Gastrodin: an ancient Chinese herbal medicine as a source for anti-osteoporosis agents via reducing reactive oxygen species.

Increased levels of reactive oxygen species (ROS) are a crucial pathogenic factor of osteoporosis. Gastrodin, isolated from the traditional Chinese herbal agent Gastrodia elata, is a potent antioxidant. We hypothesized that gastrodin demonstrates protective effects against osteoporosis by partially reducing reactive oxygen species in human bone marrow mesenchymal stem cells (hBMMSCs) and a macrophage cell line (RAW264.7 cells). We investigated gastrodin on osteogenic and adipogenic differentiation under oxidative stress in hBMMSCs. We also tested gastrodin on osteoclastic differentiation in RAW264.7 cells. Hydrogen peroxide (H2O2) was used to establish an oxidative cell injury model. Our results showed that gastrodin significantly promoted the proliferation of hBMMSCs, improved some osteogenic markers, reduced lipid generation and inhibited the mRNA expression of several adipogenic genes in hBMMSCs. Moreover, gastrodin reduced the number of osteoclasts, TRAP activity and the expression of osteoclast-specific genes in RAW264.7 cells. Gastrodin suppressed the production of reactive oxygen species in both hBMMSCs and RAW264.7 cells. In vivo, we established a murine ovariectomized (OVX) osteoporosis model. Our data revealed that gastrodin treatment reduced the activity of serum bone degradation markers, such as CTX-1 and TRAP. Importantly, it ameliorated the micro-architecture of trabecular bones. Gastrodin decreased osteoclast numbers in vivo by TRAP staining. To conclude, these results indicated that gastrodin shows protective effects against osteoporosis linking to a reduction in reactive oxygen species, suggesting that gastrodin may be useful in the prevention and treatment of osteoporosis.

Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway.

Estrogen deficiency is the main reason of bone loss, leading to postmenopausal osteoporosis, and estrogen replacement therapy (ERT) has been demonstrated to protect bone loss efficiently. Notch signaling controls proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Moreover, imperfect estrogen-responsive elements (EREs) were found in the 5'-untranslated region of Notch1 and Jagged1. Thus, we examined the molecular and biological links between estrogen and the Notch signaling in postmenopausal osteoporosis in vitro. hBMSCs were obtained from healthy women and patients with postmenopausal osteoporosis. Notch signaling molecules were quantified using real-time polymerase chain reaction (real-time PCR) and Western Blot. Luciferase reporter constructs with putative EREs were transfected into hBMSCs and analyzed. hBMSCs were transduced with lentiviral vectors containing human Notch1 intracellular domain (NICD1). We also used N-[N-(3, 5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester, a γ-secretase inhibitor, to suppress the Notch signaling. We found that estrogen enhanced the Notch signaling in hBMSCs by promoting the expression of Jagged1. hBMSCs cultured with estrogen resulted in the up-regulation of Notch signaling and increased proliferation and differentiation. Enhanced Notch signaling could enhance the proliferation and differentiation of hBMSCs from patients with postmenopausal osteoporosis (OP-hBMSCs). Our results demonstrated that estrogen preserved bone mass partly by activating the Notch signaling. Because long-term ERT has been associated with several side effects, the Notch signaling could be a potential target for treating postmenopausal osteoporosis.

MicroRNA­125b suppresses the proliferation and osteogenic differentiation of human bone marrow­derived mesenchymal stem cells.

The regressive biological function of human bone marrow­derived mesenchymal stem cells (hBMSCs) is one of the key factors resulting in the decrease of bone mass in senile osteoporosis. MicroRNAs (miRs) are non­coding small RNAs involved in various gene regulation processes. Whether any miR(s) are involved in the progression of osteoporosis by regulating the biological function of hBMSCs remains to be elucidated. The present study aimed to compare the expression levels of miR­125b in hBMSCs derived from senile osteoporotic patients with that of control (normal) subjects. A significantly upregulated expression of miR­125b in osteoporotic hBMSCs was detected. To elucidate the biological function of miR­125b in senile osteoporosis, the effects of miR­125b expression on proliferation and osteogenic differentiation of hBMSCs were assessed using gain­ and loss­of­function studies. It was evident that the overexpression of a miR­125b mimic was able to suppress the proliferative and osteogenic differentiation of senile hBMSCs. In contrast, repression of the function of miR­125b by transfection of an miR­125b inhibitor promoted the proliferation and osteogenic differentiation of hBMSCs. Furthermore, the potential target gene of miR­125b, osterix (Osx), was examined. The results of the present study strongly suggested that miR­125b may regulate osteogenic differentiation of hBMSCs through the modulation of Osx expression.

Overexpression of integrin a2 promotes osteogenic differentiation of hBMSCs from senile osteoporosis through the ERK pathway.

UNLABELLED: Osteoporosis is a major health problem affecting the aging population, especially in patients 65 years of age and older. The imbalance between bone formation and bone resorption is generally accepted as the essential mechanism leading to osteoporosis. In addition to the abnormal activation of osteoclast-mediated bone resorption, the dysfunction of bone marrow stromal cells (BMSCs) in mediating bone formation has been accepted as a major contributor to the progression of senile osteoporosis. RESULTS: In our study, senile osteoporotic hBMSCs displayed a decreasing capacity for proliferation and osteoblast differentiation, which was associated with the downregulation of integrin α2. Forced ectopic integrin α2 expression using a lentivirus vector reversed the dysfunction of senile osteoporotic hBMSCs. Additionally, the overexpression of integrin α2 upregulated the levels of Runx2 and Osterix. Mechanically, Western blot analyses revealed that integrin α2 phosphorylated ERK1/2 and the inactivation of ERK by PD98059 suppressed the osteoblastic differentiation of hBMSCs, suggesting that integrin α2 promotes osteoblast proliferation through the activation of ERK1/2 MAPK. CONCLUSION: Taken together, our results show that hBMSCs obtained from senile osteoporotic patients gradually lose their capability to differentiate along the osteogenic lineage and proliferate, which might be associated with the abnormal regulation of the integrin α2/ERK/Runx2 signaling pathway undergoing senile osteoporosis.

Protective effect of tetrahydroxystilbene glucoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells.

Oxidative stress can induce apoptosis and decrease activities of osteoblasts. 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG), is a potent antioxidant derived from a Chinese herb Polygonum multiflorum Thunb. To evaluate the protective effect provided by TSG to osteoblastic MC3T3-E1 cells, the cells were pretreated with TSG for 24h before being treated with 0.3mM hydrogen peroxide (H(2)O(2)) for 24 h, then some markers of osteoblast function and oxidative damage of the cells were examined. Our data demonstrated that TSG significantly (P< 0.05) increased cell survival, alkaline phosphatase (ALP) activity, calcium deposition, and the mRNA expression of ALP, collagen I (COL-I) and osteocalcin (OCN) in the presence of H(2)O(2). In addition, TSG decreased the production of receptor activator of nuclear factor-κB ligand (RANKL), interleukin-6 (IL-6), intracellular reactive oxygen species and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells induced by H(2)O(2). Taken together, these results demonstrated that the protective effect provided by TSG to osteoblastic MC3T3-E1 cells was mediated, at least in part, via inhibition of the release of bone-resorbing mediators and oxidative damage of the cells. Our results indicated that TSG may be effective in providing protection against osteoporosis associated with oxidative stress.