2.The Production Mechanism of TNF-alpha and IL-6 by Group IIA Phospholipase A2.
Jae Hong AHN ; Dae Won PARK ; Jin Hee KIM ; Jei Jun BAE ; Yeun Kyoung BAE ; Yoon Ki PARK
Yeungnam University Journal of Medicine 2004;21(2):177-190
BACKGROUND: Secretory phospholipase A2 (sPLA2) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 (sPLA2-IIA) has been detected in the inflammatory fluids, and its plasma level increases in the inflammatory disease. This study examined the effect of sPLA2-IIA on mouse macropahges in order to investigate the potential mechanism of sPLA2-induced inflammation. MATERIALS AND METHODS: Wild type PLA2 and mutant H48Q PLA2 were purified from HEK293 cells transfected with the corresponding plasmids, and the PLA2 activities were measured using 1-palmitoyl-2-[1- (14) C]linoleoyl-3-phosphatidylethanolamine as substrates. The TNF-alpha and IL-6 released in the supernatants were determined by ELISA. In addition, the TNF-alpha and IL-6 mRNA were analyzed by RT-PCR. RESULTS: sPLA2-IIA stimulated the production of TNF-alpha and IL-6 in a dose- and time-dependent manner. In addition, the effect of sPLA2-IIA on cytokine production from the macrophage was found to be associated with the accumulation of their specific mRNA. The mRNA levels of TNF-alpha and IL-6 peaked at 2 and 6 hours in a time-dependent manner, respectively. CONCLUSION: In conclusion, the production of proinflammatory cytokine might be mediated by the binding of sPLA2-IIA to the receptors.
Animals
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Enzyme-Linked Immunosorbent Assay
;
Fatty Acids
;
Group II Phospholipases A2*
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HEK293 Cells
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Inflammation
;
Interleukin-6*
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Macrophages
;
Mice
;
Phospholipases A2, Secretory
;
Phospholipids
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Plasma
;
Plasmids
;
RNA, Messenger
;
Tumor Necrosis Factor-alpha*
3.Effect of E54 mutation of human secreted phospholipase A2 GIIE on substrate selectivity.
Shulin HOU ; Junping BAI ; Xin LU ; Yulong ZHANG ; Tingting XU ; Jun XIE
Chinese Journal of Biotechnology 2021;37(7):2513-2521
Human secreted phospholipase A2 GIIE (hGIIE) is involved in inflammation and lipid metabolism due to its ability of hydrolyzing phospholipids. To reveal the mechanism of substrate head-group selectivity, we analyzed the effect of mutation of hGIIE on its activity and selectivity. hGIIE structural analysis showed that E54 might be related to its substrate head-group selectivity. According to the sequence alignment, E54 was mutated to alanine, phenylalanine, and lysine. Mutated genes were cloned and expressed in Pichia pastoris X33, and the enzymes with mutations were purified with 90% purity by ion exchange and molecular size exclusion chromatography. The enzymatic activities were determined by isothermal microthermal titration method. The Km of mutant E54K towards 1,2-dihexyl phosphate glycerol decreased by 0.39-fold compared with that of wild type hGIIE (WT), and the Km of E54F towards 1,2-dihexanoyl-sn-glycero-3-phosphocholine increased by 1.93-fold than that of WT. The affinity of mutant proteins with phospholipid substrate was significantly changed, indicating that E54 plays an important role in the substrate head-group selectivity of hGIIE.
Humans
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Kinetics
;
Mutation
;
Phospholipases A2, Secretory
;
Phospholipids
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Saccharomycetales
;
Substrate Specificity
4.The Effects of Moxifloxacin in Endotoxin-induced Acute Lung Injury.
Jong Hyun BAEK ; Jang Hoon LEE ; Jung Cheul LEE ; Dong Hyup LEE ; Yong Suk MOON ; Young Man LEE
The Korean Journal of Thoracic and Cardiovascular Surgery 2009;42(1):1-8
BACKGROUND: The pathophysiology of acute respiratory distress syndrome with sepsis is acute lung injury (ALI) that's' caused by endotoxin (LPS). We evaluate effects of moxifloxacin on LPS-induced ALI in a rat model. MATERIAL AND METHOD: The rats were divided into 3 groups as the control group (C), the LPS insult group (L), and the LPS+moxifloxacin treated group (L-M). ALI was induced by endotracheal instillation of E.coli LPS, then moxifloxacin was given in 30 minutes. Five hours later, we checked the lung weight/body weight ratio(the L/BW ratio), the protein & neutrophils in the bronchoalveolar lavage fluid (BALF), the myeloperoxidase (MPO) activity & the malondialdehyde (MDA) content, the expressions of cytosolic and secretory phospholipase A2 (c, sPLA2), and the morphology of the lung with using a light microscope. RESULT: The L/BW ratio, the protein content and the neutrophil count in the BALF, and the MPO activity and the MDA content in lung were significantly increased in group L compared to group C, and these factors were markedly decreased in group L-M compare to group L. The cPLA2 expression and the sPLA2 expression were increased in group L and the cPLA2 expression was decreased in group L-M. Yet the sPLA2 expression was not changed in group L-M. Morphologically, many inflammatory findings were observed in group L, but not in group L-M. CONCLUSION: Many of the inflammatory changes of ALI that were caused by LPS insult were ameliorated by moxifloxacin treatment.
Acute Lung Injury
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Animals
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Aza Compounds
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Bronchoalveolar Lavage Fluid
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Cytosol
;
Light
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Lung
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Malondialdehyde
;
Neutrophils
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Peroxidase
;
Phospholipases A2, Secretory
;
Quinolines
;
Rats
;
Respiratory Distress Syndrome, Adult
;
Sepsis
5.Moxifloxacin Ameliorates Oleic Acid-induced Acute Lung Injury by Modulation of Neutrophilic Oxidative Stress in Rats.
Byung Yong KIM ; Young Man LEE
Tuberculosis and Respiratory Diseases 2010;68(6):334-344
BACKGROUND: Based on the known immunoregulatory functions of moxifloxacin on phagocytes, the therapeutic effect of moxifloxacin on oleic acid (OA)-induced acute lung injury (ALI) was investigated. METHODS: Moxifloxacin (10 mg/kg) was given to male Sprague-Dawley rats that had been given oleic acid (OA, 30 microliter) intravenously. Five hours after OA injection, parameters demonstrating ALI were assessed to measure the effects of moxifloxacin on acute lung injury. RESULTS: The pathological findings of OA-induced ALI's was diminished by moxifloxacin. Through ultrastructural and CeCl3 EM histochemistry, moxifloxacin was confirmed to be effective in decreasing oxidative stress in the lung as well. Indices of ALI, such as lung weight/body weight ratio, protein content in bronchoalveolar lavage fluid, and lung myeloperoxidase were decreased by moxifloxacin. In diaminobenzidine immunohistochemistry, fluorescent immunohistochemistry, and Western blotting of the lung, moxifloxacin had decreased the enhanced expression of secretory phospholipase A2 (sPLA2) by OA. CONCLUSION: We concluded that moxifloxacin was effective in lessening acute inflammatory pulmonary edema caused by OA, by inhibiting the neutrophilic respiratory burst, which was initiated by the activation of sPLA2.
Acute Lung Injury
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Animals
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Aza Compounds
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Blotting, Western
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Bronchoalveolar Lavage Fluid
;
Cerium
;
Free Radicals
;
Humans
;
Immunohistochemistry
;
Lung
;
Male
;
Neutrophils
;
Oleic Acid
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Oxidative Stress
;
Peroxidase
;
Phagocytes
;
Phospholipases A2, Secretory
;
Pulmonary Edema
;
Quinolines
;
Rats
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Rats, Sprague-Dawley
;
Respiratory Burst
6.Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa.
Jacqueline LESSIG ; Uta REIBETANZ ; Jurgen ARNHOLD ; Hans-Jurgen GLANDER
Asian Journal of Andrology 2008;10(6):829-836
AIMTo determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes.
METHODSAcrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used.
RESULTSAlthough sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality.
CONCLUSIONThe ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality.
Acrosome ; drug effects ; physiology ; Acrosome Reaction ; drug effects ; Annexin A5 ; metabolism ; Anti-Bacterial Agents ; pharmacology ; Calcimycin ; pharmacology ; Ethidium ; Flow Cytometry ; Fluorescent Dyes ; Humans ; In Vitro Techniques ; Male ; Microscopy, Confocal ; Pancreatic Elastase ; metabolism ; Phosphatidylserines ; metabolism ; Phospholipases A2, Secretory ; metabolism ; Semen ; cytology ; drug effects ; Spermatozoa ; drug effects ; enzymology
7.Stimulated Bronchial Epithelial Cells Release Bioactive Lysophosphatidylcholine 16:0, 18:0, and 18:1.
Yan ZHUGE ; Yang YUAN ; Richard VAN BREEMEN ; Michael DEGRAND ; Oksana HOLIAN ; Mark YODER ; Hazel LUM
Allergy, Asthma & Immunology Research 2014;6(1):66-74
PURPOSE: In human subjects and animal models with acute and chronic lung injury, the bioactive lysophosphatidylcholine (LPC) is elevated in lung lining fluids. The increased LPC can promote an inflammatory microenvironment resulting in lung injury. Furthermore, pathological lung conditions are associated with upregulated phospholipase A2 (PLA2), the predominant enzyme producing LPC in tissues by hydrolysis of phosphatidylcholine. However, the lung cell populations responsible for increases of LPC have yet to be systematically characterized. The goal was to investigate the LPC generation by bronchial epithelial cells in response to pathological mediators and determine the major LPC species produced. METHODS: Primary human bronchial epithelial cells (NHBE) were challenged by vascular endothelial growth factor (VEGF) for 1 or 6 h, and condition medium and cells collected for quantification of predominant LPC species by high performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). The cells were analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for PLA2. The direct effects of LPC in inducing inflammatory activities on NHBE were assessed by transepithelial resistance as well as expression of interleukin-8 (IL-8) and matrix metalloproteinase-1 (MMP-1). RESULTS: VEGF stimulation of NHBE for 1 or 6 h, significantly increased concentrations of LPC16:0, LPC18:0, and LPC18:1 in condition medium compared to control. The sPLA2-selective inhibitor (oleyloxyethyl phosphorylcholine) inhibited the VEGF-induced release of LPC16:0 and LPC18:1 and PLA2 activity. In contrast, NHBE stimulated with TNF did not induce LPC release. VEGF did not increase mRNA of PLA2 subtypes sPLA2-X, sPLA2-XIIa, cPLA2-IVa, and iPLA2-VI. Exogenous LPC treatment increased expression of IL-8 and MMP-1, and reduced the transepithelial resistance in NHBE. CONCLUSIONS: Our findings indicate that VEGF-stimulated bronchial epithelial cells are a key source of extracellular LPCs, which can function as an autocrine mediator with potential to induce airway epithelial inflammatory injury.
Epithelial Cells*
;
Group X Phospholipases A2
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Humans
;
Hydrolysis
;
Interleukin-8
;
Lung
;
Lung Injury
;
Lysophosphatidylcholines*
;
Mass Spectrometry
;
Matrix Metalloproteinase 1
;
Models, Animal
;
Phosphatidylcholines
;
Phospholipases A2
;
Reverse Transcriptase Polymerase Chain Reaction
;
RNA, Messenger
;
Vascular Endothelial Growth Factor A
8.Elevated serum secretory type II phospholipase A2 in patients with coronary heart disease.
Lu YU ; Wen-bing JIANG ; Guo-sheng FU ; Guang-ming QIN ; Jun-hong WANG ; Zhao-quan HUANG
Chinese Journal of Cardiology 2006;34(9):812-815
OBJECTIVETo measure the serum level of secretory type II phospholipase A2 (sPLA2) in patients with coronary heart disease and investigate the possible relationship with IL-8 and LPA.
METHODSA total of 110 patients with acute coronary syndrome (ACS), 63 patients with stable coronary heart disease (SCHD) group and 89 non-CHD control patients were studied. Serum levels of sPLA2, IL-8, LPA and hs-CRP were measured and the correlation among these parameters was observed.
RESULTSThe levels of serum sPLA2 [(68 +/- 17) U/ml], IL-8 [(182 +/- 80) pg/ml] and LPA [(2.85 +/- 0.36) micromol/L] were significantly higher in CHD patients than those in controls [sPLA2: (55 +/- 12) U/ml; IL-8: (119 +/- 33) pg/ml; LPA: (2.34 +/- 0.36) micromol/L, all P < 0.01], and sPLA2 and IL-8 were also significantly higher in ACS patients [sPLA2: (71 +/- 18) U/ml; IL-8: (195 +/- 78) pg/ml] than those in SCHD patients [sPLA2: (63 +/- 12) U/ml; IL-8: (159 +/- 79) pg/ml, both P < 0.01]. Serum sPLA2 level was positively correlated with hs-CRP, IL-8 and LPA (r = 0.203, P = 0.007; r = 0.658, P < 0.01; r = 0.231, P = 0.005, respectively). The relative risk of having CHD is 6.248 (P < 0.01) with the sPLA2 level above 63.75 U/ml.
CONCLUSIONElevated serum sPLA2 level is a risk factor for CHD.
Adult ; Aged ; Aged, 80 and over ; C-Reactive Protein ; metabolism ; Coronary Angiography ; Coronary Disease ; blood ; diagnostic imaging ; Female ; Group II Phospholipases A2 ; Humans ; Interleukin-8 ; blood ; Lysophospholipids ; blood ; Male ; Middle Aged ; Phospholipases A ; blood ; Phospholipases A2
10.Effects of rhubarb on the immune substances secreted from intestine in mice.
Nan XU ; Xiao-Li CHEN ; Ling-Jun LU ; Sang-Zhu CIREN
China Journal of Chinese Materia Medica 2005;30(18):1441-1443
OBJECTIVETo investigate the therapeutic mechanism of rhubarb in protecting the intestinal muco-membranous barrier in the mice.
METHODBal b/c mice were divided into 2 groups, gavaged with normal saline and 10% rhubarb decoction, respectively. The animals were killed after 24 hours after the treatments. The intestinal juice was collected after intestinal lavage and centrifuged for determination of IgA, total protein, C3, high density lipoprotein, type II PLA2 activity, and content of lysozyme. At the same time, 40 mg of small intestine were incised in each mouse. Reverse transcription polymerase chain reaction (RT-PCR) and gel image analysis were performed to detect the content of the cryptdin gene expression.
RESULTThe content of IgA, total protein, the C3, lysozyme, and the type II PLA2 activity in intestinal lavaged juice exhibited the statistical differences between the two groups (P < 0.05). There were no significant difference in the ontents of HDL, cryptdin-1 and cryptdin-4 gene expression between the two groups (P > 0.05).
CONCLUSIONRhubarb could increase secretion of several immune associated substances of the mucous membrane in normal intestine, indicating a possibility to abate the injury of intestine mucus resulted from severe stress induced by trauma, burn and shock. Through above mechanisms Rhubarb may also reduce the incidence of bacterial translocation and systemic inflammatory reaction syndrome (SIRS).
Animals ; Complement C3 ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Group II Phospholipases A2 ; Immunoglobulin A ; metabolism ; Intestinal Mucosa ; metabolism ; Intestinal Secretions ; metabolism ; Intestine, Small ; metabolism ; Mice ; Mice, Inbred BALB C ; Muramidase ; metabolism ; Phospholipases A ; metabolism ; Phospholipases A2 ; Plants, Medicinal ; chemistry ; Proteins ; metabolism ; Rheum ; chemistry