Catecholamines Attenuate LPS-Induced Inflammation through ß2 Adrenergic Receptor Activation- and PKA Phosphorylation-Mediated TLR4 Downregulation in Macrophages.

Inflammation is a tightly regulated process involving immune receptor recognition, immune cell migration, inflammatory mediator secretion, and pathogen elimination, all essential for combating infection and restoring damaged tissue. However, excessive inflammatory responses drive various human diseases. The autonomic nervous system (ANS) is known to regulate inflammatory responses; however, the detailed mechanisms underlying this regulation remain incompletely understood. Herein, we aimed to study the anti-inflammatory effects and mechanism of action of the ANS in RAW264.7 cells. Quantitative PCR and immunoblotting assays were used to assess lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) expression. The anti-inflammatory effects of catecholamines (adrenaline, noradrenaline, and dopamine) and acetylcholine were examined in LPS-treated cells to identify the receptors involved. Catecholamines inhibited LPS-induced TNFα expression by activating the ß2 adrenergic receptor (ß2-AR). ß2-AR activation in turn downregulated the expression of Toll-like receptor 4 (TLR4) by stimulating protein kinase A (PKA) phosphorylation, resulting in the suppression of TNFα levels. Collectively, our findings reveal a novel mechanism underlying the inhibitory effect of catecholamines on LPS-induced inflammatory responses, whereby ß2-AR activation and PKA phosphorylation downregulate TLR4 expression in macrophages. These findings could provide valuable insights for the treatment of inflammatory diseases and anti-inflammatory drug development.

Cytoskeleton-disrupting agent cytochalasin B reduces oxidative stress caused by high glucose in the human arterial smooth muscle.

The role of cytoskeleton dynamics in the oxidative stress toward human vasculature has been unclear. The current study examined whether the cytoskeleton-disrupting agent cytochalasin B reduces oxidative stress caused by high glucose in the human arterial smooth muscle. All experiments in the human omental arteries without endothelium or the cultured human coronary artery smooth muscle cells were performed in d-glucose (5.5 mmol/L). The exposure toward d-glucose (20 mmol/L) for 60 min reduced the relaxation or hyperpolarization to an ATP sensitive K+ channel (KATP) opener levcromakalim (10-8 to 3 × 10-6 mol/L and 3 × 10-6 mol/L, respectively). Cytochalasin B and a superoxide inhibitor Tiron, restored them similarly. Cytochalasin B reduced the NADPH oxidase activity, leading to a decrease in superoxide levels of the arteries treated with high d-glucose. Also, cytochalasin B impaired the F-actin constitution and the membrane translocation of an NADPH oxidase subunit p47phox in artery smooth muscle cells treated with high d-glucose. A clinical concentration of cytochalasin B prevented human vascular smooth muscle malfunction via the oxidative stress caused by high glucose. Regulation of the cytoskeleton may be essential to keep the normal vascular function in patients with hyperglycemia.

Lidocaine Prevents Oxidative Stress-Induced Endothelial Dysfunction of the Systemic Artery in Rats With Intermittent Periodontal Inflammation.

BACKGROUND: Periodontal inflammation causes endothelial dysfunction of the systemic artery. However, it is unknown whether the use of local anesthetics during painful dental procedures alleviates periodontal inflammation and systemic endothelial function. This study was designed to examine whether the gingival or systemic injection of lidocaine prevents oxidative stress-induced endothelial dysfunction of the systemic artery in rats with intermittent periodontal inflammation caused by lipopolysaccharides (LPS). METHODS: Some rats received 1500 µg LPS injections to the gingiva during a week interval from the age of 8 to 11 weeks (LPS group). Lidocaine (3 mg/kg), LPS + lidocaine (3 mg/kg), LPS + lidocaine (1.5 mg/kg), and LPS + lidocaine (3 mg/kg, IP) groups simultaneously received gingival 1.5 or 3 mg/kg or IP 3 mg/kg injection of lidocaine on the same schedule as the gingival LPS. Isolated aortas or mandibles were subjected to the evaluation of histopathologic change, isometric force recording, reactive oxygen species, and Western immunoblotting. RESULTS: Mean blood pressure and heart rate did not differ among the control, LPS, LPS + lidocaine (3 mg/kg), and lidocaine (3 mg/kg) groups. LPS application reduced acetylcholine (ACh, 10 to 10 mol/L)-induced relaxation (29% difference at ACh 3 × 10 mol/L, P = .01), which was restored by catalase. Gingival lidocaine (1.5 and 3 mg/kg) dose dependently prevented the endothelial dysfunction caused by LPS application (24.5%-31.1% difference at ACh 3 × 10 mol/L, P = .006 or .001, respectively). Similar to the gingival application, the IP injection of lidocaine (3 mg/kg) restored the ACh-induced dilation of isolated aortas from rats with the LPS application (27.5% difference at ACh 3 × 10 mol/L, P < .001). Levels of reactive oxygen species were double in aortas from the LPS group (P < .001), whereas the increment was abolished by polyethylene glycol-catalase, gingival lidocaine (3 mg/kg), or the combination. The LPS induced a 4-fold increase in the protein expression of tumor necrosis factor-α in the periodontal tissue (P < .001), whereas the lidocaine (3 mg/kg) coadministration partly reduced the levels. Lidocaine application also decreased the protein expression of the nicotinamide adenine dinucleotide phosphate oxidase subunit p47phox, which was enhanced by the gingival LPS (5.6-fold increase; P < .001). CONCLUSIONS: Lidocaine preserved the aortic endothelial function through a decrease in arterial reactive oxygen species produced by nicotinamide adenine dinucleotide phosphate oxidase and periodontal tumor necrosis factor-α levels in rats with periodontal inflammation. These results suggest the beneficial effect of the gingival application of local anesthetics on the treatment of periodontal diseases on endothelial function of systemic arteries.

High oxygen modifies vasodilator effect of cysteine via enhanced oxidative stress and thromboxane production in the rat mesenteric artery.

Whether high oxygen is harmful to the vascular function is unclear. The present study examined if high oxygen modifies vasodilator effect of cysteine via enhanced oxidative stress and thromboxane production. Rat mesenteric arteries with endothelium at 95 or 50 % oxygen were subjected to isometric force recordings, measurement of thromboxane B2 levels, determination of superoxide and peroxynitrite levels and evaluation of NADPH oxidase subunit protein expression, respectively. L-cysteine (0.01-3 mM) constricted or dilated arteries at 95 and 50 % oxygen, respectively. Thromboxane receptor antagonist SQ-29,548 (1 µM) abolished the constriction at 95 % oxygen. L-cysteine (3 mM) increased levels of thromboxane B2 in arteries upon 95 % oxygen application. L-cysteine relaxed arteries treated with superoxide inhibitor tiron (2 mM) or NADPH oxidase inhibitor gp91ds-tat (1 µM) irrespective of the oxygen concentration while ATP-sensitive K(+) channel inhibitor glibenclamide (1 µM) and cystathionine-γ-lyase (CSE) inhibitor DL-propargylglycine (10 mM) similarly abolished the relaxation. L-cysteine (3 mM) with 95 % oxygen augmented levels of superoxide as well as nitrotyrosine within the artery, concomitantly with enhanced membrane protein expression of NADPH oxidase subunit p47phox. The higher concentration of oxygen attenuates L-cysteine-induced vasodilation via superoxide production mediated by NADPH oxidase along with thromboxane A2 production, resulting in vasoconstriction. The increased levels of superoxide, as well as peroxynitrite, coexist with the impaired vasodilation related to ATP-sensitive K(+) channels and CSE. Higher oxygen with plasma cysteine may cause oxidative stress and vasoconstrictor prostanoid production in blood vessels.

Propofol reduces liver dysfunction caused by tumor necrosis factor-α production in Kupffer cells.

PURPOSE: The present study, conducted in rats, investigated whether propofol attenuates lipopolysaccharide (LPS)-triggered liver dysfunction via regulation of tumor necrosis factor (TNF)-α production in activated Kupffer cells. METHODS: Rats received LPS (500 µg/kg) under Urethane™ sedation (1 g/kg) in combination with propofol (5 mg/kg/h) or Intralipid™ from 1 h before to 6 h after LPS administration. Some rats were treated with 10 mg/kg gadolinium chloride (GdCl3) to induce Kupffer cell depletion. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), TNF-α mRNA and protein expression, caspase-3 activation and apoptosis were evaluated in hepatocytes. Immunofluorescence staining revealed expression of the pan-macrophage marker CD68 as well as TNF-α in Kupffer cells. RESULTS: ALT and AST serum levels increased approximately four-fold in LPS-exposed rats compared with Intralipid™-treated rats at 6 h after LPS administration, whereas propofol and GdCl3 reduced the LPS-induced increases. LPS simultaneously augmented TNF-α expression in Kupffer cells, followed by increased caspase-3 activity and apoptosis in hepatocytes. Immunofluorescence staining and immunoblotting assay showed that TNF-α expression in Kupffer cells was inhibited by propofol and GdCl3, resulting in a reduction of caspase-3 activity and apoptosis in LPS-treated rat hepatocytes. CONCLUSIONS: Propofol (5 mg/kg/h) attenuated LPS-triggered liver dysfunction via inhibition of TNF-α production in activated Kupffer cells. These results suggest that propofol is capable of inhibiting inflammation-induced liver dysfunction in vivo.

Kynurenine causes vasodilation and hypotension induced by activation of KCNQ-encoded voltage-dependent K(+) channels.

Kynurenine is a potential contributor to hypotension in animal and human sepsis. The present study was designed to examine whether the voltage-dependent K(+) channels encoded by the KCNQ gene family (Kv7 channels) mediate vasodilator effects of kynurenine and whether modulation of these channels ameliorates hypotension caused by this compound. Rat aortas and mesenteric arteries or human omental arteries without endothelium were used. Some rings were incubated with the selective Kv7 channel inhibitor linopirdine (10 µM). l-Kynurenine (10 µM-1 mM) induced concentration-dependent relaxation in rat aortas and mesenteric arteries as well as human omental arteries, whereas linopirdine abolished the relaxation. l-Kynurenine (1 mM) produced hyperpolarization of vascular smooth muscle, which was reversed by linopirdine (10 µM). Wistar rats received l-kynurenine (1 mM) iv and subsequent linopirdine (10 µM) iv under 3% sevoflurane inhalation. l-Kynurenine iv caused hypotension, whereas linopirdine iv partially reversed it. In conclusion, kynurenine dilates arteries from rats as well as humans via Kv7 channels in the vascular smooth muscle. In rats, this tryptophan metabolite causes hypotension, which is partly counteracted by Kv7 channel inhibition. These results suggest that modulation of Kv7 channels may be a novel strategy to treat hypotension induced by the kynurenine.

Enhanced expression of WD repeat-containing protein 35 (WDR35) stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation.

BACKGROUND: Domoic acid (DA) is an excitatory amino acid analogue of kainic acid (KA) that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS) to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo. RESULTS: Our results showed that intraperitoneally administered DA was present in the brain and induced neurodegenerative changes including apoptosis in the CA1 region of the hippocampus. DA also increased the expression of WDR35 mRNA and protein in a dose- and time-dependent manner in the hippocampus. In experiments using glutamate receptor antagonists, the AMPA/KA receptor antagonist NBQX significantly attenuated the DA-induced increase in WDR35 protein expression, but the NMDA receptor antagonist MK-801 did not. In addition, the radical scavenger edaravone significantly attenuated the DA-induced increase in WDR35 protein expression. Furthermore, NBQX and edaravone significantly attenuated the DA-induced increase in p38 MAPK phosphorylation. CONCLUSION: In summary, our results indicated that DA activated AMPA/KA receptors and induced ROS production and p38 MAPK phosphorylation, resulting in an increase in the expression of WDR35 in vivo.

Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells.

BACKGROUND: Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved. RESULTS: Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H(2)O(2)) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H(2)O(2). Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H(2)O(2)-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H(2)O(2)-treated cells. CONCLUSIONS: In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells.

Naofen, a novel WD40-repeat protein, mediates spontaneous and tumor necrosis factor-induced apoptosis.

Naofen has recently been identified from the rat brain/spinal cord cDNA library as a substance reactive against an anti-shigatoxin (Stx)-2 antibody. Naofen mRNA is composed of 4620 nucleotides and encodes 1170 amino acids. Naofen contains four WD-repeat domains in its N-terminus and is ubiquitously distributed in many tissues of the rat. Tumor necrosis factor (TNF)-alpha enhanced the expression of naofen mRNA in HEK293 cells in a dose-dependent manner. Furthermore, naofen siRNA, which predominantly knocked down the expression of naofen mRNA, significantly reduced both TNF-alpha-induced caspase-3 activation and apoptosis in HEK293 cells. Overexpression of naofen in HEK293 cells (FLAG-NF) spontaneously induced caspase -3 activation and apoptosis, and showed extremely high susceptibility to TNF-alpha-induced apoptosis. These results indicated that naofen may function as a novel modulator activating caspase-3, and promoting TNF-alpha-stimulated apoptosis.

Regulation of hepatic branched-chain alpha-keto acid dehydrogenase kinase in a rat model for type 2 diabetes mellitus at different stages of the disease.

Branched-chain alpha-keto acid dehydrogenase (BCKDH) kinase (BDK) is responsible for the regulation of BCKDH complex, which is the rate-limiting enzyme in the catabolism of branched-chain amino acids (BCAAs). In the present study, we investigated the expression and activity of hepatic BDK in spontaneous type 2 diabetes using hyperinsulinemic Zucker diabetic fatty rats aged 9weeks and hyperglycemic, but not hyperinsulinemic rats aged 18weeks. The abundance of hepatic BDK mRNA and total BDK protein did not correlate with changes in serum insulin concentrations. On the other hand, the amount of BDK bound to the complex and its kinase activity were correlated with alterations in serum insulin levels, suggesting that hyperinsulinemia upregulates hepatic BDK. The activity of BDK inversely corresponded with the BCKDH complex activity, which was suppressed in hyperinsulinemic rats. These results suggest that insulin regulates BCAA catabolism in type 2 diabetic rats by modulating the hepatic BDK activity.

Enhanced expression of naofen in kidney of streptozotocin-induced diabetic rats: possible correlation to apoptosis of tubular epithelial cells.

BACKGROUND: Hyperglycemia/high glucose may induce apoptosis in diabetic kidney, but the mechanism is not fully understood. Naofen was found as a Shiga toxin (Stx)-2-related protein. Based on renal dysfunction in infection with Stx-producing Escherichia coli and on participation of naofen in apoptosis of human embryonic kidney cells, the present study was undertaken to investigate the mechanism of renal dysfunction in diabetes mellitus with particular reference to naofen. METHODS: In in vivo studies utilizing streptozotocin (STZ)-induced diabetic rats, and also in in vitro cultured rat kidney epithelial (NRK52E) cells, naofen messenger RNA (mRNA) and protein expressions were analyzed. Naofen mRNA location in diabetic kidney was studied by in situ hybridization. Apoptosis was assessed by caspase-3 activity assay. RESULTS: Rat diabetic kidney showed significant increases in caspase-3 activities and naofen mRNA. Naofen was mainly observed at both proximal and distal urinary tubules. Incubation of NRK52E cells in high glucose medium resulted in elevated naofen mRNA expression, whereas neither interleukin-1, interleukin-6, nor tumor necrosis factor-alpha elicited such action. Moreover, treatment of NRK52E cells with naofen small interfering RNA (siRNA) inhibited naofen mRNA expression induced by high glucose and blocked the increase in caspase-3 activity. CONCLUSIONS: These data suggest that naofen expression may be upregulated by hyperglycemia, with possible correlation to apoptosis of tubular epithelial cells and thereby to diabetic nephropathy.

Roles of naofen, a novel WD-repeat-2 protein, in the CCl4-treated livers--a possible relationship to cell proliferation.

Naofen (GenBank accession no. EF613262), a newly found intracellular protein in the WD-repeat-2 protein family, has been cloned as an anti-verotoxin II antibody immunoreactive substance, and the nucleotide- and amino acid-sequences have been clarified. The present study was undertaken to evaluate the roles of naofen especially in carbon tetrachloride (CCl4-induced cirrhosis model of rats, also in partial hepatectomy. Naofen mRNA expressions were observed from the early phases of cirrhosis development and during regenerative phases after partial hepatectomy, more remarkable in the former. Naofen immunoreactive fragments located in the vascular endothelial cells and peri-vascular spaces in normal livers especially in Glisson's areas, being strongly stained in the connective tissues 8 weeks after starting CCl4-injections, besides in the cytoplasm of hepatocytes in pseudo-lobules. In contrast, partial hepatectomy caused a small increase of naofen expressions in the whole hepatocytes, and significantly in the endothelial cells of portal veins and hepatic arterioles. Furthermore, in parallel to the degree of naofen mRNA and protein expressions, the rates of double-nuclei cells to total hepatocytes in the Glisson's areas increased in both cirrhosis and partial hepatectomy, suggesting a relationship between naofen expression and mitosis. In in-vitro studies with cell lines, vascular endothelial growth factor, a cell proliferation stimulant, increased the naofen mRNA expressions in HepG(2) cell lines, whereas paclitaxel, a cytotoxic anti-cancer drug, diminished them in NRK52E, both concentration-dependently. These results indicated that naofen immunoreactive fragments play an important role in the cell proliferation, relevant for analyzing the regenerative phases during cirrhosis developments and after partial hepatectomy.

Role of naofen, a novel WD repeat-containing protein, in reducing nitric oxide-induced relaxation.

1. Naofen, a novel WD40 repeat domain-containing protein, has recently been found in the intracellular compartment. The aim of the present study was to determine whether naofen affects thoracic aortic vascular reactivity in normotensive and hypertensive rats and whether naofen is present in the thoracic aorta. In addition, we examined whether naofen modulates acetylcholine (ACh)-stimulated nitric oxide (NO) release from the endothelium. 2. Immunohistochemistry showed greater naofen expression in endothelial cells in the DOCA-salt group compared with controls. There was increased naofen mRNA expression in deoxycorticosterone acetate (DOCA)-salt hypertensive rats compared with normotensive rats. 3. Acetylcholine-induced relaxation of rat aortic strips was decreased in DOCA-salt hypertensive rats compared with normotensive rats. Naofen-N- but not naofen-C-terminal protein caused a significant decrease in ACh-induced relaxation of aortic strips from normotensive rats. 4. Using a nitrite assay in a murine aortic endothelial cell line demonstrated that naofen-N-terminal protein, but not naofen-C-terminal protein, significantly reduced ACh-induced NO production, suggesting that naofen interferes with NO production. 5. Administration of naofen-N-terminal protein, but not naofen-C-terminal protein, significantly inhibited cyclohydrolase (GCH) I mRNA expression in a murine aortic endothelial cell line, suggesting that naofen-N-terminal protein interferes with NO synthesis by inhibiting GCH I mRNA expression. 6. The results of the present study suggest that naofen is present in vascular endothelial cells and has an inhibitory effect on ACh-induced relaxation under normotensive conditions. The findings reinforce the functional significance of naofen-N-terminal protein on rat vascular reactivity.

Phosphatidylinositol 3-kinase inhibition induces vasodilator effect of sevoflurane via reduction of Rho kinase activity.

AIMS: This study was aimed to examine whether a volatile anesthetic sevoflurane in clinical doses reduces vasoconstriction under the inhibition of phosphatidylinositol 3-kinase (PI3K) in the rat and human arteries and whether the intravenous administration of the PI3K inhibitor decreases blood pressure in rats under the sevoflurane inhalation. MATERIALS AND METHODS: Rat arteries (n=5-6) and human omental arteries (n=5-6) were subjected to isometric force recordings and western immunoblotting for Rho kinase, mitogen-activated protein kinase, and protein kinase C. Some arteries were incubated with sevoflurane (1.5% or 3%), a selective PI3K inhibitor LY294002 (3×10-6mol/L) or the combination. Mean arterial pressure (MAP) and heart rate (HR) in rats (n=7) were evaluated with or without intravenous injection of LY294002 (3×10-6mol/L) under 2% sevoflurane inhalation. KEY FINDINGS: Sevoflurane with LY294002, but not sevoflurane or LY294002 solely, inhibited the phenylephrine-induced contraction (32% to 52% decrease at phenylephrine [3×10-6mol/L] in rat arteries and [3×10-5mol/L] in human arteries). Sevoflurane (3%) only with LY294002 decreased Rho kinase activity in the rat aorta into 30%. Intravenous LY294002 reduced MAP (8.1-12.4mmHg decrease), but not HR, in rats under 2% sevoflurane inhalation. SIGNIFICANCE: Clinical sevoflurane doses with PI3K inhibition reduce the contraction of rat and human arteries ex vivo resulting from Rho kinase inhibition, and systemic blood pressure of rats in vivo. These results suggest that sevoflurane potentially causes vasodilation and hypotension in patients receiving anti-cancer therapy that inhibits PI3K.

Human serum albumin and oxidative stress in preeclamptic women and the mechanism of albumin for stress reduction.

AIMS: The present study to address one of the mechanisms in preeclampsia, examined whether levels of oxidative stress, human serum albumin, and endothelial function correlate in pregnant women and whether human serum albumin reduces levels of superoxide produced by NADPH oxidase activation in the human vascular smooth muscle cells. MATERIALS AND METHODS: Pregnant women with (Preeclampsia group, n = 33) and without preeclampsia (Normal group, n = 37) were recruited to determine levels of reactive oxygen species (serum diacron-reactive oxygen metabolite [d-ROM]), and the flow-mediated dilation (FMD). Human coronary arterial smooth muscle cells or omental arteries were subjected to evaluate isometric force recordings, levels of superoxide, western immunoblotting, and immunohistochemistry. The superoxide scavenging assay was also performed in a cell-free system. KEY FINDINGS: Women in the preeclampsia group demonstrated lower FMD and higher serum d-ROM values than those in the normal group. There were the inverse correlations between serum levels of d-ROM and the degree of FMD and between serum levels of albumin and those of d-ROM. D-glucose reduced the levcromakalim-induced dilation of human omental arteries, and it increased levels of superoxide and the recruitment of the NADPH oxidase subunit p47phox in human coronary arterial smooth muscle cells. Human serum albumin (0.05 to 0.5 g/dL) prevented these alterations whereas it exerted no superoxide scavenging effect. SIGNIFICANCE: Serum albumin relates to oxidative stress inversely, but to the endothelial function positively, in pregnant women. Human serum albumin appears to reduce oxidative stress via NADPH oxidase inhibition in the human vascular smooth muscle, indicating that the serum level may be a critical determinant of vascular oxidative stress in some human diseases.

Branched-chain amino acids alleviate hepatic steatosis and liver injury in choline-deficient high-fat diet induced NASH mice.

BACKGROUND: For successful treatment for nonalcoholic steatohepatitis (NASH), it may be important to treat the individual causative factors. At present, however, there is no established treatment for this disease. Branched-chain amino acids (BCAAs) have been used to treat patients with decompensated cirrhosis. AIM: In order to elucidate the mechanisms responsible for the effects of BCAAs on hepatic steatosis and disease progression, we investigated the effects of BCAA supplementation in mice fed a choline-deficient high-fat diet (CDHF), which induces NASH. METHODS: Male mice were divided into four groups that received (1) choline-sufficient high fat (HF) diet (HF-control), (2) HF plus 2% BCAA in drinking water (HF-BCAA), (3) CDHF diet (CDHF-control), or (4) CDHF-BCAA for 8weeks. We monitored liver injury, hepatic steatosis and cholesterol, gene expression related to lipid metabolism, and hepatic fat accumulation. RESULTS: Serum alanine aminotransferase (ALT) levels and hepatic triglyceride (TG) were significantly elevated in CDHF-control relative to HF-control. Liver histopathology revealed severe steatosis, inflammation, and pericellular fibrosis in CDHF-control, confirming the NASH findings. Serum ALT levels and hepatic TG and lipid droplet areas were significantly lower in CDHF-BCAA than in CDHF-control. Gene expression and protein level of fatty acid synthase (FAS), which catalyzes the final step in fatty acid biosynthesis, was significantly decreased in CDHF-BCAA than in CDHF-control (P<0.05). Moreover, hepatic total and free cholesterol of CDHF-BCAA was significantly lower than those of CDHF-control. CONCLUSIONS: BCAA can alleviate hepatic steatosis and liver injury associated with NASH by suppressing FAS gene expression and protein levels.

Sevoflurane Inhalation Accelerates the Long-Term Memory Consolidation via Small GTPase Overexpression in the Hippocampus of Mice in Adolescence.

Sevoflurane exposure impairs the long-term memory in neonates. Whether the exposure to animals in adolescence affects the memory, however, has been unclear. A small hydrolase enzyme of guanosine triphosphate (GTPase) rac1 plays a role in the F-actin dynamics related to the synaptic plasticity, as well as superoxide production via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. The current study was designed to examine whether sevoflurane exposure to mice in early adolescence modifies the long-term learning ability concomitantly with the changes in F-actin constitution as well as superoxide production in the hippocampus according to the levels of rac1 protein expression. Four-week-old mice were subjected to the evaluation of long-term learning ability for three days. On day one, each mouse was allowed to enter a dark chamber for five min to acclimatization. On day two, the procedure was repeated with the addition of an electric shock as soon as a mouse entered the dark chamber. All mice subsequently inhaled 2 L/min air with (Sevoflurane group) and without (Control group) 2.5% sevoflurane for three hours. On day three, each mouse was placed on the platform and retention time, which is the latency to enter the dark chamber, was examined. The brain removed after the behavior test, was used for analyses of immunofluorescence, Western immunoblotting and intracellular levels of superoxide. Sevoflurane exposure significantly prolonged retention time, indicating the enhanced long-term memory. Sevoflurane inhalation augmented F-actin constitution coexisting with the rac1 protein overexpression in the hippocampus whereas it did not alter the levels of superoxide. Sevoflurane exposure to 4-week-old mice accelerates the long-term memory concomitantly with the enhanced F-actin constitution coexisting with the small GTPase rac1 overexpression in the hippocampus. These results suggest that sevoflurane inhalation may amplify long-term memory consolidation via the increased cytoskeleton constitution in the hippocampus of animals in early adolescence.

Intermittent local periodontal inflammation causes endothelial dysfunction of the systemic artery via increased levels of hydrogen peroxide concomitantly with overexpression of superoxide dismutase.

BACKGROUND: The present study was designed to examine whether the intermittent local periodontal inflammation induces endothelial dysfunction of the systemic artery caused by oxidative stress and if increased levels of hydrogen peroxide coexisted with overexpression of superoxide dismutase (SOD) as well as NADPH oxidase contribute to the oxidative stress. METHODS: The rats in lipopolysaccharides (LPS) group received 1500µg LPS injection to bilateral gingiva of the lower jaw a week interval from eight- to eleven-week-old. Isolated mandibles or aortas were subjected to the evaluation of histopathological changes, isometric force recordings, reactive oxygen species using 2',7'-dichlorofluorescin diacetate (10(-5)mol/L) and protein expression of NADPH oxidase subunits and SOD, respectively. RESULTS: Mandible sections demonstrated the periodontal inflammation only in the LPS group at three days, but not seven days, after the LSP injection. Acetylcholine (10(-9) to 10(-5)mol/L)-induced relaxation was reduced only in aortas from the LPS group. Gp91ds-tat and PEG-catalase restored the impaired dilation in arteries from the LPS group. Levels of reactive oxygen species were enhanced in aortas from the LPS group, whereas the increment was abolished by the treatment with gp91-ds-tat or PEG-catalase. Expression of a NADPH oxidase subunit p47phox and CuZn-SOD increased in the LPS group. CONCLUSIONS: The intermittent local periodontal inflammation induces systemic endothelial dysfunction caused by overproduction of reactive oxygen species in the systemic artery of rats and that overexpression of CuZn-SOD as well as a NADPH oxidase cytosolic subunit contributes to increased levels of hydrogen peroxide in blood vessels of this animal model.

A role of anti-verotoxin antibody immunoreactive peptide, Virp5, from rat spinal cord.

An anti-verotoxin 2 (VT2) antibody immunoreactive 5-kDa polypeptide (Virp5), has been obtained through screening of the rat spinal cord cDNA library with the aid of anti-VT2 antibody. Virp5 was mainly expressed in the central nervous system, liver and kidney, and localized at glia-like cells and nerve fibers in the central nervous system, vascular endothelial cells and hepatic cells in the liver, as well as epithelial cells of distal tubules in the kidney. Intravenous administration of purified Virp5 elicited a dose-dependent increase in blood pressure. These results suggest that Virp5 commonly exists in the body, being partly playing a role in regulating the blood pressure.

A role for L-glutamate ionotropic receptors in the development of rat neurogenic pulmonary edema.

The present study was undertaken to evaluate possible roles of L-glutamate ionotropic receptors in neurogenic pulmonary edema. Perfusion of L-glutamate into the fourth ventricles of rats increased nitric oxide (NO) signals in the efflux solution concentration-dependently, significantly reducing both the occurrence and severity of neurogenic pulmonary edema. This effect was completely reversed by prior intracisternal injection of an NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), or an N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine maleate (MK-801), and partially by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a 2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA)/kainic acid receptor antagonist. Administration of MK-801 or CNQX alone, without L-glutamate, almost completely prevented neurogenic pulmonary edema development. These results suggest that endogenous L-glutamate may facilitate underlining disease process, whereas L-glutamate exogenously applied into the fourth ventricle may have an inhibitory action via release of NO, through ionotropic receptors.