and wLL-H (152 mg/kg) groups. Drugs were administered orally once daily for 9 weeks. wLL inhibited the formation of TRAP-positive osteoclasts; TRAP activity; pit formation; transcription factors (the nuclear factor of activated T-cell cytoplasmic 1 and c-fos); and osteoclast-related genes such as TRAP, carbonic anhydrase II, cathepsin K, osteoclast-associated receptor, and the d2 isoform of the vacuolar ATPase Vo domain. Also, wLL prevented loss of the trabecular area in the OVX femur without change of estrogen level. These results indicate that wLL is able to inhibit osteoclastogenesis and protect bone loss in the OVX-induced osteoporosis model without the influence of hormones like estrogen.
Highlights
Osteoporosis is a systemic skeletal disease characterized by a loss of bone mass and deterioration of the bone microarchitecture; it results in fractures caused by bone weakening [1]
The differentiation and activation of osteoclasts cannot take place without the receptor activator nuclear factor-kappa B ligand (RANKL), which is produced by osteoblasts [7]
An alpha-minimal essential medium (α-MEM), penicillinstreptomycin solution, and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY); recombinant mouse RANKL was purchased from Peprotehc (London, UK); an osteo assay surface plate was obtained from Corning, Inc. (Corning, NY); a cell titer 96 aqueous nonradioactive cell proliferation assay kit was obtained from Promega (Madison, WI); antiactin and anti-c-fos were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-NFATc1 was purchased from BD pharmingen (San Diego, CA); a reverse-transcription kit was purchased from Invitrogen (Carlsbad, CA); and the taq polymerase was purchased from Kapa Biosystem (Wilmington, MA)
Summary
Osteoporosis is a systemic skeletal disease characterized by a loss of bone mass and deterioration of the bone microarchitecture; it results in fractures caused by bone weakening [1]. On the other hand, is maintained by the turnover of bone resorption by osteoclasts and bone formation by osteoblasts [2]. Menopause, and the abuse of steroids such as glucocorticoid cause osteoporosis by inhibiting sufficient bone turnover [1, 3]. Osteoclasts are multinucleated cells formed by the fusion and differentiation of mononucleated hematopoietic cells; they play a major role in bone turnover and pathological conditions like osteoporosis [4]. The differentiation and activation of osteoclasts cannot take place without the receptor activator nuclear factor-kappa B ligand (RANKL), which is produced by osteoblasts [7]. The interaction of RANKL and its receptor, RANK, induces the expression of the nuclear factor of activated T cells (NFATc1) through tumor necrosis factor receptor-associated factor 6 (TRAF6) and c-fos [8]. NFATc1, as a master regulator of osteoclastogenesis, induces the tartrate- resistant acid phosphatase (TRAP), carbonic anhydrase II (CA II), cathepsin K (CTK), and osteoclastassociated receptor (OSCAR) concerned with osteoclastogenesis and the d2 isoform of vacuolar ATPase Vo domain (ATP6v0d2) relative with cell fusion [9, 10]
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