Huai-Chia Chang

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.

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.

Cytoskeleton Interruption in Human Hepatoma HepG2 Cells Induced by Ketamine Occurs Possibly through Suppression of Calcium Mobilization and Mitochondrial Function

Ketamine is an intravenous anesthetic agent often used for inducing and maintaining anesthesia. Cytoskeletons contribute to the regulation of hepatocyte activity of drug biotransformation. In this study, we attempted to evaluate the effects of ketamine on F-actin and microtubular cytoskeletons in human hepatoma HepG2 cells and its possible molecular mechanisms. Exposure of HepG2 cells to ketamine at ≤100 μM, which corresponds to clinically relevant concentrations for 1, 6, and 24 h, did not affect cell viability. Meanwhile, administration of therapeutic concentrations of ketamine obviously interrupted F-actin and microtubular cytoskeletons. In parallel, levels of intracellular calcium concentration- and time-dependently decreased after ketamine administration. Analysis by confocal microscopy further revealed that ketamine suppressed calcium mobilization from an extracellular buffer into HepG2 cells. Exposure to ketamine decreased cellular ATP levels. The mitochondrial membrane potential and complex I NADH dehydrogenase activity were both reduced after ketamine administration. Ketamine did not change the production of actin or microtubulin mRNA in HepG2 cells. Consequently, ketamine-caused cytoskeletal interruption led to suppression of CYP3A4 expression and its metabolizing activity. Therefore, this study shows that therapeutic concentrations of ketamine can disrupt F-actin and microtubular cytoskeletons possibly through suppression of intracellular calcium mobilization and cellular ATP synthesis due to down-regulation of the mitochondrial membrane potential and complex I enzyme activity. Such disruption of the cytoskeleton may lead to reductions in CYP3A4 activity in HepG2 cells.

2,6-Diisopropylphenol protects osteoblasts from oxidative stress-induced apoptosis through suppression of caspase-3 activation.

Abstract: 2,6‐Diisopropylphenol is an intravenous anesthetic agent used for induction and maintenance of anesthesia. Since it is similar to α‐tocopherol, 2,6‐diisopropylphenol may have antioxidant effects. Osteoblasts play important roles in bone remodeling. In this study, we attempted to evaluate the protective effects of 2,6‐diisopropylphenol on oxidative stress‐induced osteoblast insults and their possible mechanisms, using neonatal rat calvarial osteoblasts as the experimental model. Clinically relevant concentrations of 2,6‐diisopropylphenol (3 and 30 μM) had no effect on osteoblast viability. However, 2,6‐diisopropylphenol at 300 μM time‐dependently caused osteoblast death. Exposure to sodium nitroprusside (SNP), a nitric oxide donor, increased amounts of nitrite in osteoblasts. 2,6‐Diisopropylphenol did not scavenge basal or SNP‐releasing nitric oxide. Hydrogen peroxide (HP) enhanced levels of intracellular reactive oxygen species in osteoblasts. 2,6‐Diisopropylphenol significantly reduced HP‐induced oxidative stress. Exposure of osteoblasts to SNP and HP decreased cell viability time‐dependently. 2,6‐Diisopropylphenol protected osteoblasts from SNP‐ and HP‐induced cell damage. Analysis by a flow cytometric method revealed that SNP and HP induced osteoblast apoptosis. 2,6‐Diisopropylphenol significantly blocked SNP‐ and HP‐induced osteoblast apoptosis. Administration of SNP and HP increased caspase‐3 activities. However, 2,6‐diisopropylphenol significantly decreased SNP‐ and HP‐enhanced caspase‐3 activities. This study shows that a therapeutic concentration of 2,6‐diisopropylphenol can protect osteoblasts from SNP‐ and HP‐induced cell insults, possibly via suppression of caspase‐3 activities.

Comparison of the relative activities of inducing platelet apoptosis stimulated by various platelet-activating agents

Apoptosis-like events are known to occur in anuclear platelets. Although the mechanisms responsible for these events are still not completely understood, studies suggested that some platelet agonists can activate platelet apoptosis. However, the relative activities of various platelet agonists in inducing apoptosis have not yet been investigated. In the present study we explored this issue, and attempted to identify the correlation between platelet activation and apoptosis. In a platelet aggregation study, there were no significant differences respectively stimulated by arachidonic acid (AA; 100 µM), ADP (20 µM), collagen (10 µg/mL), thrombin (0.1 U/mL), U46619 (10 µM), and A23187 (5 µM). In a subsequent study, we fixed these concentrations of agonists to further compare their relative activities in inducing platelet apoptosis. Our results found that thrombin, U46619, and A23187 possess stronger activities than the other agonists in inducing platelet apoptosis (i.e., phosphatidylserine exposure, mitochondrial membrane potential depolarization, eukaryotic initiation factor (eIF)2α, and caspase activation). On the other hand, AA induced no apoptotic events in platelets. Based on this approach, we demonstrated for the first time that thrombin, U46619, and A23187, but not AA, possess stronger activity in inducing platelet apoptosis. In addition, we also found that platelet activation might not necessarily be associated with the occurrence of platelet apoptosis. The in vivo physiological function of the apoptotic machinery in platelets is not yet clearly understood, and needs to be further investigated in the future.

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.

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.