Tyng-Guey Chen

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.

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.

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.

Propofol protects against nitrosative stress-induced apoptotic insults to cerebrovascular endothelial cells via an intrinsic mitochondrial mechanism

BACKGROUND Cerebrovascular endothelial cells (CECs), major component cells of the blood-brain barrier, can be injured by oxidative stress. Propofol can protect cells from oxidative injury. The aim of this study was to evaluate the effects of propofol on nitrosative stress-induced insults to CECs and its possible mechanisms. METHODS Primary CECs isolated from mouse cerebral capillaries were exposed to2 nitric oxide (NO) donors: sodium nitroprusside (SNP) or S-nitrosoglutathione (GSNO). Cellular NO levels, cell morphologies, and cell viabilities were analyzed. DNA fragmentation and apoptotic cells were quantified using flow cytometry. Proapoptotic Bcl2-antagonist-killer (Bak) and cytochrome c were immunodetected. Bak translocation was analyzed using confocal microscopy. Caspases-9 and -3 activities were measured fluorometrically. Permeability of the CEC monolayer was assayed by measuring the transendothelial electrical resistance. RESULTS Exposure of CECs to SNP increased cellular NO levels and simultaneously decreased cell viability (P < .01). Meanwhile, treatment of CECs with propofol at a therapeutic concentration (50 μM) decreased SNP-induced cell death (P < .01). SNP induced DNA fragmentation and cell apoptosis, but propofol decreased the cell injury (P < .01). Sequentially, propofol decreased SNP-enhanced Bak levels and translocation from the cytoplasm to mitochondria (P < .05). Exposure of CECs to propofol attenuated GSNO-induced cell death, apoptosis, and caspase-3 activation (P < .01). Additionally, propofol protected CECs against SNP-induced disruption of the CEC monolayer (P < .05). Consequently, SNP-enhanced cascade activation of caspases-9 and -3 was decreased by propofol (P < .01). CONCLUSION This study suggested that propofol at a therapeutic concentration can protect against nitrosative stress-induced apoptosis of CECs due to downregulation of the intrinsic Bak-mitochondrion-cytochrome c-caspase protease pathway.

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.

Adverse postoperative outcomes in surgical patients with immune thrombocytopenia

Patients with immune thrombocytopenia (ITP) are likely to have various medical co‐morbidities, yet their global features regarding adverse postoperative outcomes and use of medical resources when undergoing major surgery are unknown. The objective of this study was to validate whether ITP is an independent risk factor for adverse postoperative outcomes, and to explore the potential clinical predictors of outcomes after major surgery among patients with ITP.

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.

Modulation of Cytochrome P450-dependent Monooxygenases in Streptozotocin-induced Diabetic Hamster: II. Reverse Role of Insulin in P450 Activity and Defluorination

BACKGROUND Metabolic activities of cytochrome (cyt) P450-dependent monooxygenase could be modulated by diabetic state in experimental diabetic animals. The purpose of this study is to validate the effect of insulin on the modulation of the metabolic activity of cyt P450 and the defluorination ability to inhalational anesthetics in diabetic animals. METHODS Diabetic state in golden Syrian hamsters was achieved by intraperitoneal injection of streptozotocin 40 mg/kg once a day for 4 days. After stabilization of diabetic state for 6 weeks, a regimen of insulin treatment given subcutaneously was carried out. Metabolic activities of cyt P450 were assessed by the reaction with benzo(a) pyrene, pentoxyresorufin, aniline and erythromycin (specific substrates). The metabolic activities of cyt 1A1, 2B1, 2E1 and 3A4 respectively in a NADPH-generating system in microsomal preparations of the diabetic hamsters were observed before and after insulin treatment, and were compared with the control group. The ability of defluorination was evaluated by measuring the free fluoride metabolites after incubating the microsomes with enflurane in diabetic and insulin-treated hamsters. Contents of cyt P450 isozymes were measured by electrophoresis and immunoblotting before and after insulin treatment. Pathological features of hepatocytes in diabetic hamsters were evaluated microscopically before and after insulin treatment. RESULTS The defluorination of enflurane and activity of aniline hydroxylase (cyt 2E1) were successfully induced by diabetic state (P < 0.01). The pentoxyresorufin O-dealkylase (cyt 2B1) was inhibited nearly 50% in the diabetic hamster liver when compared with that of control (P < 0.01). While the activities of benzo(a)pyrene hydroxylase (cyt 1A1) and the erythromycin N-demethylase (cyt 3A4) were basically unaffected by diabetes, alterations in content of cyt P450 were parallel to the alterations in enzyme activities. Microscopically, diabetes induced vacuolization with fatty droplets in the hepatocytes. After treatment with insulin injection, the enzyme activities, protein content and pathologic features returned to the baseline similar to the control. CONCLUSIONS Our data demonstrated that under diabetic state, metabolic activities of cyt P450 and its extent of defluorination would be polymorphically modulated. After administration of insulin, the activities of cyt P450 and defluorination of enflurane returned to baseline as the blood sugar level had been normalized. This could remind the clinicians of the importance of insulin treatment in the potential drug-to-drug interactions in the diabetic patients.

Modulation of Cytochrome P-450 Dependent Monooxygenases in Streptozotocin-Induced Diabetic Hamster: I. Effects of Propofol on Defluorination and Cytochrome P-450 Activities

BACKGROUND Diabetes mellitus could induce polymorphic alterations of metabolic activities of cytochrome P-450 dependent monooxygenases in chemical-induced diabetic animals. The purpose of this study is to define the functional impact of clinical concentrations of propofol on the metabolic activities of cytochrome P-450 in the diabetic animals. METHODS In order to validate the effect of propofol on cytochrome P-450 activities, especially the cytochrome P-450 2E1 and its defluorination activity, we applied NADPH-generating system to measure the metabolizing activities of cytochrome P-450 isozymes of streptozotocin-induced diabetic hamsters within the microsomes preincubated with various concentrations of propofol. The extent of defluorination and activity of cytochrome P-450 2E1 were assessed by reacting the propofol-treated microsomes in NADPH-generating system with enflurane and aniline as substrates respectively. Drug metabolizing activities of cytochrome 1A1, 2B1, and 3A4 were evaluated by metabolizing specific substrates, benzo(a)pyrene, pentoxyresorufin and erythromycin, within the microsomes of diabetic hamsters preincubated with various concentrations of propofol. RESULTS The hepatic and renal defluorination of enflurane was significantly inhibited by 0.05 and 0.10 mM propofol in the microsomes of diabetic hamster (P < 0.05). The activities of aniline hydroxylase (cytochrome 2E1), pentoxyresorufin O-dealkylase (cytochrome 2B1) and benzo(a)pyrene hydroxylase (cytochrome 1A1) were inhibited by propofol in a concentration-dependent manner from 0.05 to 0.10 mM. However, propofol showed no significant effect to the erythromycin N-demethylase (cytochrome 3A4) at its concentration of 0.05-0.10 mM in the diabetic hamsters. CONCLUSIONS Our data demonstrated that propofol in therapeutic concentrations of 0.05 and 0.10 mM, could inhibit both liver and kidney defluorination and cytochrome P-450s activities of the diabetic hamsters in vitro of different extent. This should remind clinicians of propofols potential drug-to-drug interactions in the diabetic patients.