Biliary phosphatidylcholine and lysophosphatidylcholine profiles in sclerosing cholangitis

AIM:To analyze phospholipid profiles in intrahepatic bile from patients with primary sclerosing cholangitis(PSC)and secondary sclerosing cholangitis(SSC).METHODS:Intrahepatic bile specimens collected via endoscopic retrograde cholangiography from 41 patients were analyzed.Fourteen of these patients were diagnosed with PSC,10 with SSC,11 with choledocholithiasis or no identifiable biliary disease,and 6 with cholangiocellular carcinoma(CCC).Bile acid,cholesterol,protein,and bilirubin contents as well as pancreas lipase activity in bile were determined by biochemical methods.Phosphatidylcholine(PC)and lysophosphatidylcholine(LPC)species were quantified using nanoelectrospray ionization tandem mass spectrometry.RESULTS:Bile from all the examined patient groups showed a remarkably similar PC and LPC species composition,with only minor statistical differences.Total biliary PC concentrations were highest in controls(8030±1843 mol/L)and lowest in patients with CCC(1969±981 mol/L)(P=0.005,controls vs SSC and CCC,respectively,P<0.05).LPC contents in bile were overall low(4.2%±1.8%).Biliary LPC/PC ratios and ratios of biliary PC to bilirubin,PC to cholesterol,PC to protein,and PC to bile acids showed no intergroup differences.CONCLUSION:PC and LPC profiles being similar in patients with or without sclerosing cholangitis,these phospholipids are likely not of major pathogenetic importance in this disease group.     

葛根素对溶血磷脂酰胆碱诱导血管平滑肌细胞基质金属蛋白酶-9表达的影响

目的：观察葛根素对血管平滑肌细胞基质金属蛋白酶-9（MMP-9）表达的影响,探讨葛根素抗动脉粥样硬化（AS）的作用机制。方法：用不同浓度（1.0～10.0mg/L）的溶血磷脂酰胆碱（LPC）孵育体外培养的大鼠胸主动脉平滑肌细胞24h,观察培养上清液中MMP-9含量的变化。不同浓度（10^-6～10^-4mol/L）的葛根素预先孵育平滑肌细胞4h,然后加入LPC（5.0mg/L）作用24h,用酶联免疫吸附法（ELISA）检测上清液中MMP-9含量,实时荧光定量PCR（real-time PCR）检测平滑肌细胞MMP-9 mRNA的表达,蛋白印迹法（Western blot）检测NF-κB p65蛋白的表达。结果：LPC能明显增加上清液中MMP-9的含量,而预先应用不同浓度的葛根素干预后,MMP-9含量、mRNA表达以及NF-κB p65蛋白的表达水平均明显降低,并且具有剂量依赖性。结论：LPC通过激活NF-κB p65而使平滑肌细胞MMP-9表达增强,而葛根素对上述效应具有抑制作用。     

Cytosolic phospholipase A2 as a molecular target for the radiosensitization of ovarian cancer

In ovarian cancer, the molecular targeted chemotherapeutics could increase the efficiency of low-dose radiotherapy while decreasing injury to adjusted organs. In irradiated A2780 human ovarian carcinoma cells, cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF(3) prevented activation of pro-survival Akt signaling and enhanced cell death. The potential molecular mechanisms of this effect could involve signaling through lysophosphatidic acid receptors. In the heterotopic A2780 tumor model using nude mice, cPLA(2) inhibition significantly delayed tumor growth compared to treatment with radiation or vehicle alone. These results identify cPLA(2) as a molecular target to enhance the therapeutic ratio of radiation in ovarian cancer.     

Intracellular Ca- and PKC-dependent upregulation of T-type Ca channels in LPC-stimulated cardiomyocytes

Lysophosphatidylcholine (LPC) accumulation in intracellular and/or interstitial space in cardiomyocytes may underlie as a mechanism for tachycardia and various arrhythmias during cardiac ischemia, which is usually accompanied by elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The present study was therefore designed to investigate possible mechanisms responsible for [Ca²⁺]_i elevation by LPC focusing on T-type Ca²⁺ channel current (I_Ca.T). LPC as well as phorbol 12-myristate 13-acetate (PMA) significantly accelerated the beating rates of neonatal rat cardiomyocytes. Augmentation of I_Ca.T by LPC was dependent on the intracellular Ca²⁺ concentration: an increase of I_Ca.T was significantly larger in high [Ca²⁺]_i condition (pCa = 7) than those in low [Ca²⁺]_i condition (pCa = 11). In heterologous expression system by use of human cardiac Ca_V3.1 and Ca_V3.2 channels expressed in HEK293 cells, LPC augmented Ca_V3.2 channel current (I_Cav3.2) in a concentration-dependent manner but not Ca_V3.1 channel current (I_Cav3.1). Augmentation of I_Cav3.2 by LPC was highly [Ca²⁺]_i dependent: I_Cav3.2 was unchanged when pCa was 11 but was markedly increased when [Ca²⁺]_i was higher than 10⁻¹⁰ M (pCa ≤ 10) by LPC application (10-50 μM). A specific inhibitor of protein kinase Cα (Ro-32-0432) attenuated the increase of I_Cav3.2 by LPC. LPC stimulates I_Ca.T in a [Ca²⁺]_i-dependent manner via PKCα activation, which may play a role in triggering arrhythmias in pathophysiological conditions of the heart.     

An enzymatic assay for lysophosphatidylcholine concentration in human serum and plasma

OBJECTIVES: Several methods for measuring lysophosphatidylcholine (LPC) concentrations have been reported. However, these methods are not practical because they are either too complicated and/or too time-consuming for LPC determinations in human serum and plasma. DESIGN AND METHODS: We have developed a new enzymatic LPC assay, which uses lysophospholipase, glycerophosphorylcholine phosphodiesterase and choline oxidase, and which determines the quantities of hydrogen peroxide generated in the presence of peroxidase using an oxidative chromogenic reagent and 4-aminoantipyrine. RESULTS: Various samples were mixed with LPC assay reagents, and their changes in absorbance were measured. The present method produced a linear calibration line between LPC concentration and absorbance change. It also measured only LPC, and not other phospholipids such as phosphatidylcholine, sphingomyelin and lysophosphatidic acid. The within-run and between-run coefficients of variation were 0.3-0.7% and 0.7%, respectively. The recovery of exogenous LPC added to control serum was 99.5-102.1%. The correlation coefficient obtained in a comparison with a method for analyzing fatty acids was 0.9122. CONCLUSIONS: The present method is simple, specific for LPC, and can be applied with an automatic analyzer. It may also be useful for further studies of the biological functions of LPC as well as clinical applications in various disorders.     

The effect of phospholipase A2 on bacterial translocation in a cell culture model

 The activity of phospholipase (PL)A₂ is elevated in the intestinal epithelia of patients with inflammatory bowel disease (IBD). Recently, we reported that lysophosphatidylcholine (L-PC), the PLA₂ hydrolysis product of phosphatidylcholine (PC), stimulates bacterial translocation (BT) in an enterocyte cell-culture model. These two observations stimulated us to examine the effects of extracellular PLA₂ on intestinal epithelial permeability. Human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell-culture system. Monolayer integrity and tight-junction permeability were measured by dextran blue (DB) permeability and transepithelial electric resistance (TEER). Monolayers were treated with PC, L-PC, or PLA₂ with and without PC. The magnitude of BT was determined 2 h after treatment by adding Escherichia coli to the apical chamber followed by quantitatively culturing basal chamber samples. Thin-layer chromatography (TLC) was utilized to verify PLA₂ hydrolysis of PC to L-PC. Statistical analysis was performed by one-way analysis of variance. The magnitude of BT across monolayers pretreated with PLA₂ + PC significantly increased compared to either PC or PLA₂ (6.83 ± 0.069, 2.41 ± 0.46, and 3.06 ± 1.14 log10 colony forming units/ml, respectively, P < 0.05). Absence of DB-permeability in any group confirmed monolayer integrity. TLC of PL samples harvested from the apical monolayer surface confirmed PC hydrolysis. PLA₂ mediates hydrolysis of PC to L-PC when both are applied to the apical surface of cultured enterocyte monolayers, resulting in increased BT and increased TEER with no damage to monolayer integrity. These observations may have implications in the pathogenesis and treatment strategies for IBD.     

Lysophosphatidylcholine regulates human microvascular endothelial cell expression of chemokines

The role of lysophosphatidylcholine (LPC) in the induction of MCP-1, IL-8 and RANTES, which are chemotactic factors to monocytes, neutrophils and lymphocytes, respectively, by human vascular endothelial cells (EC), was examined. LPC induced the expression of MCP-1 and IL-8 in a concentration- and time-dependent manner in microvascular EC (MVEC) and in large vessel EC from aorta, pulmonary artery and umbilical vein. LPC also induced RANTES in MVEC but not in large vessel EC. Signaling pathways responsible for LPC induction of chemokines were examined in MVEC. LPC and TNFalpha, a cytokine secreted in sites of inflammation, additively stimulated RANTES expression. LPC did not augment TNFalpha induction of MCP-1 or IL-8. A platelet-activating factor receptor antagonist (BN52021) failed to block LPC induction of MVEC chemokines, but the G(i)-protein inhibitor pertussis toxin partially blocked LPC induction of RANTES and IL-8. LPC activated multiple kinases in MVEC; it increased the phosphorylation of ERK1/2, AKT and p38 MAP kinase in a time-dependent manner. An inhibitor of the MAPK/ERK pathway, PD98059, blocked the phosphorylation of ERK1/2 and RANTES induction by LPC, but augmented IL-8 induction. LY294002, a specific inhibitor of phosphoinositide 3 kinase (PI3 kinase), blunted the phosphorylation of AKT and inhibited LPC induction of RANTES more strongly than IL-8. Inhibition of p38 MAP kinase pathway by SB202190 also blocked LPC-induced expression of IL-8 and RANTES. Our results suggest that LPC induction of chemokines in MVEC is distinct from that in large vessel EC, and required the activities of MAP kinases and PI3 kinase for the induction of RANTES and IL-8. We speculate that the presence of LPC, a bioactive lipid product of phospholipase A(2) (PLA(2)) and a constituent of oxidized low-density lipoprotein, can differentially influence the chemotaxis of particular leukocyte subpopulations during inflammation.     

串联质谱法在过氧化物酶体病筛查中的应用

目的：探讨串联质谱法检测极长链酰基肉碱（VLCAC）和溶血磷脂酰胆碱（LPC）在过氧化物酶体病筛查中的价值。方法：选取2017年1月至2021年3月以发育迟缓等神经系统异常就诊于上海市儿童医院，根据临床症状、磁共振成像和基因检测结果明确诊断为X-连锁肾上腺脑白质营养不良（X-ALD）患儿14例和脑肝肾综合征（ZS）患儿4例。另选取同年龄段体检儿童200名为健康对照组。使用含稳定同位素内标的溶剂萃取所有对象干血斑标本中的VLCAC和LPC，直接采用串联质谱法检测二十碳酰基肉碱（C20）、二十二碳酰基肉碱（C22）、二十四碳酰基肉碱（C24）、二十六碳酰基肉碱（C26）、二十碳溶血磷脂酰胆碱（C20:0-LPC）、二十二碳溶血磷脂酰胆碱（C22:0-LPC）、二十四碳溶血磷脂酰胆碱（C24:0-LPC）和二十六碳溶血磷脂酰胆碱（C26:0-LPC）水平，并计算C24/C20、C24/C22、C26/C20、C26/C22、C24:0-LPC/C20:0-LPC、C24:0-LPC/C22:0-LPC、C26:0-LPC/C20:0-LPC、C26:0-LPC/C22:0-LPC比值。采用Kruskal-Wallis H检验和Mann-Whitney U检验比较各组间VLCAC和LPC各指标检测值及比值，采用偏最小二乘法和变量投影重要度权重评分分析各指标对判断疾病的贡献度。 结果：除C24:0-LPC/C20:0-LPC外，所有指标和比值在各组间差异均有统计学意义（ P<0.05或 P<0.01）；X-ALD组与健康对照组、ZS组与健康对照组间，各指标有不同程度的差异，但X-ALD组与ZS组间差异无统计学意义（ P>0.05）；偏最小二乘法分析显示X-ALD和ZS组与健康对照组能够完全分离，C26的变量投影重要度值最大。 结论：串联质谱法检测VLCAC和LPC可作为过氧化物酶体病筛查的方法，其中C26或可作为诊断敏感指标。