Hong-Yang Zhang

Ginsenoside-Rb2 displays anti-osteoporosis effects through reducing oxidative damage and bone-resorbing cytokines during osteogenesis

Reactive oxygen species (ROS) are a significant pathogenic factor of osteoporosis. Ginsenoside-Rb2 (Rb2), a 20(S)-protopanaxadiol glycoside extracted from ginseng, is a potent antioxidant that generates interest regarding the bone metabolism area. We tested the potential anti-osteoporosis effects of Rb2 and its underlying mechanism in this study. We produced an oxidative damage model induced by hydrogen peroxide (H2O2) in osteoblastic MC3T3-E1 cells to test the essential anti-osteoporosis effects of Rb2in vitro. The results indicated that treatment of 0.1 to 10μM Rb2 promoted the proliferation of MC3T3-E1 cells, improved alkaline phosphatase (ALP) expression, elevated calcium mineralization and mRNA expressions of Alp, Col1a1, osteocalcin (Ocn) and osteopontin (Opn) against oxidative damage induced by H2O2. Importantly, Rb2 reduced the expression levels of receptor activator of nuclear factor kappa-B ligand (RANKL) and IL-6 and inhibited the H2O2-induced production of ROS. The in vivo study indicated that the Rb2 administered for 12weeks partially decreased blood malondialdehyde (MDA) activity and elevated the activity of reduced glutathione (GSH) in ovariectomized (OVX) mice. Moreover, Rb2 improved the micro-architecture of trabecular bones and increased bone mineral density (BMD) of the 4th lumbar vertebrae (L4) and the distal femur. Altogether, these results demonstrated that the potential anti-osteoporosis effects of Rb2 were linked to a reduction of oxidative damage and bone-resorbing cytokines, which suggests that Rb2 might be effective in preventing and alleviating osteoporosis.

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.

Glucocorticoids: Dose-related effects on osteoclast formation and function via reactive oxygen species and autophagy

Whether glucocorticoids directly enhance or interrupt osteoclastogenesis is still a controversial subject. In this study, we ascertained the dose-dependent positive effects of glucocorticoids on osteoclastogenesis in vivo and in vitro as well as investigated the mechanism in vitro. As the dose of glucocorticoids increased, osteoclastogenesis was stimulated at 0.1 μM, a peak was achieved at 1 μM and a corresponding decrease occurred at 10 μM. Reactive oxygen species (ROS), which play a crucial role in osteoclastogenesis, and autophagy flux activity, a cellular recycling process, were consistently up-regulated along with the dose-dependent effects of the glucocorticoids on osteoclast formation and function. N-acetyl-cysteine (NAC), a ROS scavenger, abrogated the effects of the glucocorticoids on autophagy and osteoclastogenesis. Moreover, 3-methyladenine (3-MA), an autophagy inhibitor, interrupted osteoclastogenesis stimulation by the glucocorticoids. These results implied that with glucocorticoid administration, ROS and autophagy, as a downstream factor of ROS, played vital roles in osteoclast formation and function. 3-MA administration did not enhance ROS accumulation, so that autophagy had no effect on ROS induced by glucocorticoids. Our investigation demonstrated that glucocorticoids had dose-dependent positive effects on osteoclast formation and function via ROS and autophagy. These results provide support for ROS and autophagy as therapeutic targets in glucocorticoid-related bone loss diseases such as glucocorticoid-induced osteoporosis.

Dose-Dependent Effect of Estrogen Suppresses the Osteo-Adipogenic Transdifferentiation of Osteoblasts via Canonical Wnt Signaling Pathway

Fat infiltration within marrow cavity is one of multitudinous features of estrogen deficiency, which leads to a decline in bone formation functionality. The origin of this fat is unclear, but one possibility is that it is derived from osteoblasts, which transdifferentiate into adipocytes that produce bone marrow fat. We examined the dose-dependent effect of 17β-estradiol on the ability of MC3T3-E1 cells and murine bone marrow-derived mesenchymal stem cell (BMMSC)-derived osteoblasts to undergo osteo-adipogenic transdifferentiation. We found that 17β-estradiol significantly increased alkaline phosphatase activity (P<0.05); calcium deposition; and Alp, Col1a1, Runx2, and Ocn expression levels dose-dependently. By contrast, 17β-estradiol significantly decreased the number and size of lipid droplets, and Fabp4 and PPARγ expression levels during osteo-adipogenic transdifferentiation (P<0.05). Moreover, the expression levels of brown adipocyte markers (Myf5, Elovl3, and Cidea) and undifferentiated adipocyte markers (Dlk1, Gata2, and Wnt10b) were also affected by 17β-estradiol during osteo-adipogenic transdifferentiation. Western blotting and immunostaining further showed that canonical Wnt signaling can be activated by estrogen to exert its inhibitory effect of osteo-adipogenesis. This is the first study to demonstrate the dose-dependent effect of 17β-estradiol on the osteo-adipogenic transdifferentiation of MC3T3-E1 cells and BMMSCs likely via canonical Wnt signaling. In summary, our results indicate that osteo-adipogenic transdifferentiation modulated by canonical Wnt signaling pathway in bone metabolism may be a new explanation for the gradually increased bone marrow fat in estrogen-inefficient condition.

17β-Estradiol inhibits ER stress-induced apoptosis through promotion of TFII-I-dependent Grp78 induction in osteoblasts.

Although many studies have suggested that estrogen prevents postmenopausal bone loss partially due to its anti-apoptosis effects in osteoblasts, the underlying mechanism has not been fully elucidated. In the present study, we found that 17β-estradiol (17β-E2), one of the primary estrogens, inhibited endoplasmic reticulum (ER) stress-induced apoptosis in MC3T3-E1 cells and primary osteoblasts. Interestingly, 17β-E2-promoted Grp78 induction, but not CHOP induction in response to ER stress. We further confirmed that Grp78-specific siRNA reversed the inhibition of 17β-E2 on ER stress-induced apoptosis by activating caspase-12 and caspase-3. Moreover, we found that 17β-E2 markedly increased the phosphorylated TFII-I levels and nuclear localization of TFII-I in ER stress conditions. 17β-E2 stimulated Grp78 promoter activity in a dose-dependent manner in the presence of TFII-I and enhanced the binding of TFII-I to the Grp78 promoter. In addition, 17β-E2 notably increased phosphorylated ERK1/2 levels and Ras kinase activity in MC3T3-E1 cells. The ERK1/2 activity-specific inhibitor U0126 remarkably blocked 17β-E2-induced TFII-I phosphorylation and Grp78 expression in response to ER stress. Together, 17β-E2 protected MC3T3-E1 cells against ER stress-induced apoptosis by promoting Ras-ERK1/2-TFII-I signaling pathway-dependent Grp78 induction.

Ophiopogonin D: A new herbal agent against osteoporosis

Excessive reactive oxygen species (ROS) play an important role in the development of osteoporosis. Ophiopogonin D (OP-D), isolated from the traditional Chinese herbal agent Radix Ophiopogon japonicus, is a potent anti-oxidative agent. We hypothesized that OP-D demonstrates anti-osteoporosis effects via decreasing ROS generation in mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells and a macrophage cell line RAW264.7 cells. We investigated OP-D on osteogenic and osteoclastic differentiation under oxidative status. Hydrogen peroxide (H2O2) was used to establish an oxidative damage model. In vivo, we established a murine ovariectomized (OVX) osteoporosis model. Then, we searched the molecular mechanism of OP-D against osteoporosis. Our results revealed that OP-D significantly promoted the proliferation of MC3T3-E1 cells and improved some osteogenic markers. Moreover, OP-D reduced TRAP activity and the mRNA expressions of osteoclastic genes in RAW264.7 cells. OP-D suppressed ROS generation in both MC3T3-E1 and RAW264.7 cells. OP-D treatment reduced the activity of serum bone degradation markers, including CTX-1 and TRAP. Further research showed that OP-D displayed anti-osteoporosis effects via reducing ROS through the FoxO3a-β-catenin signaling pathway. In summary, our results indicated that the protective effects of OP-D against osteoporosis are linked to a reduction in oxidative stress via the FoxO3a-β-catenin signaling pathway, suggesting that OP-D may be a beneficial herbal agent in bone-related disorders, such as osteoporosis.

Ginsenoside-Rb2 inhibits dexamethasone-induced apoptosis through promotion of GPR120 induction in bone marrow-derived mesenchymal stem cells.

Apoptosis of bone marrow-derived mesenchymal stem cells (BMMSCs) is an essential pathogenic factor of osteoporosis. Ginsenoside-Rb2 (Rb2), a 20(S)-protopanaxadiol glycoside extracted from ginseng, is a potent treatment for bone loss, which raises interest regarding the bone metabolism area. In the present study, we found that dose-response Rb2 inhibited high dosage of dexamethasone (Dex)-induced apoptosis in primary murine BMMSCs. Interestingly, Rb2 promoted GPR120 induction, which is the unsaturated long-chain fatty acid receptor. We further confirmed that GPR120-specific ShRNA reversed the inhibition of Rb2 on Dex-induced apoptosis by activating caspase-3 and reducing cell viability. In addition, Rb2 notably increased phosphorylated ERK1/2 levels and Ras kinase activity dependently through the GPR120. The ERK1/2 activity-specific inhibitor U0126 remarkably blocked the Rb2-induced antiapoptotic effect in response to Dex-induced apoptosis. Together, dose-response Rb2 protected BMMSCs against Dex-induced apoptosis dependently by inducing GPR120 promoted Ras-ERK1/2 signaling pathway. Therefore, in the prevalence of the abuse of Dex in the clinic, our findings suggest for the first time that Rb2 is not only a key to understand the link between Chinese medicine and the pathology of osteoporosis but also an underlying target for the treatment of bone complications in the foreseeable future.

Tetramethylpyrazine Protects Against Glucocorticoid-Induced Apoptosis by Promoting Autophagy in Mesenchymal Stem Cells and Improves Bone Mass in Glucocorticoid-Induced Osteoporosis Rats

Glucocorticoid-induced osteoporosis (GIOP) is a widespread clinical complication due to the common use of glucocorticoids. Excess glucocorticoids induce apoptosis of bone marrow-derived mesenchymal stem cells (BMSCs), which have been shown to play an increasingly important role in the pathogenesis and therapy of osteoporosis. Tetramethylpyrazine (TMP), an extract from one of the most recognized herbs in traditional Chinese medicine (Chuanxiong), has been reported to have antiapoptotic properties. In this study, we tested whether TMP protects rat BMSCs following exposure to glucocorticoids in vitro and in vivo. We treated BMSCs with different concentrations of TMP (50, 100, or 200 μM) and exposed them to 10-6 M dexamethasone (Dex) for 48 h in vitro. Our data showed that TMP inhibited Dex-induced cytotoxicity and protected BMSCs from apoptosis. Interestingly, further results demonstrated that TMP prevented apoptosis in BMSCs by promoting autophagy in an AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway-dependent manner. In addition, calcein fluorescence double labeling and microcomputed tomography scanning indicated that 12 weeks of TMP administration augmented bone formation and protected trabecular bone mass in GIOP rats. We also discovered that first-passage BMSCs isolated from the TMP treatment group had a lower rate of apoptosis and a higher light chain 3 (LC3)-II/LC3-I ratio than the GIOP group. Our findings demonstrate for the first time that TMP can protect BMSCs from exposure to excess glucocorticoids by promoting autophagy through AMPK/mTOR pathway and might be an effective agent for the prevention and treatment of GIOP.

Disturbed MEK/ERK signaling increases osteoclast activity via the Hedgehog-Gli pathway in postmenopausal osteoporosis.

Postmenopausal osteoporosis is a worldwide health problem and is characterized by increased and activated osteoclasts. However, the mechanism by which osteoclasts are dysregulated in postmenopausal osteoporosis is not fully understood. In this study, we found that the Hedgehog-Gli pathway was upregulated in postmenopausal osteoporotic osteoclasts and that 17β-estradiol both inhibited osteoclastogenesis and induced osteoclast apoptosis by downregulating Hedgehog-Gli signaling. Furthermore, we demonstrated that the Hedgehog-Gli pathway was negatively regulated by MEK/ERK signaling and that this effect was Sonic Hedgehog (SHH)-dependent and was partially blocked by an anti-SHH antibody. Moreover, we found that the stimulatory effect of Hedgehog signaling on osteoclastogenesis and the inhibitory effect on osteoclast apoptosis were dependent on the Gli family of transcription factors. The pathways and molecules that contribute to the regulation of osteoclastogenesis and apoptosis represent potential new strategies for designing molecular drugs for the treatment of postmenopausal osteoporosis.

Intramembranous ossification and endochondral ossification are impaired differently between glucocorticoid-induced osteoporosis and estrogen deficiency-induced osteoporosis.

A fracture is the most dangerous complication of osteoporosis in patients because the associated disability and mortality rates are high. Osteoporosis impairs fracture healing and prognosis, but how intramembranous ossification (IO) or endochondral ossification (EO) during fracture healing are affected and whether these two kinds of ossification are different between glucocorticoid-induced osteoporosis (GIOP) and estrogen deficiency-induced osteoporosis (EDOP) are poorly understood. In this study, we established two bone repair models that exhibited repair via IO or EO and compared the pathological progress of each under GIOP and EDOP. In the cortical drill-hole model, which is repaired through IO, osteogenic differentiation was more seriously impaired in EDOP at the early stage than in GIOP. In the periosteum scratch model, in which EO is replicated, chondrocyte hypertrophy progression was delayed in both GIOP and EDOP. The in vitro results were consistent with the in vivo results. Our study is the first to establish bone repair models in which IO and EO occur separately, and the results strongly describe the differences in bone repair between GIOP and EDOP.