Yu-Ting Tai

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.

Lipoteichoic acid-induced TNF-α and IL-6 gene expressions and oxidative stress production in macrophages are suppressed by ketamine through downregulating Toll-like receptor 2-mediated activation oF ERK1/2 and NFκB.

Lipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that ketamine has anti-inflammatory and antioxidant effects on gram-negative LPS-induced macrophage activation. In this study, we further evaluated the effects of ketamine on the regulation of LTA-induced TNF-&agr; and IL-6 gene expressions and oxidative stress production in macrophages and its possible mechanisms. Exposure of macrophages to a therapeutic concentration of ketamine (100 &mgr;M) inhibited LTA-induced TNF-&agr; and IL-6 expressions at protein or mRNA levels. In parallel, ketamine at 100 &mgr;M reduced LTA-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Sequentially, ketamine reduced the LTA-triggered translocation of nuclear factor-&kgr;B (NF&kgr;B) from the cytoplasm to nuclei and its transactivation activity. Pretreatment with PD98059, an inhibitor of ERK, decreased LTA-enhanced NF&kgr;B activation and TNF-&agr; and IL-6 mRNA syntheses. Cotreatment with ketamine and PD98059 synergistically suppressed the LTA-induced translocation and transactivation of NF&kgr;B and biosyntheses of TNF-&agr; and IL-6 mRNA. Application of toll-like receptor 2 (TLR2) small interfering RNA (si)RNA into macrophages decreased the levels of this receptor, and simultaneously ameliorated LTA-augmented NF&kgr;B transactivation and consequent production of TNF-&agr; and IL-6 mRNA. Cotreatment with ketamine and TLR2 siRNA synergistically lowered TNF-&agr; and IL-6 mRNA syntheses in LTA-activated macrophages. Ketamine and TLR2 siRNA could reduce the LTA-induced increases in production of nitrite and intracellular reactive oxygen species in macrophages, and their combination had better effects than a single exposure. Thus, this study shows that one possible mechanism involved in ketamine-induced inhibition of LTA-induced TNF-&agr; and IL-6 gene expressions and oxidative stress production is through downregulating TLR2-mediated phosphorylation of ERK1/2 and the subsequent translocation and transactivation of NF&kgr;B.

Propofol specifically inhibits mitochondrial membrane potential but not complex I NADH dehydrogenase activity, thus reducing cellular ATP biosynthesis and migration of macrophages

Abstract: Propofol is a widely used intravenous anesthetic agent. Our previous study showed that a therapeutic concentration of propofol can modulate macrophage functions. Mitochondria play critical roles in the maintenance of macrophage activities. This study attempted to evaluate further the effects of mitochondria on the propofol‐induced suppression of macrophage functions using mouse macrophage‐like Raw 264.7 cells as the experimental model. Macrophages were exposed to a clinically relevant concentration of propofol for 1, 6, and 24 h. Analysis by the Trypan blue exclusion method revealed that propofol was not cytotoxic to macrophages. Exposure of macrophages to propofol did not affect mitochondrial NADH dehydrogenase activity of complex I. However, analysis of flow cytometry showed that propofol significantly decreased the mitochondrial membrane potential of macrophages. Cellular levels of ATP in macrophages were significantly reduced after propofol administration. In parallel with the dysfunction of mitochondria, the chemotactic analysis showed that exposure to propofol significantly inhibited the migration of macrophages. This study shows that a therapeutic concentration of propofol can specifically reduce the mitochondrial membrane potential, but there is no such effect on complex I NADH dehydrogenase activity. Modulation of the mitochondrial membrane potential may decrease the biosynthesis of cellular ATP and thus reduce the chemotactic activity of macrophages. This study provides in vitro data to validate mitochondrial dysfunction as a possible critical cause for propofol‐induced immunosuppression of macrophage functions.

MOLECULAR MECHANISMS OF PROPOFOL-INVOLVED SUPPRESSION OF NO BIOSYNTHESIS AND INDUCIBLE INOS GENE EXPRESSION IN LPS-STIMULATED MACROPHAGE-LIKE RAW 264.7 CELLS

Propofol (PPF), a widely used intravenous anesthetic agent, has been reported to have immunosuppressive and antioxidative effects. NO plays crucial roles in mediating inflammatory reactions. This study was designed to evaluate the effects of PPF on regulation of iNOS and its possible signal-transducing mechanisms in LPS-activated macrophage-like Raw 264.7 cells. Exposure of Raw 264.7 cells to LPS significantly increased nitrite production, but PPF reduced such enhancement in concentration- and time-dependent manners. In parallel, treatment of Raw 264.7 cells with a clinically relevant concentration of PPF (50 &mgr;M) significantly inhibited iNOS mRNA and protein production in LPS-activated Raw 264.7 cells. Propofol at 50 &mgr;M decreased the LPS-caused augmentation in nuclear c-Jun levels. An electrophoretic mobility shift assay revealed that PPF significantly decreased the binding affinity of the nuclear extracts from LPS-treated Raw 264.7 cells to activator protein 1 (AP-1) consensus DNA elements. A reporter gene assay further showed that PPF ameliorated the transactivated activity of AP-1 in LPS-stimulated Raw 264.7 cells. LPS sequentially increased phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 4 and c-Jun N-terminal kinase (JNK) 1/JNK-2. Meanwhile, a therapeutic concentration of PPF significantly decreased the activation of these two protein kinases. Application of Toll-like receptor 4 (TLR-4) siRNA to Raw 264.7 cells decreased cellular TLR-4 levels and LPS-caused activation of MEK-4. Cotreatment with PPF and TLR-4 siRNA synergistically lowered the LPS-induced increased biosynthesis of iNOS mRNA and nitrite. Therefore, a clinically relevant concentration of PPF can inhibit NO production and iNOS gene expression in LPS-activated Raw 264.7 cells. The suppressive mechanisms may occur through sequential down-regulation of TLR-4/MEK-4/JNK-1/JNK-2/AP-1 activation.

Lipopolysaccharide stimulates syntheses of toll-like receptor 2 and surfactant protein-A in human alveolar epithelial A549 cells through upregulating phosphorylation of MEK1 and ERK1/2 and sequential activation of NF-κB.

Surfactant proteins (SPs) and toll-like receptors (TLRs) contribute to regulation of sepsis-induced acute lung injury. Lipopolysaccharide (LPS) is one of the major causes of septic shock. This study was designed to evaluate the effects of LPS on the regulation of tlr-2 and sp-a gene expression in human alveolar epithelial A549 cells and the possible mechanisms. Exposure of A549 cells to LPS increased the expressions of TLR2 and SP-A mRNA and protein in time-dependent manners. A search using a bioinformatic approach found that there are several nuclear factor kappa-B (NF-κB)-DNA-binding motifs in the promoter region of the tlr2 and sp-a genes. Immunoblotting analyses revealed that exposure to LPS time-dependently enhanced the translocation of NF-κB from the cytoplasm to nuclei. Analyses of an electrophoretic mobility shift assay further showed that LPS augmented the transactivation activity of NF-κB to its consensus oligonucleotides in A549cells. Sequentially, treatment of A549 cells with LPS increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38-mitogen-activated protein kinase (p38MAPK), and MAPK kinase-1 (MEK1). Pretreatment with PD98059, an inhibitor of ERK1/2, significantly decreased LPS-induced TLR2 and SP-A mRNA expression.

Nitric oxide induces osteoblast apoptosis through a mitochondria-dependent pathway.

Abstract: Osteoblasts contribute to bone remodeling. Nitric oxide can regulate osteoblast activities. In this study, we attempted to evaluate the pathophysiological effects of nitric oxide on osteoblasts and its possible mechanism using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside, a nitric oxide donor, decreased alkaline phosphatase activities and cell viability in a concentration‐ and time‐dependent manner. Apoptotic analysis revealed that sodium nitroprusside time‐dependently increased the percentages of osteoblasts undergoing apoptosis. Administration of sodium nitroprusside reduced the mitochondrial membrane potential of osteoblasts. In parallel with the mitochondrial dysfunction, levels of intracellular reactive oxygen species and cytochrome c were significantly elevated following sodium nitroprusside administration. Exposure of osteoblasts to sodium nitroprusside significantly increased caspase‐3 activity. Results of this study show that nitric oxide, decomposed from sodium nitroprusside, can induce osteoblast apoptosis through a mitochondrion‐dependent cascade that causes mitochondrial dysfunction, release of intracellular reactive oxygen species and cytochrome c from mitochondria to cytoplasm, and activation of caspase‐3.

Resveratrol attenuates high-fat diet-induced disruption of the blood-brain barrier and protects brain neurons from apoptotic insults.

The blood-brain barrier (BBB) maintains brain microenvironment. Our previous study showed that oxidized low-density lipoprotein (oxLDL) can damage the BBB by inducing apoptosis of cerebrovascular endothelial cells. This study was aimed at evaluating the effects of resveratrol on high-fat diet-induced insults to the BBB and brain neurons. Exposure of mice to a high-fat diet for 8 weeks increased levels of serum total cholesterol (146 ± 13) and LDL (68 ± 8), but resveratrol decreased such augmentations (119 ± 6; 45 ± 8). Permeability assays showed that a high-fat diet induced breakage of the BBB (88 ± 21). Meanwhile, resveratrol alleviated this interruption (16 ± 6). Neither resveratrol nor a high-fat diet caused the death of cerebrovascular endothelial cells. Instead, exposure to a high-fat diet disrupted the polymerization of occludin and zonula occludens (ZO)-1, but resveratrol significantly attenuated those injuries. Neither a high-fat diet nor resveratrol changed the levels of occludin or ZO-1 in brain tissues. Resveratrol protected brain neurons against high-fat diet-induced caspase-3 activation and genomic DNA fragmentation. This study shows that resveratrol can attenuate the high-fat diet-induced disruption of the BBB via interfering with occludin and ZO-1 tight junctions, and protects against apoptotic insults to brain neurons.

GATA-2 Transduces LPS-Induced il-1β Gene Expression in Macrophages via a Toll-Like Receptor 4/MD88/MAPK-Dependent Mechanism

Lipopolysaccharide (LPS) is a critical factor for inducing acute lung injury. GATA-2, a transcription factor, contributes to the control of cell activity and function. Exposure of RAW 264.7 cells to LPS induced interleukin (IL)-1β mRNA and protein expression and GATA-2 translocation from the cytoplasm to nuclei in concentration- and time-dependent manners. A bioinformatic search revealed that GATA-2-specific binding elements exist in the 5’-promoter region of the il-1β gene. LPS could enhance the transactivation activity of GATA-2 in macrophages. Knocking-down translation of GATA-2 mRNA using RNA interference significantly alleviated LPS-induced IL-1β mRNA and protein expression. As to the mechanism, transfection of toll-like receptor (TLR) 4 small interfering (si)RNA into macrophages concurrently decreased LPS-caused increases in nuclear GATA-2 levels. Sequentially, treatment with myeloid differentiation factor 88 (MyD88) siRNA decreased LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) kinase 1/2 and subsequent translocation of GATA-2. Reducing MAPK activities using specific inhibitors simultaneously decreased GATA-2 activation. Furthermore, exposure of primary macrophages to LPS significantly increased the transactivation activities of GATA-2 and IL-1β mRNA and protein expression. Transfection of GATA-2 siRNA inhibited LPS-induced IL-1β mRNA expression. Results of this study show that LPS induction of il-1β gene expression in macrophages is mediated by GATA-2 via activation of TLR4, MyD88, and MAPKs.

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

BackgroundLipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS), could induce surfactant protein-A (SP-A) production in human alveolar epithelial (A549) cells.ObjectivesIn this study, we further evaluated the effect of LTA on SP-A biosynthesis and its possible signal-transducing mechanisms.MethodsA549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2 (ERK1/2), and mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1 were determined.ResultsExposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11–7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA.ConclusionsTherefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway.

Propofol inhibits renal cytochrome P450 activity and enflurane defluorination in vitro in hamsters

Purpose: To determine the effect of propofol on renal cytochrome P450 activity and defluorination of enflurane.Methods: Renal microsomes were prepared by homogenization and differential centrifugation from pooled hamster kidneys. Defluorination of enflurane was assessed by measuring free fluoride metabolites after reacting enflurane with renal microscomes incubated with various concentrations, 0.05 – 1.0 mmol·L−1 propofol in the NADPH-generating system. Drug metabolizing activities of renal cytochrome P450 mono-oxygenase enzymes were evaluated within microsomes preincubated with propofol and reacted with the specific marker substrates, aniline, benzo(a)pyrene, erythromycin and pentoxyresorufin, for cytochrome P450 2E1, 1A1, 3A4 and 2B1, respectively.Results: Renal defluorination of enflurance was inhibited by clinical concentrations, 0.05 mmol·L−1 of propofol (P <0.05). Dose-dependent inhibition of defluorination, aniline and benzo(a)pyrene hydroxlase within kidney microsomes was related to propofol concentration. Propofol demonstrated a profound inhibition of renal pentoxyresorufin dealkylase activity even at low concentrations, 0.05 mmol·L−1 (P<0.01). Propofol did not exhibit inhibition of erythromycin N-demethylation of kidney microsomes except at high concentration, 1.0 mmol·L−1. Spectral analyses of key coenzymes of renal cytochrome P450 monooxygenase, cytochrome b5 and cytochromec reductase, demonstrated an inhibition when incubated with high concentrations of propofol (P<0.05).Conclusion: In anin vitro study in an NADPH-generating system of hamster kidney microsomes, propofol, in clinical concentrations, exhibited a broad-spectrum of inhibition to renal monooxygenase activities and enflurane defluorination.RésuméObjectif: Déterminer l’effet du propofol sur l’activité du cytochrome P450 rénal et sur la défluoration de l’enflurane.Méthode: Les microsomes rénaux ont été préparés par homogénéisation et centrifugation différentielle de reins de hamsters. La défluoration de l’enflurane a été évaluée en mesurant les métabolites de fluorure libres après la réaction avec les microsomes rénaux incubés dans diverses concentrations, 0,05 – 1,0 mmol·L−1, de propofol dans un système générateur de NADPH. Les activités métabolisantes de médicament des enzymes rénales cytochrome P450 mono-oxygénase ont été évaluées dans les microsomes préincubés avec du propofol et qui ont réagi avec des substrats de marqueurs spécifiques, aniline, benzo(a)pyrène, érythromycine et pentoxyrésorufine, pour les cytochromes P450, 2E1, 1A1, 3A4 et 2B1, respectivement.Résultats: La défluoration rénale de l’enflurance a été inhibée par les concentrations cliniques, 0,05 mmol·L−1 de propofol (P<0,05). L’inhibition dose-dépendante de la défluoration, de l’aniline et de la benzo(a)pyrène hydroxylase dans les microsomes rénaux était reliée à la concentration de propofol. Le propofol a démontré une importante inhibition de l’activité de la pentoxyrésorufine dealdylase rénale même avec une faible concentration, 0,05 mmol·L−1 (P<0,01). L’emploi de propofol n’a pas montré d’inhibition de la N-déméthylation de l’érythromycine des microsomes du rein, sauf avec une forte concentration, 1,0 mmol·L−1. Les analyses spectrales des coenzymes clés de la cytochrome P450-mono-oxygénase rénale, de la cytochrome b5 etc-réductase ont démontré une inhibition dans une incubation avec de fortes concentrations de propofol (P<0,05).Conclusion: Une étudein vitro de microsomes rénaux de hamster, réalisée avec un système producteur de NADPH, a montré que le propofol en concentrations cliniques affiche une inhibition à large spectre des activités de la mono-oxygénase rénale et de la défluoration de l’enflurane.