Ji-Yeon Yu

Inhibitory effects of quercetin on aflatoxin B1-induced hepatic damage in mice.

Aflatoxin B(1) (AFB(1))-mediated hepatic damage is involved in production of AFB(1)-8,9-epoxide-bound DNA adducts and this is also affected by a pro-oxidant potential of the toxin. In this study we investigated the effects of quercetin on AFB(1)-treated HepG2 cells. We also examined the biochemical mechanisms associated with the effects of quercetin on AFB(1)-mediated liver damage in mice. Our results revealed that quercetin and isorhamnetin inhibit production of reactive oxygen species and cytotoxicity, and block the decrease of reduced glutathione (GSH) levels in AFB(1)-treated HepG2 cells. Isorhamnetin have inhibitory ability on lipid peroxidation stronger than quercetin in the cells. Oral supplementation with quercetin decreased serum lactate dehydrogenase levels, increased hepatic GSH levels and superoxide dismutase activity, and reduced lipid peroxidation in both the liver and kidney in AFB(1)-treated mice. However, quercetin did not show a significant reduction on serum levels of alkaline phosphate, alanine aminotransferase and aspartate aminotransferase that were increased in AFB(1)-treated mice. HPLC analysis revealed that quercetin in plasma is mainly present as glucoronides and/or sulfates of quercetin. Collectively, it is suggested that quercetin does not directly protect against AFB(1)-mediated liver damage in vivo, but exerts a partial role in promoting antioxidative defense systems and inhibiting lipid peroxidation.

Mycotoxin zearalenone induces AIF- and ROS-mediated cell death through p53- and MAPK-dependent signaling pathways in RAW264.7 macrophages.

Zearalenone (ZEN) is commonly found in many food commodities and is known to cause reproductive disorders and genotoxic effects. However, the mode of ZEN-induced cell death of macrophages and the mechanisms by which ZEN causes cytotoxicity remain unclear. The present study shows that ZEN treatment reduces viability of RAW264.7 cells in a dose-dependent manner. ZEN causes predominantly necrotic and late apoptotic cell death. ZEN treatment also results in the loss of mitochondrial membrane potential (MMP), mitochondrial changes in Bcl-2 and Bax proteins, and cytoplasmic release of cytochrome c and apoptosis-inducing factor (AIF). Pre-treatment of the cells with either z-VAD-fmk or z-IETD-fmk does not attenuate ZEN-mediated cell death, whereas catalase suppresses the ZEN-induced decrease in viability in RAW264.7 cells. Treating the cells with c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), or p53 inhibitor prevented ZEN-mediated changes, such as MMP loss, cellular reactive oxygen species (ROS) increase, and cell death. JNK or p38 MAPK inhibitor inhibited mitochondrial alterations of Bcl-2 and Bax proteins with attendant decreases in cellular ROS levels. Knockdown of AIF via siRNA transfection also diminished ZEN-induced cell death. Further, adenosine triphosphate was markedly depleted in the ZEN-exposed cells. Collectively, these results suggest that ZEN induces cytotoxicity in RAW264.7 cells via AIF- and ROS-mediated signaling, in which the activations of p53 and JNK/p38 play a key role.

Anti-Inflammatory Activities of Licorice Extract and Its Active Compounds, Glycyrrhizic Acid, Liquiritin and Liquiritigenin, in BV2 Cells and Mice Liver

This study provides the scientific basis for the anti-inflammatory effects of licorice extract in a t-BHP (tert-butyl hydrogen peroxide)-induced liver damage model and the effects of its ingredients, glycyrrhizic acid (GA), liquiritin (LQ) and liquiritigenin (LG), in a lipopolysaccharide (LPS)-stimulated microglial cell model. The GA, LQ and LG inhibited the LPS-stimulated elevation of pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and interleukin (IL)-6 in BV2 (mouse brain microglia) cells. Furthermore, licorice extract inhibited the expression levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in the livers of t-BHP-treated mice models. This result suggested that mechanistic-based evidence substantiating the traditional claims of licorice extract and its three bioactive components can be applied for the treatment of inflammation-related disorders, such as oxidative liver damage and inflammation diseases.

Cellular mechanisms of the cytotoxic effects of the zearalenone metabolites α-zearalenol and β-zearalenol on RAW264.7 macrophages

Zearalenone (ZEN) and its metabolites are commonly found in many food commodities and are known to cause reproductive disorders and genotoxic effects. The major ZEN metabolites are α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). Although many studies have demonstrated the cytotoxic effects of these metabolites, the mechanisms by which α-ZOL or β-ZOL mediates their cytotoxic effects appear to differ according to cell type and the exposed toxins. We evaluated the toxicity of α-ZOL and β-ZOL on RAW264.7 macrophages and investigated the underlying mechanisms. β-ZOL not only more strongly reduced the viability of cells than did α-ZOL, but it also induced cell death mainly by apoptosis rather than necrosis. The ZEN metabolites induced loss of mitochondrial membrane potential (MMP), mitochondrial changes in Bcl-2 and Bax proteins, and cytoplasmic release of cytochrome c and apoptosis-inducing factor (AIF). Use of an inhibitor specific to c-Jun N-terminal kinase (JNK), p38 kinase or p53, but not pan-caspase or caspase-8, decreased the toxin-induced generation of reactive oxygen species (ROS) and also attenuated the α-ZOL- or β-ZOL-induced decrease of cell viability. Antioxidative enzyme or compounds such as catalase, acteoside, and (E)-1-(3,4-dihydroxyphenethyl)-3-(4-hydroxystyryl)urea suppressed the ZEN metabolite-mediated reduction of cell viability. Further, knockdown of AIF via siRNA transfection diminished the ZEN metabolite-induced cell death. Collectively, these results suggest that the activation of p53, JNK or p38 kinase by ZEN metabolites is the main upstream signal required for the mitochondrial alteration of Bcl-2/Bax signaling pathways and intracellular ROS generation, while MMP loss and nuclear translocation of AIF are the critical downstream events for ZEN metabolite-mediated apoptosis in macrophages.

Continuously Generated H2O2 Stimulates the Proliferation and Osteoblastic Differentiation of Human Periodontal Ligament Fibroblasts

Numerous studies have shown that hydrogen peroxide (H2O2) inhibits proliferation and osteoblastic differentiation in bone‐like cells. Human periodontal ligament fibroblasts (PLF) are capable of differentiating into osteoblasts and are exposed to oxidative stress during periodontal inflammation. However, the cellular responses of PLF to H2O2 have not been identified. In this study, we examined how H2O2 affects the viability and proliferation of PLF by exposing the cells to glucose oxidase (GO) or direct addition of H2O2. We also explored the effects of GO on the osteoblastic differentiation of PLF and the mechanisms involved. The viability and proliferation in PLF were increased with the addition of 10 mU/ml GO but not by volumes greater than 15 mU/ml or by H2O2 itself. GO‐stimulated DNA synthesis was correlated with the increase in cyclin E protein levels in the cells. Osteoblastic differentiation of PLF was also augmented by combined treatment with GO, as evidenced by the increases in alkaline phosphatase activity, mineralization, collagen synthesis, and osteocalcin content in the cells. The inductions of runt‐related transcription factor 2 and osterix mRNA and proteins were further increased in PLF incubated in combination with GO compared to those in untreated cells. These results demonstrate that the continuous presence of H2O2 stimulates the proliferation of PLF and augments their potential to differentiate into osteoblasts through the up‐regulation of bone‐specific transcription factors. Collectively, we suggest that H2O2 may elicit the functions of PLF in maintaining the dimensions of the periodontal ligament and in mediating a balanced metabolism in alveolar bone. J. Cell. Biochem. 113: 1426–1436, 2012.

Inhibitory effect of fenofibrate on neointima hyperplasia via G0/G1 arrest of cell proliferation

We have previously reported that fenofibrate displayed a potent antithrombotic effect by the inhibition of platelet aggregation. The present study was designed to investigate the effects of fenofibrate on the neointimal hyperplasia and its possible molecular mechanism. Neointimal hyperplasia was measured in balloon-inflated-induced vascular injury model of male Sprague-Dawley rats and cell proliferation was measured in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Fenofibrate-treated group showed a significant reduction in neointimal formation (0.07±0.04mm(2)) from the control (0.13±0.04mm(2)). Fenofibrate significantly inhibited platelet-derived growth factor (PDGF)-BB-induced cell counting and [(3)H]-thymidine incorporation into DNA. Fenofibrate suppressed the PDGF-BB-inducible progression through G(0)/G(1) to S phase of cell cycle. Moreover, fenofibrate inhibited not only phosphorylation of retinoblastoma (Rb) protein and expression of cyclin D/E, CDK 2/4 and proliferating cell nuclear antigen (PCNA) proteins but also mitogen-activated protein kinase (MAPK) signaling pathways such as ERK 1/2, p38 and JNK phosphorylation. In conclusion, the present study demonstrates that fenofibrate significantly inhibits neointimal formation via G(0)/G(1) arrest of PDGF-BB-induced cell proliferation in association with the inhibition of MAPK, which resulted in the downregulation of expressions of cyclin D/E, CDK 2/4 and PCNA proteins, suggesting that fenofibrate may be useful for individuals with a high risk of thrombotic or cardiovascular diseases.

Chemoprevention of a flavonoid fraction from Rhus verniciflua Stokes on aflatoxin B1‐induced hepatic damage in mice

Since aflatoxin B1 (AFB1)‐mediated hepatic damage is related to the production of AFB1‐8,9‐epoxide and reactive oxygen species, bioactive compounds having antioxidant potentials are suggested to be capable of reducing AFB1‐induced toxicity. We previously purified a mixture of flavonoids that we named RCMF (Rhus verniciflua Stokes chloroform–methanol fraction), from a traditional Korean food additive and herbal medicine. RCMF exhibited various biological effects, including antioxidant and antitumor activities. In this study, we examined whether RCMF protects against AFB1‐induced liver injury using in vitro and in vivo systems. Pretreatment of HepG2 cells with RCMF significantly reduced AFB1‐stimulated production of ROS and malondialdehyde (MDA) to the control levels. RCMF also prevented the reduction in HepG2 cell viability caused by AFB1. Oral administration of RCMF to mice significantly suppressed an AFB1‐induced increase in serum levels of alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase. It also prevented MDA formation and blocked decreases in glutathione levels and superoxide dismutase activities in the livers of AFB1‐treated mice. In addition, RCMF supplementation prevented an AFB1‐induced decrease in serum titers of IgA and IgG1. Collectively, these results suggest that RCMF attenuates AFB1‐mediated damage to the liver, and that this effect is at least partially related to the restoration of antioxidant defense systems and an increase in AFB1–GSH conjugate formation. Copyright

Continuous presence of H2O2 induces mitochondrial-mediated, MAPK- and caspase-independent growth inhibition and cytotoxicity in human gingival fibroblasts

The continuous generation of reactive oxygen species (ROS) is one of the most important events that occur during periodontal inflammation. Hydrogen peroxide (H(2)O(2)) is widely used in dental clinics. Many investigators have tried to elucidate the exact effect of H(2)O(2) on human gingival fibroblasts (HGFs). These studies have shown that H(2)O(2) induces growth inhibition and apoptosis in cells. However, the mechanisms involved in H(2)O(2)-induced cell death in HGFs are not completely understood. In this study, we examine how continuously generated H(2)O(2) affects the viability and proliferation of HGFs using glucose oxidase (GO). We also explored the mechanisms by which the continuous presence of H(2)O(2) induces cell death. GO treatment not only inhibited HGF growth and proliferation, but it also induced cell death in HGFs without typical apoptotic features such as nuclear DNA laddering. This GO-mediated cytotoxicity was proportional to the levels of intracellular ROS that were generated, rather than proportional to changes of cellular antioxidant activities. GO treatment also resulted in the loss of mitochondrial membrane potential and the relocation of mitochondrial apoptogenic factors. There was also an acute and severe depletion of cellular ATP levels. However, none of the pharmacological inhibitors specific for mitogen-activated protein kinases (MAPKs) or pancaspase prevented GO-induced cell death. Treatment with either catalase or acteoside significantly attenuated the GO-mediated cytotoxicity in the HGFs, thereby suggesting a protective effect of antioxidants against ROS-mediated gingival damage. Here we demonstrate that continuously generated H(2)O(2) not only inhibits the viability and proliferation of HGFs, but also causes pyknotic/necrotic cell death through mitochondrial stress-mediated, MAPK- and caspase-independent pathways.

A phenolic acid phenethyl urea compound inhibits lipopolysaccharide-induced production of nitric oxide and pro-inflammatory cytokines in cell culture

We previously used the Curtius rearrangement to synthesize various phenolic acid phenethyl urea compounds from phenolic acids and demonstrated their beneficial anti-oxidant and anti-cancer effects. Here, we investigated the effects of one of these synthetic compounds, (E)-1-(3,4-dihydroxystyryl)-3-(4-hydroxyphenethyl)urea (DSHP-U), on nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, and cytokine secretion in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. DSHP-U suppressed LPS-induced NO production and iNOS expression at a concentration of 50 microM and inhibited LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase. Inhibitors of phosphorylated (p)-ERK and p-p38, but not of p-JNK, reduced LPS-stimulated NO production. DSHP-U also prevented the nuclear translocation of the Rel A (p65) subunit and DNA-NF-kappaB binding by suppressing IkappaBalpha phosphorylation and by the degradation of IkappaBalpha in LPS-stimulated cells. Furthermore, DSHP-U decreased the production of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6 in LPS-treated macrophages. However, the LPS-stimulated expression of LPS receptors, such as Toll-like receptor 4, myeloid differentiation factor-2, and CD14, was unchanged after DSHP-U treatment at significantly high levels. Our data suggest that DSHP-U blocks NO and inflammatory cytokine production in LPS-stimulated macrophages and that these effects are mainly mediated through the inhibition of the ERK/p38- and NF-kappaB signaling pathways.

The protective effects of paclitaxel on platelet aggregation through the inhibition of thromboxane A2 synthase

Paclitaxel is an anticancer drug used in the treatment of ovarian, breast, head and neck, lung, and prostate cancer. We investigated the anti-platelet activity of paclitaxel in vitro as well as a possible anti-platelet mechanism. Paclitaxel inhibited washed rabbit platelet aggregation induced by collagen in a concentration-dependent manner, with an IC50 of 59.7 ± 3.5. However, it had little effect on platelet aggregation mediated by arachidonic acid, U46619, a thromboxane (TX) A2 mimic, or thrombin, suggesting that paclitaxel may strongly inhibit collagenmediated signal transduction. In accordance with these findings, paclitaxel blocked collageninduced cytosolic calcium mobilization, arachidonic acid liberation, and serotonin secretion. In addition, it inhibited arachidonic acid-mediated platelet aggregation by about 37% by interfering with TXA2 synthase as measured by the formation of arachidonic acid-mediated TXA2 and prostaglandin D2, as well as cyclooxygenase-1 and TXA2 synthase activity assays. Taken together, these results point to a cellular mechanism for the anti-platelet activity of paclitaxel through the inhibition of TXA2 synthase and cytosolic calcium mobilization. This may contribute to the beneficial effects of paclitaxel on the cardiovascular system.