Kwang Sik Suh

Soybean ethanol extract increases the function of osteoblastic MC3T3-E1 cells.

To investigate the bioactivities of soybean, which act on bone metabolism, we studied the effect of a soybean ethanol extract on the activity of osteoblast MC3T3-E1 cells. Soy extract (0.01-0.1 g/l) dose-dependently increased survival (P<0.05) and DNA synthesis (P<0.05) of MC3T3-E1 cells. In addition, soy extract (0.05 g/l) increased alkaline phosphatase activity (P<0.05) and collagen synthesis (P<0.05) of MC3T3-E1 cells. Moreover, the anti-estrogen tamoxifen eliminated the stimulation of MC3T3-E1 cells on the proliferation, ALP activity and collagen synthesis by soy extract, indicating that the main action of the soy extract on osteoblastic MC3T3-E1 cells is similar to that of estrogen effects. Treatment with soy extract prevented apoptosis, as assessed by a one-step sandwich immunoassay and DNA gel electrophoresis studies. This effect may be associated with the activation of the estrogen receptor, since we observed soy extract-mediated survival against apoptosis was blocked by the estrogen receptor antagonist tamoxifen in cells, further supporting a receptor-mediated mechanism of cell survival. These results suggest that osteoblast function is promoted by soy extract and that the estrogen receptor is involved in the response, thereby playing an important role in bone remodeling. In conclusion, soy extract has a direct stimulatory effect on bone formation in cultured osteoblastic cell in vitro. Presumably, dietary soy products are useful in the prevention of osteoporosis.

Kaempferol protects HIT-T15 pancreatic beta cells from 2-deoxy-D-ribose-induced oxidative damage

During the progression of Type 2 diabetes, glucose toxicity is likely to contribute importantly to progressive beta cell failure. Oxidative stress is an important aspect of glucose toxicity in pancreatic beta cells, and reducing sugars, such as 2‐deoxy‐D‐ribose (dRib), produce reactive oxygen species. Furthermore, many of the biological properties of flavonoids are likely to be related to their antioxidant and free‐radical scavenging abilities. Accordingly, in the present study, we investigated whether kaempferol (a flavonol) protects beta cells from dRib‐induced oxidative damage. HIT‐T15 cells were cultured with various concentrations of dRib for 24h. Cell survivals, amounts of reactive oxygen species (ROS) generated, apoptosis, and lipid peroxidation were measured. dRib was found to dose‐dependently reduce cell survival and to markedly increase intracellular ROS levels, apoptosis, and lipid peroxidation. However, kaempferol (10 µM) suppressed dRib (20 mM) induced intracellular ROS, apoptosis, and lipid peroxidation. So, we demonstrate that kaempferol reduces dRib‐mediated beta cell damage interfering with ROS metabolism and protective effects against lipid peroxidation. Our findings indicate that kaempferol protects HIT‐T15 cells from dRib‐induced associated oxidative damage. Copyright

Methylglyoxal induces oxidative stress and mitochondrial dysfunction in osteoblastic MC3T3-E1 cells

Abstract Methylglyoxal is a reactive dicarbonyl compound produced by glycolytic processing and identified as a precursor of advanced glycation end products. The elevated methylglyoxal levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of methylglyoxal on the function of osteoblastic MC3T3-E1 cells. The data indicated that methylglyoxal decreased osteoblast differentiation and induced osteoblast cytotoxicity. Pretreatment of MC3T3-E1 cells with aminoguanidine (a carbonyl scavenger), Trolox (an antioxidant), and cyclosporin A (a blocker of the mitochondrial permeability transition pore) prevented methylglyoxal-induced cytotoxicity in MC3T3-E1 cells. However, BAPTA/AM (an intracellular Ca2+ chelator) and dantrolene (an inhibitor of endoplasmic reticulum Ca2+ release) did not reverse the cytotoxic effect of methylglyoxal. Methylglyoxal increased the formation of intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation in osteoblastic MC3T3-E1 cells. Methylglyoxal also decreased the mitochondrial membrane potential and intracellular ATP and nitric oxide levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity contributes to the cytotoxicity of methylglyoxal. Furthermore, the results demonstrated that methylglyoxal induced protein adduct formation, inactivation of glyoxalase I, and activation of glyoxalase II. Aminoguanidine reversed all aforementioned effects of methylglyoxal. Taken together, these data support the notion that high methylglyoxal concentrations have detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction.

Xanthohumol modulates the expression of osteoclast-specific genes during osteoclastogenesis in RAW264.7 cells.

RANKL has been shown to play a critical role in osteoclast formation and bone resorption. Thus, agents that suppress RANKL signaling have a potential to suppress bone loss. In this study, we examined the ability of xanthohumol, a structurally simple prenylated chalcone, to suppress RANKL signaling during osteoclastogenesis in RAW264.7 cells. Xanthohumol markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclasts formation, and resorption-pit formation. In experiments to elucidate its mechanism of action, xanthohumol was found to suppress RANKL-induced expression of TRAF6, GAB2, ERK, c-Src, PI3K, and Akt genes. Moreover, RANKL-induced expressions of c-Fos and NFATc1, which are crucial transcription factors for osteoclastogenesis, were reduced by treatment with xanthohumol. Xanthohumol also inhibited RANKL-induced expression of bone-resorption related osteoclast-specific genes (carbonic anhydrase II, TCIRG, CLCN7, OSTM1, cathepsin K, and MMP-9). These data demonstrate that xanthohumol inhibits osteoclastogenesis by modulating RANKL signaling and may be useful for the prevention of bone-destructive diseases such as osteoporosis, arthritis and periodontitis.

Protective effect of albiflorin against oxidative-stress-mediated toxicity in osteoblast-like MC3T3-E1 cells

Albiflorin isolated from Paeoniae Radix was investigated for its ability to protect against antimycin A-induced osteoblast toxicity in the MC3T3-E1 cell line. MC3T3-E1 cells showed significantly reduced viability, increased apoptosis and lactate dehydrogenase release, elevated ROS/RNS levels, and decreased mitochondrial function after exposure to antimycin A. Pretreatment with albiflorin reversed the loss of cell viability in antimycin A-treated cultures. Similarly, pretreatment with albiflorin before antimycin A resulted in decreased apoptosis and lactate dehydrogenase release, decreased ROS/RNS levels, and increased mitochondrial function compared to antimycin A-treated cultures. In addition, albiflorin increased the mineralization reduced by antimycin A. Albiflorin reduced antimycin A-induced mitochondrial cytochrome c loss and cardiolipin peroxidation, conferring protection against ROS. These results confirmed the crucial role of cytochrome c and cardiolipin in the underlying mechanistic action of albiflorin. Therefore, the results suggest that albiflorin enhances mitochondrial function to suppress antimycin A-induced oxidative damage via the preservation of cytochrome c and cardiolipin. All of these data indicate that albiflorin may reduce or prevent osteoblast degeneration in osteoporosis.

Liquiritigenin restores osteoblast damage through regulating oxidative stress and mitochondrial dysfunction.

We investigated the protective effect of liquiritigenin, one of the flavonoids present in Glycyrrhizae radix, against antimycin A‐induced mitochondrial dysfunction in MC3T3‐E1 osteoblast cells. Osteoblastic MC3T3‐E1 cells were pre‐incubated with liquiritigenin before treatment with antimycin A, and markers of mitochondrial function and oxidative damage were examined. In addition, the effects of liquiritigenin on the activation of phosphoinositide 3‐kinase (PI3K) were examined in MC3T3‐E1 cells. Liquiritigenin protected MC3T3‐E1 cells from antimycin A‐induced cell death. However, the PI3K inhibitor, LY294002, significantly attenuated liquiritigenin‐mediated cell survival, indicating the involvement of PI3K in the cytoprotective effect of liquiritigenin. Pretreatment with liquiritigenin prior to antimycin A exposure significantly reduced antimycin A‐induced PI3K inactivation, mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Liquiritigenin also reduced mitochondrial superoxide generation, nitrotyrosine production, and cardiolipin peroxidation during mitochondrial complex inhibition with antimycin A. Taken together, the results of this study show that modulation of PI3K, antioxidant effects, and the attenuation of mitochondrial dysfunction by liquiritigenin represent an important mechanism for its protection of osteoblasts against cytotoxicity resulting from mitochondrial oxidative stress. Copyright

Inhibitory effect of paeoniflorin on methylglyoxal-mediated oxidative stress in osteoblastic MC3T3-E1 cells

PURPOSE Methylglyoxal (MG) has been suggested to be one major source of intracellular reactive carbonyl compounds. In the present study, the effect of paeoniflorin on MG-induced cytotoxicity was investigated using osteoblastic MC3T3-E1 cells. METHODS Osteoblastic MC3T3-E1 cells were pre-incubated with paeoniflorin before treatment with MG, and markers of oxidative damage and mitochondrial function were examined. RESULTS Pretreatment of MC3T3-E1 cells with paeoniflorin prevented the MG-induced cell death and formation of intracellular reactive oxygen species, cardiolipin peroxidation, and protein adduct in osteoblastic MC3T3-E1 cells. In addition, paeoniflorin increased glutathione level and restored the activity of glyoxalase I to almost the control level. These findings suggest that paeoniflorin provide a protective action against MG-induced cell damage by reducing oxidative stress and by increasing MG detoxification system. Pretreatment with paeoniflorin prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss. Additionally, the nitric oxide and nuclear respiratory factor 1 levels were significantly increased by paeoniflorin, suggesting that paeoniflorin may induce mitochondrial biogenesis. Paeoniflorin treatment decreased the levels of proinflammatory cytokines such as TNF-α and IL-6. CONCLUSIONS These findings indicate that paeoniflorin might exert its therapeutic effects via upregulation of glyoxalase system and mitochondrial function. Taken together, paeoniflorin may prove to be an effective treatment for diabeteic osteopathy.

Effect of Cilostazol on the Neuropathies of Streptozotocin-Induced Diabetic Rats

Objectives This study examined the effect of cilostazol, a potent phosphodiesterase inhibitor, on the progression of neuropathies associated with streptozotocininduced diabetes mellitus in Sprague-Dawley rats. Methods Eight weeks after streptozotocin treatment, a pelleted diet containing 0.03% cilostazol (15mg/kg body weight) was given for four weeks. Body weight, blood glucose level, motor nerve conduction velocity(MNCV), myelinated fiber density and size distribution of sciatic nerves were compared between age-matched normal rats (Group 1), control diabetic rats (Group 2) and cilostazol-treated diabetic rats (Group 3). Results Body weight was significantly reduced and blood glucose level was significantly increased in diabetic rats (Group 2 and 3) compared to normal rats. MNCV and cAMP content of sciatic nerves were significantly reduced in diabetic rats 12 weeks after streptozotocin treatment. Myelinated fiber size and density were also significantly reduced, and thickening of the capillary walls and duplication of the basement membranes of the endoneural vessels were observed in the diabetic rats. Whereas both body weight and blood glucose level of Group 3 did not differ significantly from those of Group 2, cilostazol treatment significantly increased MNCV and cAMP content of sciatic nerves in Group 3 but not to the levels observed in Group 1. MNCV positively correlated with cAMP content of sciatic nerves (r=0.86; p < 0.001). Cilostazol treatment not only restored myelinated fiber density and size distribution but reversed some of the vascular abnormalities. Conclusion These findings suggest that a reduced cAMP content in motor nerves may be involved in the development of diabetic neuropathy, and that cilostazol may prevent the progression of diabetic neuropathy by restoring functional impairment and morphological changes of peripheral nerves.

Chrysanthemum zawadskii extract protects osteoblastic cells from highly reducing sugar-induced oxidative damage

In this study, Chrysanthemum zawadskii extract (CZE) was investigated to determine its effects on 2-deoxy-D-ribose (dRib)-induced oxidative damage and cellular dysfunction in the MC3T3-E1 mouse osteoblastic cell line. Osteoblastic cells were treated with the highly reducing sugar, dRib, in the presence or absence of CZE. Cell viability, apoptosis and reactive oxygen species (ROS) production were subsequently examined. It was observed that dRib reduced cell survival, while it markedly increased the intracellular levels of ROS and apoptosis. However, pre-treatment of the cells with CZE attenuated all the dRib-induced effects. The antioxidant, N-acetyl-L-cysteine (NAC), also prevented dRib-induced oxidative cell damage. In addition, treatment with CZE resulted in a significant increase in alkaline phosphatase (ALP) activity and collagen content, as well as in the expression of genes associated with osteoblast differentiation [ALP, collagen, osteopontin (OPN), osteoprotegerin (OPG), bone sialoprotein (BSP), osteocalcin (OC) and bone morphogenetic protein (BMP)2, BMP4 and BMP7]. In mechanistic studies of the antioxidative potential of CZE, we found that CZE reversed the dRib-induced decrease in the expression of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT)1 and AKT2 genes, which are master regulators of survival-related signaling pathways. CZE also upregulated the gene expression of the antioxidant enzymes, superoxide dismutase (SOD)2, SOD3 and glutathione peroxidase 4 (GPx4), which was inhibited by dRib. Taken together, these results suggest that CZE attenuates dRib-induced cell damage in osteoblastic cells and may be useful for the treatment of diabetes-associated bone disease.

Sciadopitysin protects osteoblast function via its antioxidant activity in MC3T3-E1 cells

Age-related osteoblast dysfunction is the main cause of age-related bone loss in both men and women. In the present study, the effect of sciadopitysin, a type of biflavonoids, on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. Sciadopitysin caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, mineralization, and glutathione content in the cells (P<0.05). Sciadopitysin also decreased the production of tumor necrosis factor-a (TNF-α) induced by antimycin A, a mitochondrial electron transport inhibitor. We investigated the protective effects of sciadopitysin on antimycin A-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused a significant reduction in osteoblast dysfunction. However, pretreatment with sciadopitysin prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, adenosine triphosphate (ATP) loss, reactive oxygen species (ROS) release, and nitrotyrosine increase, suggesting that sciadopitysin may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, sciadopitysin increased phosphorylation of cAMP-response element-binding protein (CREB) inhibited by antimycin A. Our results demonstrate that sciadopitysin may reduce or prevent osteoblasts degeneration.