Lysophosphatidylcholine induces azurophil granule translocation via Rho/Rho kinase/F-actin polymerization in human neutrophils

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.

Glycine induces enhancement of bactericidal activity of neutrophils

Severe bacterial infections are frequently accompanied by depressed neutrophil functions. Thus, agents that increase the microbicidal activity of neutrophils could add to a direct antimicrobial therapy. Lysophosphatidylcholine augments neutrophil bactericidal activity via the glycine (Gly)/glycine receptor (GlyR) α2/ TRPM2/p38 mitogen-activated protein kinase (MAPK) pathway. However, the direct effect of glycine on neutrophil bactericidal activity was not reported. In this study, the effect of glycine on neutrophil bactericidal activity was examined. Glycine augmented bactericidal activity of human neutrophils (EC50 = 238 μM) in a strychnine (a GlyR antagonist)-sensitive manner. Glycine augmented bacterial clearance in mice, which was also blocked by strychnine (0.4 mg/kg, s.c.). Glycine enhanced NADPH oxidase-mediated reactive oxygen species (ROS) production and TRPM2-mediated [Ca2+ ]i increase in neutrophils that had taken up E. coli. Glycine augmented Lucifer yellow uptake (fluid-phase pinocytosis) and azurophil granule-phagosome fusion in neutrophils that had taken up E. coli in an SB203580 (a p38 MAPK inhibitor)-sensitive manner. These findings indicate that glycine augments neutrophil microbicidal activity by enhancing azurophil granule-phagosome fusion via the GlyRα2/ROS/calcium/ p38 MAPK pathway. We suggest that glycine could be a useful agent for increasing neutrophil bacterial clearance.

Changes in plasma lipoxin A4, resolvins and CD59 levels after ischemic and traumatic brain injuries in rats

Ischemic and traumatic brain injuries are the major acute central nervous system disorders that need to be adequately diagnosed and treated. To find biomarkers for these acute brain injuries, plasma levels of some specialized pro-resolving mediators (SPMs, i.e., lipoxin A4 [LXA4], resolvin [Rv] E1, RvE2, RvD1 and RvD2), CD59 and interleukin (IL)-6 were measured at 0, 6, 24, 72, and 168 h after global cerebral ischemic (GCI) and traumatic brain injuries (TBI) in rats. Plasma LXA4 levels tended to increase at 24 and 72 h after GCI. Plasma RvE1, RvE2, RvD1, and RvD2 levels showed a biphasic response to GCI; a significant decrease at 6 h with a return to the levels of the sham group at 24 h, and again a decrease at 72 h. Plasma CD59 levels increased at 6 and 24 h post-GCI, and returned to basal levels at 72 h post-GCI. For TBI, plasma LXA4 levels tended to decrease, while RvE1, RvE2, RvD1, and RvD2 showed barely significant changes. Plasma IL-6 levels were significantly increased after GCI and TBI, but with different time courses. These results show that plasma LXA4, RvE1, RvE2, RvD1, RvD2, and CD59 levels display differential responses to GCI and TBI, and need to be evaluated for their usefulness as biomarkers.

Changes in plasma lipoxin A4, resolvins and CD59 levels after ischemic and traumatic brain injuries in rats

Ischemic and traumatic brain injuries are the major acute central nervous system disorders that need to be adequately diagnosed and treated. To find biomarkers for these acute brain injuries, plasma levels of some specialized pro-resolving mediators (SPMs, i.e., lipoxin A4 [LXA4], resolvin [Rv] E1, RvE2, RvD1 and RvD2), CD59 and interleukin (IL)-6 were measured at 0, 6, 24, 72, and 168 h after global cerebral ischemic (GCI) and traumatic brain injuries (TBI) in rats. Plasma LXA4 levels tended to increase at 24 and 72 h after GCI. Plasma RvE1, RvE2, RvD1, and RvD2 levels showed a biphasic response to GCI; a significant decrease at 6 h with a return to the levels of the sham group at 24 h, and again a decrease at 72 h. Plasma CD59 levels increased at 6 and 24 h post-GCI, and returned to basal levels at 72 h post-GCI. For TBI, plasma LXA4 levels tended to decrease, while RvE1, RvE2, RvD1, and RvD2 showed barely significant changes. Plasma IL-6 levels were significantly increased after GCI and TBI, but with different time courses. These results show that plasma LXA4, RvE1, RvE2, RvD1, RvD2, and CD59 levels display differential responses to GCI and TBI, and need to be evaluated for their usefulness as biomarkers.

Lipidomic analysis of plasma lipids composition changes in septic mice

A lipidomic study on extensive plasma lipids in bacterial peritonitis (cecal ligation and puncture, CLP)-induced sepsis in mice was done at 24 h post-CLP. The effects of administration of lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), compounds known to have beneficial effects in CLP, on the sepsis-induced plasma lipid changes were also examined. Among the 147 plasma lipid species from 13 lipid subgroups (fatty acid [FA], LPA, LPC, lysophosphatidylethanolamine [LPE], phosphatidic acid [PA], phosphatidylcholine [PC], phosphatidylethanolamine [PE], phosphatidylinositol [PI], monoacylglyceride [MG], diacylglyceride [DG], triacylglyceride [TG], sphingomyelin [SM], and ceramide [Cer]) analyzed in this study, 40 and 70 species were increased, and decreased, respectively, in the CLP mice. Treatments with LPC and LPA affected 14 species from 7 subgroups, and 25 species from 9 subgroups, respectively. These results could contribute to finding the much needed reliable biomarkers of sepsis.

N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca²⁺ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca²⁺]ᵢ) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca²⁺]ᵢ in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca²⁺]ᵢ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca²⁺]ᵢ in human neutrophils was observed. In Ca²⁺-free buffer, NAC- and cysteine-induced [Ca²⁺]ᵢ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca²⁺]ᵢ in human neutrophils occur through Ca²⁺ influx. NAC- and cysteine-induced [Ca²⁺]ᵢ increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na⁺-free HEPES, both NAC and cysteine induced a marked increase in [Ca²⁺]ᵢ in human neutrophils, arguing against the possibility that Na⁺-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca²⁺]ᵢ increasing activity. Our results show that NAC and cysteine induce [Ca²⁺]ᵢ increase through Ca²⁺ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 microM) and SKF-96365 (20 microM) signifi cantly inhibited K49-PLA2-induced [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

Protective effects of Acanthopanax divaricatus extract in mouse models of Alzheimer's disease

BACKGROUND: Acanthopanax divaricatus var. albeofructus (ADA) extract has been reported to have anti-oxidant, immunomodulatory, and anti-mutagenic activity. MATERIALS/METHODS: We investigated the effects of ADA extract on two mouse models of Alzheimer's disease (AD); intracerebroventricular injection of beta-amyloid peptide (Abeta) and amyloid precursor protein/presenilin 1 (APP/PS1)-transgenic mice. RESULTS: Intra-gastric administration of ADA stem extract (0.25 g/kg, every 12 hrs started from one day prior to injection of Abeta1-42 until evaluation) effectively blocked Abeta1-42-induced impairment in passive avoidance performance, and Abeta1-42-induced increase in immunoreactivities of glial fibrillary acidic protein and interleukin (IL)-1alpha in the hippocampus. In addition, it alleviated the Abeta1-42-induced decrease in acetylcholine and increase in malondialdehyde levels in the cortex. In APP/PS1-transgenic mice, chronic oral administration of ADA stem extract (0.1 or 0.5 g/kg/day for six months from the age of six to 12 months) resulted in significantly enhanced performance of the novel-object recognition task, and reduced amyloid deposition and IL-1beta in the brain. CONCLUSIONS: The results of this study suggest that ADA stem extract may be useful for prevention and treatment of AD.

Protective effects of Acanthopanax divaricatus extract in mouse models of Alzheimer's disease

BACKGROUND: Acanthopanax divaricatus var. albeofructus (ADA) extract has been reported to have anti-oxidant, immunomodulatory, and anti-mutagenic activity. MATERIALS/METHODS: We investigated the effects of ADA extract on two mouse models of Alzheimer's disease (AD); intracerebroventricular injection of beta-amyloid peptide (Abeta) and amyloid precursor protein/presenilin 1 (APP/PS1)-transgenic mice. RESULTS: Intra-gastric administration of ADA stem extract (0.25 g/kg, every 12 hrs started from one day prior to injection of Abeta1-42 until evaluation) effectively blocked Abeta1-42-induced impairment in passive avoidance performance, and Abeta1-42-induced increase in immunoreactivities of glial fibrillary acidic protein and interleukin (IL)-1alpha in the hippocampus. In addition, it alleviated the Abeta1-42-induced decrease in acetylcholine and increase in malondialdehyde levels in the cortex. In APP/PS1-transgenic mice, chronic oral administration of ADA stem extract (0.1 or 0.5 g/kg/day for six months from the age of six to 12 months) resulted in significantly enhanced performance of the novel-object recognition task, and reduced amyloid deposition and IL-1beta in the brain. CONCLUSIONS: The results of this study suggest that ADA stem extract may be useful for prevention and treatment of AD.

Scant Extracellular NAD Cleaving Activity of Human Neutrophils is Down-Regulated by fMLP via FPRL1

Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences. It is yet to be elucidated whether intact human neutrophils have any extracellular NAD cleaving activity. In this study, with a simple fluorometric assay utilizing 1,N6-ethenoadenine dinucleotide (etheno-NAD) as the substrate, we have shown that intact peripheral human neutrophils have scant extracellular etheno-NAD cleaving activity, which is much less than that of mouse bone marrow neutrophils, mouse peripheral neutrophils, human monocytes and lymphocytes. With high performance liquid chromatography (HPLC), we have identified that ADP-ribose (ADPR) is the major extracellular metabolite of NAD degradation by intact human neutrophils. The scant extracellular etheno-NAD cleaving activity is decreased further by N-formyl-methionine-leucine-phenylalanine (fMLP), a chemoattractant for neutrophils. The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist. These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.

Lysophosphatidylcholine, Oxidized Low-Density Lipoprotein and Cardiovascular Disease in Korean Hemodialysis Patients: Analysis at 5 Years of Follow-up

Although oxidized low-density lipoprotein (LDL) and lysophosphatidylcholine (LPC) have been proposed as important mediators of the atherosclerosis, the long-term contribution to the risk of cardiovascular disease (CVD) in hemodialysis patients has not been evaluated. This study investigated the relation between oxidized LDL and LPC levels with long term risk of CVD. Plasma oxidized LDL and LPC levels were determined in 69 Korean hemodialysis patients as a prospective observational study for 5 yr. During the observation period, 18 cardiovascular events (26.1%) occurred including 6 deaths among the hemodialysis patients. The low LPC level group ( 254 microM/L) (P = 0.01). However, serum levels of oxidized LDL were not significantly different between groups with and without CVD. In adjusted Cox analysis, previous CVD, (hazard ratio [HR], 5.68; 95% confidence interval [CI], 1.94-16.63, P = 0.002) and low LPC level (HR, 3.45; 95% CI, 1.04-11.42, P = 0.04) were significant independent risk factors for development of CVD. It is suggested that low LPC, but not oxidized LDL, is associated with increased risk of CVD among a group of Korean hemodialysis patients.

Protective Effect of Decursinol on Mouse Models of Sepsis: Enhancement of Interleukin-10

The effects of decursinol on various models of sepsis were investigated. Intra-peritoneal pretreatment of mice with various doses of decursinol (1~100 mg/kg) effectively suppressed lethality induced in three mouse models of experimental sepsis, i.e., lipopolysaccharide (LPS)/D-galactosamine (GalN), high-dose LPS (20 mg/kg), and cecal ligation and puncture (CLP). Intra-peritoneal pretreatment of mice with decursinol (50 mg/kg) markedly enhanced the LPS/GalN-induced increase of plasma interleukin-10 (IL-10) levels, without affecting plasma TNF-alpha, IL-6 and IL-12 levels. These results suggest that decursinol could be effective for prevention or treatment of sepsis.

Effects of Ginseng Saponin on the Stress-Induced Plasma Corticosterone Levels in Mice / 신경정신의학

OBJECTIVES: The present study was designed to investigate the effect of ginseng saponin and its major active metabolite on the HPA axis under acute stress-i.c.v. injection stress, and immobilization stress, and to examine whether nitric oxide is involved in the mechanism of ginseng saponin on the HPA axis under acute stress. METHODS: In the experiment to study the effect of ginseng on HPA axis during stress, various dose of GTS were injected intracerebroventricularly(i.c.v.) or intraperitoneally(i.p.). Plasma corticosterone levels were measured 30 min after the i.c.v. injection stress. Immobilization stress was applied for 30 min and then blood was cellected for the assays of plasma corticosterone levels immediately after the completion of immobilization stress. To determine the active ginsenosides that can affect the stressinduced plasma corticosterone levels, various dose of each gisendosides(Rb1, Rb2, Rc, Re, Rf, Rg1, 20(S)-Rg3, and 20(R)-Rg3) were injected i.c.v. or i.p.. In the experiment to determine the involvement of the nitric oxide in the inhibitory effect of ginseng on the HPA, NG-Nitro-L-arginine methyl ester(L-NAME) and ginsenosides were coadministered i.c.v. or i.p., and plasma corticosterone levels were measured 30 min after stress was applied. RESULTS: First, the present study showed that ginseng total saponin, ginsenoside Rg3(S form), and ginsenoside Rc administered i.c.v. attenuated the intracerebroventricular injection stress-induced increase in plasma corticosterone levels, and these effects were removed by nitric oxide co-injection. Second, ginseng total saponin and ginsenoside Rc administered i.p. attenuated the immobilization stress-induced increase in plasma corticosterone levels, but ginsenoside Rg3(S form) did not attenuate the immobilization stress-induced increase in plasma corticosterone levels. The attenuative effects of ginseng total saponin and ginsenoside Rc in the immobilization stress-induced increase in plasma corticosterone levels were not affected by L-NAME co-injection. CONCLUSION: This study suggests that ginseng saponin attenuated stress-induced increase in plasma corticosterone levels and these effects were mediated by different mechanisms according to the components of ginseng saponin, and routes of administration.

Effect of kainic acid on the phosphorylation of mitogen activated protein kinases in rat hippocampus

In rat hippocampus, kainic acid (KA; 10 mg/kg; i.p.) increased the phosphorylated forms of ERK1/2 (p-ERK1/2) and Jun kinase1 (p-JNK1), but not p-JNK2 and p38 (p-p38). The preadministration with cycloheximide (CHX; 5 mg/kg; i.p.) inhibited KA-induced increase of p-JNK1, but not p-ERK1/2. Surprisingly, the phosphorylated upstream MAP kinase kinases (p-MKKs) were not correlated with their downstream MAP kinases. The basal p-MKK1/2 levels were completely abolished by KA, which were reversed by CHX. In addition, p-MKK4 and p-MKK3/6 levels were enhanced by CHX alone, but were attenuated by KA. Thus, our results showed that KA increased the p-ERK and p-JNK levels in rat hippocampus, which were not parallel with their classical upstreamal kinases.

Effect of Fentanyl on the TNF-alpha and IL-1beta Level during Global Ischemia/reperfusion in Rats / 대한마취과학회지

BACKGROUND: To reduce surgical stress, fentanyl is frequently used for neurosurgical procedure where focal and/or global ischemia may occur. However, the effect of fentanyl on the cytokine level during ischemia/reperfusion is still uncertain. The goal of this study was to evaluate the effect of fentanyl infusion on the proinflammatory cytokine, TNF-alpha and IL-1beta, levels during global cerebral ischemia/reperfusion (I/R) in rats using the intracerebral microdialysis technique. METHODS: Forty male S-D rats weighing 280 320 g were randomly assigned to four groups. Group 1: no fentanyl infusion and only I/R, Group 2: 1.5 ng/ml of fentanyl infusion during I/R, Group 3: 3.0 ng/ml of fentanyl infusion during I/R (n = 10 in each group). Rats were anesthetized with a intraperitoneal injection of pentobarbital (50 mg/kg), intubated and ventilated with room air using an animal ventilator. Two femoral arteries and one femoral vein were cannulated with PE-50 tubing for hemorrhagic hypotension, drug infusion and hydration. Both carotid arteries were dissected and a sling was placed for brain ischemia. The head was fixed on a stereotaxic device and a small burrhole was made for probe insertion. A CMA-12 probe was inserted into the left hippocampal CA-1 region according to the guidelines. Artificial CSF was run from the inserted microdialysis probe and infused with or without fentanyl at 3 microliter/min using a microinjection syringe pump during I/R. Ischemia was induced by clamping the carotid arteries while hemorrhagic hypotension for 17 min via the femoral artery and reperfusion were accomplished by the unclamping of the sling and reinfusing the blood via the femoral artery. Nasopharyngeal and rectal temperatures were maintained within the normal range during the whole procedure. After 2 hours of stabilization, the microdialysate was collected every 17 min just before (control) and during I/R and stored at 80oC until analysis using HPLC. RESULTS: During global I/R, TNF-alpha and IL-1 beta significantly increased at reperfusion (R5) compared to the control value (P < 0.05). However, in both cases of fentanyl infusion, TNF-alpha and IL-1 beta did not increase compared to the control value. CONCLUSIONS: Fentanyl inhibited the increase of proinflammatory cytokine TNF-alpha and IL-1 beta levels during global cerebral ischemia/reperfusion in rats.