Ruei-Ming Chen

Nitric oxide from both exogenous and endogenous sources activates mitochondria-dependent events and induces insults to human chondrocytes

During inflammation, overproduction of nitric oxide (NO) can damage chondrocytes. In this study, we separately evaluated the toxic effects of exogenous and endogenous NO on human chondrocytes and their possible mechanisms. Human chondrocytes were exposed to sodium nitroprusside (SNP), an NO donor, or a combination of lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) as the exogenous and endogenous sources of NO, respectively. Administration of SNP or a combination of LPS and IFN‐γ in human chondrocytes increased cellular NO levels but decreased cell viability. Exposure to exogenous or endogenous NO significantly induced apoptosis of human chondrocytes. When treated with exogenous or endogenous NO, the mitochondrial membrane potential time‐dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase‐3 activity and consequently induced DNA fragmentation. Suppression of caspase‐3 activation by Z‐DEVD‐FMK decreased NO‐induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S‐nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase‐3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N‐monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO‐induced alterations in cellular Cyt c amounts, caspase‐3 activity, DNA fragmentation, and cell apoptosis. Results of this study show that NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria‐dependent mechanism. J. Cell. Biochem. 101: 1520–1531, 2007.

Ketamine inhibits tumor necrosis factor-α and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 microM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 microM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-alpha and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-alpha and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 microM) significantly inhibited LPS-induced TNF-alpha and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-alpha and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-alpha and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated sequential activations of c-Jun N-terminal kinase and activator protein-1.

Propofol suppresses macrophage functions and modulates mitochondrial membrane potential and cellular adenosine triphosphate synthesis

Background Propofol is an intravenous anesthetic agent that may impair host defense system. The aim of this study was to evaluate the effects of propofol on macrophage functions and its possible mechanism. Methods Mouse macrophage-like Raw 264.7 cells were exposed to propofol, at 3, 30 (a clinically relevant concentration), and 300 &mgr;m. Cell viability, lactate dehydrogenase, and cell cycle were analyzed to determine the cellular toxicity of propofol to macrophages. After administration of propofol, chemotactic, phagocytic, and oxidative ability and interferon-&ggr; mRNA production were carried out to validate the potential effects of propofol on macrophage functions. Mitochondrial membrane potential and cellular adenosine triphosphate levels were also analyzed to evaluate the role of mitochondria in propofol-induced macrophage dysfunction. Results Exposure of macrophages to 3 and 30 &mgr;m propofol did not affect cell viability. When the administered concentration reached 300 &mgr;m, propofol would increase lactate dehydrogenase release, cause arrest of cell cycle in G1/S phase, and lead to cell death. In the 1-h-treated macrophages, propofol significantly reduced macrophage functions of chemotactic and oxidative ability in a concentration-dependent manner. However, the suppressive effects were partially or completely reversed after 6 and 24 h. Propofol could reduce phagocytic activities of macrophages in concentration- and time-dependent manners. Exposure of macrophages to lipopolysaccharide induced the mRNA of interferon-&ggr;, but the induction was significantly blocked by propofol. Propofol concentration-dependently decreased the membrane potential of macrophage mitochondria, but the effects were descended with time. The levels of cellular adenosine triphosphate in macrophages were also reduced by propofol. Conclusions A clinically relevant concentration of propofol can suppress macrophage functions, possibly through inhibiting their mitochondrial membrane potential and adenosine triphosphate synthesis instead of direct cellular toxicity.

Anti-Inflammatory and Antioxidative Effects of Propofol on Lipopolysaccharide-Activated Macrophages

Abstract: Sepsis is a serious and life‐threatening syndrome that often occurs in intensive care unit (ICU) patients. During sepsis, inflammatory cytokines and nitric oxide (NO) can be overproduced, causing tissue and cell injury. Propofol is an intravenous agent used for sedation of ICU patients. Our previous study showed that propofol has immunosuppressive effects on macrophage functions. This study was designed to evaluate the anti‐inflammatory and antioxidative effects of propofol on the biosyntheses of tumor necrosis factor α (TNF‐α), interleukin 1β (IL‐1β), IL‐6, and NO in lipopolysaccharide (LPS)‐ activated macrophages. Exposure to a therapeutic concentration of propofol (50 μM), LPS (1 ng/mL), or a combination of these two drugs for 1, 6, and 24 h was not cytotoxic to the macrophages. ELISA revealed that LPS increased macrophage TNF‐α, IL‐1β, and IL‐6 protein levels in a time‐dependent manner, whereas propofol significantly reduced the levels of LPS‐enhanced TNF‐α, IL‐1β, and IL‐6 proteins. Data from RT‐PCR showed that LPS induced TNF‐α, IL‐1β, and IL‐6 mRNA, but propofol inhibited these effects. LPS also increased NO production and inducible nitric oxide synthase (iNOS) expression in macrophages. Exposure of macrophages to propofol significantly inhibited the LPS‐induced NO biosynthesis. The present study shows that propofol, at a therapeutic concentration, has anti‐inflammatory and antioxidative effects on the biosyntheses of TNF‐α, IL‐1β, IL‐6, and NO in LPS‐activated macro‐phages and that the suppressive effects are exerted at the pretranslational level.

MicroRNA-210 targets antiapoptotic Bcl-2 expression and mediates hypoxia-induced apoptosis of neuroblastoma cells

MicroRNAs (miRNAs) can regulate cell survival and death by targeting apoptosis-related gene expression. miR-210 is one of the most hypoxia-sensitive miRNAs. In this study, we evaluated the roles of miR-210 in hypoxia-induced insults to neural cells. Treatment of neuro-2a cells with oxygen/glucose deprivation (OGD) induced cell apoptosis in a time-dependent manner. In parallel, OGD time-dependently increased cellular miR-210 levels. Knocking down miR-210 expression using specific antisenses significantly attenuated OGD-induced neural apoptosis. Concurrently, OGD increased hypoxia-inducible factor (HIF)-1α mRNA and protein syntheses. Pretreatment with YC-1, an inhibitor of HIF-1α, reduced OGD-caused cell death. Sequentially, OGD specifically decreased antiapoptotic Bcl-2 mRNA and protein levels in neuro-2a cells. A search by a bioinformatic approach revealed that miR-210-specific binding elements exist in the 3′-untranslated region of Bcl-2 mRNA. Application of miR-210 antisenses simultaneously alleviated OGD-involved inhibition of Bcl-2 mRNA expression. In comparison, overexpression of miR-210 synergistically diminished OGD-caused inhibition of Bcl-2 mRNA expression and consequently induced greater cellular insults. Taken together, this study shows that OGD can induce miR-210 expression through activating HIF-1α. And miR-210 can mediate hypoxia-induced neural apoptosis by targeting Bcl-2.

Apoptotic insults to human HepG2 cells induced by S-(+)-ketamine occurs through activation of a Bax-mitochondria-caspase protease pathway

BACKGROUND Ketamine is widely used as an i.v. anaesthetic agent and as a drug of abuse. Hepatocytes contribute to the metabolism of endogenous and exogenous substances. This study evaluated the toxic effects of S-(+)-ketamine and possible mechanisms using human hepatoma HepG2 cells as the experimental model. METHODS HepG2 cells were exposed to S-(+)-ketamine. Cell viability and the release of lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GPT) were measured to determine the toxicity of S-(+)-ketamine to HepG2 cells. Cell morphology, DNA fragmentation, and apoptotic cells were analysed to evaluate the mechanism of S-(+)-ketamine-induced cell death. Amounts of Bax, an apoptotic protein, and cytochrome c in the cytoplasm or mitochondria were quantified by immunoblotting. Cellular adenosine triphosphate levels were analysed using a bioluminescence assay. Caspases-3, -9, and -6 were measured fluorometrically. RESULTS Exposure of HepG2 cells to S-(+)-ketamine increased the release of LDH and GPT, but decreased cell viability (all P<0.01). S-(+)-Ketamine time-dependently caused shrinkage of HepG2 cells. Exposure to S-(+)-ketamine led to significant DNA fragmentation and cell apoptosis (P=0.003 and 0.002). S-(+)-Ketamine increased translocation of Bax from the cytoplasm to mitochondria, but decreased the mitochondrial membrane potential and cellular adenosine triphosphate levels (all P<0.01). Sequentially, cytosolic cytochrome c levels and activities of caspases-9, -3, and -6 were augmented after S-(+)-ketamine administration (all P<0.001). Z-VEID-FMK, an inhibitor of caspase-6, alleviated the S-(+)-ketamine-induced augmentation of caspase-6 activity, DNA fragmentation, and cell apoptosis (all P<0.001). CONCLUSIONS This study shows that S-(+)-ketamine can induce apoptotic insults to human HepG2 cells via a Bax-mitochondria-caspase protease pathway. Thus, we suggest that S-(+)-ketamine at a clinically relevant or an abused concentration may induce liver dysfunction possibly due to its toxicity to hepatocytes.

Resveratrol Protects against Oxidized LDL-Induced Breakage of the Blood-Brain Barrier by Lessening Disruption of Tight Junctions and Apoptotic Insults to Mouse Cerebrovascular Endothelial Cells

Cerebrovascular endothelial cells (CEC) comprise the blood-brain barrier (BBB). In a previous study, we showed that oxidized LDL (oxLDL) can induce apoptosis of mouse CEC. Resveratrol possesses chemopreventive potential. This study aimed to evaluate the effects of resveratrol on oxLDL-induced insults to mouse CEC and its possible mechanisms. Exposure of mouse CEC to 200 μmol/L oxLDL for 1 h did not cause cell death but significantly altered the permeability and transendothelial electrical resistance of the cell monolayer. However, resveratrol completely normalized such injury. As for the mechanisms, resveratrol completely protected oxLDL-induced disruption of F-actin and microtubule cytoskeletons as well as occludin and zona occludens-1 (ZO-1) tight junctions. The oxLDL-induced decreases in the mitochondrial membrane potential and intracellular ATP levels were normalized by resveratrol. Exposure of mouse CEC to 200 μmol/L oxLDL for 24 h elevated oxidative stress and simultaneously induced cell apoptosis. However, resveratrol partially protected against oxLDL-induced CEC apoptosis. The oxLDL-induced alterations in levels of Bcl-2, Bax, and cytochrome c were completely normalized by resveratrol. Consequently, resveratrol partially decreased oxLDL-induced activation of caspases-9 and -3. Therefore, in this study, we show that resveratrol can protect against oxLDL-induced damage of the BBB through protecting disruption of the tight junction structure and apoptotic insults to CEC.

Nitric Oxide Modulates Pro- and Anti-inflammatory Cytokines in Lipopolysaccharide-Activated Macrophages

BACKGROUND Sepsis is a serious and life-threatening syndrome that occurs in intensive care unit patients. Lipopolysaccharide (LPS) has been implicated as one of major causes of sepsis. Nitric oxide (NO) and cytokines are involved in sepsis-induced inflammatory responses. This study is aimed at evaluating the effects of NO on the modulation of pro- and anti-inflammatory cytokines in LPS-activated macrophages and its possible mechanism. METHODS N-Monomethyl arginine (NMMA), an inhibitor of NO synthase, was used in this study to suppress NO production. Mouse macrophage-like Raw 264.7 cells were exposed to LPS, NMMA, or a combination of NMMA and LPS. Cell viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide assay. The amounts of nitrite, an oxidative product of NO, in the culture medium were quantified according to the Griess reaction method. Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction were carried out to determine the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-10 in macrophages. RESULTS Exposure of macrophages to LPS, NMMA, and a combination of NMMA and LPS for 24 hours did not affect cell viability. LPS significantly increased the amounts of nitrite in macrophages (p < 0.01). Treatment with NMMA decreased LPS-enhanced nitrite (p < 0.01) in a concentration-dependent manner. Analyses of enzyme-linked immunosorbent assays and reverse-transcriptase polymerase chain reaction revealed that LPS significantly induced TNF-alpha, IL-1 beta, and IL-10 proteins and mRNA (p < 0.01). A combined treatment with NMMA and LPS significantly blocked LPS-induced TNF-alpha and IL-1 beta (p < 0.01), but synergistically enhanced LPS-induced IL-10 (p < 0.05) protein and RNA. CONCLUSION This study has shown that NO suppression can inhibit LPS-induced TNF-alpha and IL-1 beta but enhance IL-10, and the modulation occurs at a pretranslational level.

Honokiol traverses the blood-brain barrier and induces apoptosis of neuroblastoma cells via an intrinsic bax-mitochondrion-cytochrome c-caspase protease pathway

Neuroblastomas, an embryonic cancer of the sympathetic nervous system, often occur in young children. Honokiol, a small-molecule polyphenol, has multiple therapeutic effects and pharmacological activities. This study was designed to evaluate whether honokiol could pass through the blood-brain barrier (BBB) and induce death of neuroblastoma cells and its possible mechanisms. Primary cerebral endothelial cells (CECs) prepared from mouse brain capillaries were cultured at a high density for 4 days, and these cells formed compact morphologies and expressed the ZO-1 tight-junction protein. A permeability assay showed that the CEC-constructed barrier obstructed the passing of FITC-dextran. Analyses by high-performance liquid chromatography and the UV spectrum revealed that honokiol could traverse the CEC-built junction barrier and the BBB of ICR mice. Exposure of neuroblastoma neuro-2a cells and NB41A3 cells to honokiolinduced cell shrinkage and decreased cell viability. In parallel, honokiol selectively induced DNA fragmentation and cell apoptosis rather than cell necrosis. Sequential treatment of neuro-2a cells with honokiol increased the expression of the proapoptotic Bax protein and its translocation from the cytoplasm to mitochondria. Honokiol successively decreased the mitochondrial membrane potential but increased the release of cytochrome c from mitochondria. Consequently, honokiol induced cascade activation of caspases-9, -3, and -6. In comparison, reducing caspase-6 activity by Z-VEID-FMK, an inhibitor of caspase-6, simultaneously attenuated honokiol-induced DNA fragmentation and cell apoptosis. Taken together, this study showed that honokiol can pass through the BBB and induce apoptotic insults to neuroblastoma cells through a Bax-mitochondrion-cytochrome c-caspase protease pathway. Therefore, honokiol may be a potential candidate drug for treating brain tumors.

Ketamine reduces nitric oxide biosynthesis in human umbilical vein endothelial cells by down-regulating endothelial nitric oxide synthase expression and intracellular calcium levels.

Objective:Ketamine, an intravenous anesthetic agent, can modulate vascular tone. Nitric oxide (NO), constitutively produced in endothelial cells, contributes to vasoregulation. In this study, we attempted to evaluate the effects of ketamine on NO biosynthesis in human umbilical vein endothelial cells and its possible mechanism. Design:Controlled laboratory study Settings:Research laboratory in a universal hospital. Subjects:Human umbilical vein endothelial cells prepared from human umbilical cord veins were exposed to 1, 10, 100, and 1000 &mgr;M ketamine for 1, 6, and 24 hrs. Measurements and Main Results:Exposure to 1, 10, and 100 &mgr;M ketamine for 1, 6, and 24 hrs was not cytotoxic to human umbilical vein endothelial cells. However, ketamine at 1000 &mgr;M significantly caused cell apoptosis. A therapeutic concentration of ketamine (100 &mgr;M) time-dependently reduced the levels of nitrite in human umbilical vein endothelial cells. Immunoblot analysis revealed that ketamine time-dependently decreased endothelial NO synthase protein production in human umbilical vein endothelial cells. Results of an assay by reverse-transcription polymerase chain reaction showed that ketamine significantly inhibited levels of endothelial NO synthase messenger RNA. Ketamine time-dependently reduced bradykinin-enhanced intracellular calcium concentrations. Analysis by confocal microscopy further demonstrated the suppressive effects of ketamine on bradykinin-induced calcium mobilization. Conclusions:A clinically relevant concentration of ketamine can reduce NO biosynthesis. The suppressive mechanisms occur not only by pretranslational inhibition of eNOS expression but also by a posttranslational decrease in endothelial NO synthase activity due to a reduction in intracellular calcium levels.