Catalpol protects against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytotoxicity in osteoblastic MC3T3-E1 cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental contaminant that produces a wide variety of adverse effects in humans. Catalpol, a major bioactive compound enriched in the dried root of Rehmannia glutinosa, is a major iridoid glycoside that alleviates bone loss. However, the detailed mechanisms underlying the effects of catalpol remain unclear. The present study evaluated the effects of catalpol on TCDD-induced cytotoxicity in osteoblastic MC3T3-E1 cells. Catalpol inhibited TCDD-induced reduction in cell viability and increases in apoptosis and autophagic activity in osteoblastic MC3T3-E1 cells. Additionally, pretreatment with catalpol significantly decreased the nitric oxide and nitrite levels compared with a control in TCDD-treated cells and significantly inhibited TCDD-induced increases in the levels of cytochrome P450 1A1 and extracellular signal-regulated kinase. Pretreatment with catalpol also effectively restored the expression of superoxide dismutase and extracellular signal-regulated kinase 1 and significantly enhanced the expression of glutathione peroxidase 4 and osteoblast differentiation markers, including alkaline phosphatase and osterix. Taken together, these findings demonstrate that catalpol has preventive effects against TCDD-induced damage in MC3T3-E1 osteoblastic cells.

Protective effects of honokiol against methylglyoxal-induced osteoblast damage.

Honokiol is an active compound isolated from Magnolia officinalis that has been used without notable side effects in traditional medicine. We investigated the effects of honokiol against methylglyoxal (MG)-induced cytotoxicity in MC3T3-E1 osteoblast cells and the possible molecular mechanism(s) involved. The results showed that honokiol alleviated MG-induced cell death and the production of intracellular ROS, mitochondrial superoxide, cardiolipin peroxidation, and inflammatory cytokines. MG induction of the soluble receptor for advanced glycation end product (AGE) was reduced by pretreatment with honokiol. Furthermore, honokiol increased the levels of Nrf2 and increased the levels of glutathione and the activity of glyoxalase I. Pretreatment with honokiol prior to MG exposure reduced MG-induced mitochondrial dysfunction and alleviated MG-induced reduction of nitric oxide and PGC1α levels, suggesting that honokiol may induce mitochondrial biogenesis. It was concluded that honokiol could be useful in the attenuation of MG-induced cell damage.

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.

Protective effect of liquiritigenin against methylglyoxal cytotoxicity in osteoblastic MC3T3-E1 cells.

Methylglyoxal (MG), a reactive dicarbonyl compound, is a metabolic byproduct of glycolysis and elevated MG levels contribute to diabetic complications. Glycation reactions of MG with amino acids can induce oxidative stress, leading to subsequent cytotoxicity. In the present study, the effect of liquiritigenin on MG-induced cytotoxicity was investigated using osteoblastic MC3T3-E1 cells. Pretreatment of MC3T3-E1 cells with liquiritigenin prevented the MG-induced cell death and production of protein adduct, intracellular reactive oxygen species, mitochondrial superoxide, cardiolipin peroxidation, and TNF-α in osteoblastic MC3T3-E1 cells. In addition, liquiritigenin increased the activity of glyoxalase I inhibited by MG. These findings suggest that liquiritigenin provides a protective action against MG-induced cell damage by reducing oxidative stress and by increasing MG detoxification. Pretreatment with liquiritigenin prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss. Additionally, the nitric oxide and PGC-1α levels were significantly increased by liquiritigenin, suggesting that liquiritigenin may induce mitochondrial biogenesis. Our findings indicate that liquiritigenin might exert its therapeutic effects via enhancement of glyoxalase I activity and mitochondrial function, and anti-oxidant and anti-inflammatory activities. Taken together, liquiritigenin has potential as a preventive agent against the development of diabetic osteopathy related to MG-induced oxidative stress in diabetes.

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.

The protective effects of Achyranthes bidentata root extract on the antimycin A induced damage of osteoblastic MC3T3-E1 cells.

Achyranthes bidentata (A. bidentata) Blume is a medicinal herb with the property of strengthening bones and muscles and ensuring proper downward flow of blood in terms of the therapeutic theory of traditional medicine. In the present study, the effect of A. bidentata root extract (AE) on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. AE caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, and mineralization in the cells (P < 0.05). AE also decreased the production of TNF-α, IL-6, and RANKL induced by antimycin A, mitochondrial electron transport inhibitor. Exposure of MC3T3-E1 cells to antimycin A caused significant reduction of cell viability and mineralization. However, pretreatment with AE prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, ATP loss, ROS release, and nitrotyrosine increase, suggesting that AE may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, AE increased the phosphorylation of cAMP-response element-binding protein inhibited by antimycin A. Our study demonstrates that A. bidentata could significantly prevent osteoblast damage in aged patients.

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.

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.

Apigenin attenuates 2-deoxy-D-ribose-induced oxidative cell damage in HIT-T15 pancreatic ß-cells.

Glucose toxicity contributes to progressive ß-cell failure and the development of overt diabetes. Oxidative stress is an important aspect of glucose toxicity in pancreatic ß-cells. We investigated whether the flavonoid apigenin protects pancreatic ß-cells from 2-deoxy-D-ribose (dRib)-induced oxidative cell damage. HIT-T15 pancreatic ß-cells were cultured with or without apigenin in the presence of dRib. Time- and dose-dependent cell viability was monitored using a cell counting kit (CCK-8), while the induction of apoptosis was analyzed using a cell death enzyme-linked immunosorbent assay (ELISA) kit. Mitochondrial membrane potential (ΔΨ(m)) was determined using the JC-1 kit. Intracellular oxidative stress was measured by fluorometric analysis of DCFH oxidation using 2',7'-dichlorofluorescin diacetate (DCFH-DA) as the probe. In addition, the DNA binding activity of the oxidative stress-related transcriptional factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) were analyzed. dRib reduced cell survival and ΔΨ(m), while it markedly increased intracellular levels of reactive oxygen species (ROS), apoptosis, and the activity of the oxidative stress-related transcription factors NF-κB and AP-1. However, pretreatment of cells with apigenin attenuated all the dRib-induced effects. The anti-oxidants, N-acetyl-L-cysteine (NAC) and alpha lipoic acid (ALA), also prevented both dRib-induced oxidative damage and activation of NF-κB and AP-1. Taken together, these results suggest that apigenin attenuates dRib-induced cell damage in pancreatic ß-cells via oxidative stress-related signaling.

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 microM) 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.

Prooxidative effects of green tea polyphenol (-)-epigallocatechin-3-gallate on the HIT-T15 pancreatic beta cell line.

Epigallocatechin-3-gallate (EGCG) is the main polyphenolic constituent in green tea and is believed to function as an antioxidant. However, increasing evidence indicates that EGCG produces reactive oxygen species (ROS) and subsequent cell death. In this study, we investigated the prooxidative effects of EGCG on the HIT-T15 pancreatic beta cell line. Dose-dependent cell viability was monitored with the cell counting kit-8 assay, while the induction of apoptosis was analyzed by a cell death ELISA kit and comet assay. Extracellular H(2)O(2) was determined using the Amplex Red Hydrogen Peroxide Assay Kit. Intracellular oxidative stress was measured by fluorometric analysis of 2',7'-dichlorofluorescin (DCFH) oxidation using DCFH diacetate (DA) as the probe. Treatment with EGCG (5-100 microM) decreased the viability of pancreatic beta cells, caused concomitant increases in apoptotic cell death, and increased the production of H(2)O(2) and ROS. Catalase, the iron-chelating agent diethylenetriaminepentaacetic acid, and the Fe(II)-specific chelator o-phenanthroline all suppressed the effects of EGCG, indicating the involvement of both H(2)O(2) and Fe(II) in the mechanism of action of EGCG. The antioxidant N-acetyl-cysteine and alpha-lipoic acid also suppressed the effects of EGCG. Furthermore, EGCG did not scavenge exogenous H(2)O(2), but rather, it synergistically increased H(2)O(2)-induced oxidative cell damage in pancreatic beta cells. Together, these findings suggest that in the HIT-T15 pancreatic beta cell line, EGCG mediated the generation of H(2)O(2), triggering Fe(II)-dependent formation of a highly toxic radical that in turn induced oxidative cell damage.

Ligularia fischeri leaf extract suppresses proinflammatory mediators in SW982 human synovial cells.

Synovial hyperplasia is a hallmark of rheumatoid arthritis (RA) and is regarded as a major destructive element of articular bone and cartilage. This pathological process is accompanied by the production of proinflammatory cytokines and matrix metalloproteinases (MMPs) in synoviocytes. The present study was conducted to analyse the effects of Ligularia fischeri extract (LF) on inflammatory functions in the SW982 human synovial cell system. When cells were exposed to LF, LF had a significant inhibitory effect on the production of TNF-alpha, IL-6 and MMP-3 by SW982 cells (p < 0.05). The mitogen-activated protein kinases (MAPKs) represent an attractive target for RA because they can regulate MMP and cytokine expression. The effects of LF on the activation of MAPKs and transcription factors were also examined in SW982 cells by ELISA assay. IL-1beta-induced JNK and p38 activation was inhibited by LF, and LF significantly reduced the DNA-binding activity of transcription factors NF-kappaB and AP-1. Taken together, these results suggest that LF modulates the inflammatory process involved in arthritis by suppressing the expression of various genes by inhibiting NF-kappaB and/or AP-1 activities.

Effect of Scutellariae radix extract on the high glucose-induced apoptosis in cultured vascular endothelial cells.

Endothelial cell apoptosis has been postulated as the initial trigger of the progression of microvascular disease in patients with diabetes mellitus. To investigate the role of Scutellariae radix extract, we examined its effect on the endothelial cell proliferation using the [3H]-thymidine incorporation method. Scutellariae radix extract significantly stimulated endothelial cell proliferation in a dose-dependent manner. We focused on the protective action of Scutellariae radix extract on the endothelial cell apoptosis induced by high glucose concentrations. Determination of endothelial cell apoptosis was performed using DNA gel electrophoresis, terminal deoxynuclotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and an ELISA kit. Exposure of vascular endothelial cell to high glucose (16.7 mM) for 72 h resulted in a significant increase in apoptosis, compared with the normal glucose concentrations (5.5 mM). Scutellariae radix extract inhibited high glucose-induced endothelial cell apoptosis. This result suggests that Scutellariae radix extract may contribute to antiapoptotic action against vascular endothelial cells, resulting in a beneficial effect in preventing diabetes-associated microvascular complications.