ERK1/2; IL-1β

Lysophosphatidylcholine (LPC) induced HUVEC injury in a concentration-dependent manner. LPC induced the overproduction of NO and ROS in HUVECs and LPC-induced HUVEC injury is significantly inhibited by the eNOS inhibitor (L-NAME) and the antioxidants (p < 0.05). Conclusions: These findings suggest that LPC induces the overproduction of NO, which may increase the oxidative stress on endothelial cells and lead to endothelial cell injury. [1]

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There is evidence for the presence of lysophosphatidylcholine (lysoPC) in oxidatively modified low density lipoprotein, human plasma and in atherosclerotic lesions. We studied the effect of lysoPC on the cytokine production by human monocytes. Among all the cytokines tested (IL-8, TNF alpha, MCP-1 and IL-1beta), we found that lysoPC most consistently stimulated human monocytes to produce IL-1beta in a dose and time dependent manner. Adherent monocytes were exposed to lysoPC in cell culture medium containing 0.5% bovine serum albumin. When exposed to lysoPC from 12.5 to 75 microM, the cellular content of IL-1beta increased 2-4 fold. Up to a concentration of 50 microM no cytotoxic effect could be seen. Over 50 microM there was evidence of toxicity. The level of IL-1beta reached its highest level at 24 h and then declined. At 48 h, the cell associated IL-1beta was low, but still the lysoPC stimulated cells produced 4.1 times more IL-1beta than controls. Also the IL-1beta mRNA was augmented by lysoPC in parallel with the IL-1beta protein levels. The stimulatory effect of lysoPC was dependent on its chain length. There was no effect on IL-1beta production when the acyl chain was shorter than 16. We also found that saturated lysoPC 18:0 stimulated IL-1beta production more than the monounsaturated lysoPC 18:1. Thus, the lysoPC in oxidatively modified LDL may stimulate the production of IL-1beta in macrophages, which may contribute to the inflammatory response in atherosclerotic tissue. [2]

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Lysophosphatidylcholine (lysoPC) is a component of oxidized low density lipoprotein (LDL) and is involved in the pathogenesis of atherosclerosis and inflammation. Previous studies demonstrated that lysoPC can induce various protein kinases including tyrosine kinases, protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in vascular endothelial cells. However, the role of lysoPC-activated kinases remains undefined. In this study, we examined the effect of lysoPC on apoptosis and investigated the role of lysoPC-activated protein kinases in human umbilical vein endothelial cells (HUVEC). The presence of apoptosis was evaluated by morphological criteria, MTT assay, and electrophoresis of DNA fragments showing the characteristic apoptotic ladder, TUNEL analysis, and quantified as the proportion of hypodiploid cells by flow cytometry. The lysoPC induced apoptosis in a time- and dose-dependent manner. It stimulated the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and p38-MAPK in HUVEC. The use of specific pharmacologic inhibitors indicated that the p38-MAPK-signaling pathway (SB203580) is required for lysoPC-induced apoptotic signals. Furthermore, lysoPC-induced apoptosis was inhibited by DEVD-FMK (a caspas-3/CPP32 inhibitor), suggesting involvement of an important segment in the apoptosis. These results demonstrate that lysoPC induces apoptosis in human endothelial cells through a p38-MAPK-dependent pathway [3].

Sepsis represents a major cause of death in intensive care units. Here we show that administration of lysophosphatidylcholine (LPC), an endogenous lysophospholipid, protected mice against lethality after cecal ligation and puncture (CLP) or intraperitoneal injection of Escherichia coli. In vivo treatment with LPC markedly enhanced clearance of intraperitoneal bacteria and blocked CLP-induced deactivation of neutrophils. In vitro, LPC increased bactericidal activity of neutrophils, but not macrophages, by enhancing H(2)O(2) production in neutrophils that ingested E. coli. Incubation with an antibody to the LPC receptor, G2A, inhibited LPC-induced protection from CLP lethality and inhibited the effects of LPC in neutrophils. G2A-specific antibody also blocked the inhibitory effects of LPC on certain actions of lipopolysaccharides (LPS), including lethality and the release of tumor necrosis factor-alpha (TNF-alpha) from neutrophils. These results suggest that LPC can effectively prevent and treat sepsis and microbial infections [4].

Neutrophil bactericidal activity. [4]
Neutrophils were incubated at 37 °C on 13-mm plastic cover slips in 60-mm plastic culture dishes (106 neutrophils per cover slip; 6–8 cover slips per dish) for 1 h, and nonadherent cells were removed. Neutrophils were then incubated with 106 opsonized E. coli cells for 1 h. After washing out unengulfed E. coli, the number of viable bacteria in neutrophils was determined before and after further incubation with 30 μM 18:0 Lysophosphatidylcholine (LPC) or vehicle for 1 h. The percentage of bacteria killed was calculated as 100 × (1 – CFUs after LPC exposure/CFUs before LPC exposure)46. The supernatants were collected to measure H2O2 production (Fig. 4c). For experiments with G2A-specific antibody, blood neutrophils were incubated with either G2A- specific antibody (1 μg/ml) or normal goat IgG (1 μg/ml) during exposure to E. coli for 1 h, and during the subsequent exposure to LPC for 1 h (Supplementary Methods online).

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Cytokine release from phagocytes in vitro. [4]
Blood neutrophils and peritoneal macrophages were incubated with LPS (100 ng/ml) for 3 h and 6 h, respectively, in the presence or absence of various concentrations of 18:0 Lysophosphatidylcholine (LPC). In some experiments, blood neutrophils were preincubated with either G2A-specific antibody (1 μg/ml) or normal goat IgG (1 μg/ml) for 30 min before the addition of 30 μM 18:0 LPC to the medium. LPS (100 ng/ml) was added to the cells 30 min later, and TNF-α in the medium was measured 3 h after the addition of LPS.

Objective: To determine whether Lysophosphatidylcholine (LPC) induces endothelial cell injury by altering the production of nitric oxide (NO) and thereby increasing reactive oxygen species (ROS). Methods: Human umbilical vein endothelial cells (HUVECs) were cultured and exposed to LPC, LPC with N(G)-nitro-l-arginine methyl ester (L-NAME), LPC with antioxidants. LPC-induced cell injury and viability were determined using LDH and Resazurin assays. The Mann-Whitney U test was used for statistical analysis[1].

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Transmission electron microscopy [3]
After HUVEC were treated for 24 h with 75 μM Lysophosphatidylcholine (lysoPC), cells were harvested by collecting both the detached cells and the adherent cells, and then used for electron microscopy studies. Cells were washed with PBS before fixation in 200 mM glutaraldehyde in phosphate-buffered saline (PBS). Cells were further processed for electron microscopy examination using standard embedding and sectioning procedures.

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TUNEL analysis [3]
After HUVEC were treated for 24 h with 75 μM Lysophosphatidylcholine (lysoPC), cells were harvested by collecting both the detached cells and the adherent cells. The apoptotic cells were labeled using terminal deoxyribonucleotide transferase according to the manufacturer's protocol.

Measurement of cytokine and Lysophosphatidylcholine (LPC) levels.[4]
For measurement of CLP-induced cytokines in peritoneal lavage fluids, mice were given 18:0 Lysophosphatidylcholine (LPC) at 2 h, 16 h, 28 h and 40 h after CLP. Peritoneal lavage fluid (∼2 ml recovered from each mouse) was collected at various times between 4 h and 72 h after CLP. For measurement of LPS-induced plasma cytokines, mice were given 18:0 LPC 30 min after injection of LPS, and plasma was collected 1 h (for TNF-α and IL-1β) or 5.5 h (for IFN-γ) later. Concentrations of cytokines were measured with an enzyme-linked immunoassay kit. Plasma LPC concentrations were assayed as described previously44, based on the standard curve for 18:0 LPC.

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Measurement of H2O2.[4]
Neutrophils isolated from CLP mice were stimulated with PMA (100 ng/ml) for 1 h (Fig. 3b). Blood neutrophils and peritoneal macrophages in fresh phenol red–free RPMI 1640 (supplemented with 5% FBS) were incubated with various Lysophosphatidylcholine (LPC)s at a concentration of 30 μM for 2 h (Fig. 4e). In some experiments, blood neutrophils were preincubated with either G2A-specific antibody (1 μg/ml) or normal goat IgG (1 μg/ml) for 0.5 or 1 h. 18:0 LPC was then added to the medium at a final concentration of 30 μM, and H2O2 production was assayed 2 h after the addition of LPC. H2O2 was measured in the supernatants with an H2O2 assay kit (Oxis International). The G2A-specific antibody (M-20) and normal goat IgG were dialyzed overnight in PBS before use.

[1]. Lysophosphatidylcholine induces endothelial cell injury by nitric oxide production through oxidative stress . The Journal of Maternal-Fetal & Neonatal Medicine, 2009, 22(4): 325-331.

[2]. Lysophosphatidylcholine induces the production of IL-1β by human monocytes . Atherosclerosis, 1998, 137(2): 351-357.

[3]. Lysophosphatidylcholine induces apoptosis in human endothelial cells through a p38-mitogen-activated protein kinase-dependent mechanism . Atherosclerosis, 2002, 161(2): 387-394.

[4]. Therapeutic effects of lysophosphatidylcholine in experimental sepsis . Nature medicine, 2004, 10(2): 161-167.

1-acetyl-sn-glycero-3-phosphocholine is a 1-O-acyl-sn-glycero-3-phosphocholine.
Lysophosphatidylcholine has been reported in Ferula tenuisecta, Urtica dioica, and other organisms with data available.
Derivatives of PHOSPHATIDYLCHOLINES obtained by their partial hydrolysis which removes one of the fatty acid moieties.
See also: Lysophosphatidylcholine, soybean (annotation moved to).

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The involvement of MAPK, especially JNK and p38-MAPK, in apoptosis has been demonstrated; however, the roles of JNK and p38-MAPK in apoptosis are controversial. For example, TNFα-induced apoptosis is dependent on JNK activity in the monocytic cell line U937 but not in fibroblasts, suggesting that consequences of JNK activation vary considerably among cell types. In endothelial cells, Yue et al. recently reported that TL1, a novel TNFα-like cytokine, induces apoptosis through both JNK and p38-MAPK pathways. Consistent with their findings, we found that lysoPC phosphorylates p38-MAPK and a selective p38-MAPK inhibitor SB203580 resulted in significant inhibition of lysoPC-induced apoptosis. Taken together, the p38-MAPK pathway may play a substantial role in endothelial apoptosis induced by certain stimuli. Interestingly, the PKC inhibitor calphostin C and GF109203X showed no effect on lysoPC-induced apoptosis whereas PKC down-regulation by PDBu enhanced it, suggesting the involvement of phorbol ester-sensitive PKC isoforms in the apoptosis. Finally, a caspase inhibitor DEVD-FMK significantly inhibited lysoPC-induced apoptosis, suggesting the importance of the caspase family of protease in the apoptosis. In addition, DEVD-FMK had no effect on p38-MAPK phosphorylation, suggesting that p38MAPK may not act as a downstream molecule for the caspase 3/CPP32 in this apoptotic pathway. Further investigations are required for an understanding of the precise intracellular signaling mechanism of lysoPC-induced apoptosis in endothelial cells.
In summary, we report that lysoPC induces apoptosis in endothelial cells through a p38-MAPK-dependent pathway. Since apoptosis of endothelial cells may be associated with the progression of atherosclerosis, our findings suggest another possible mechanism for the atherogenic effects of lysoPC. An understanding of the mechanisms involved in endothelial apoptosis may help to provide new strategies for modifying the pathophysiology of atherosclerosis. [3]

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It was recently reported that plasma LPC is significantly decreased in septic patients, and that patients who die of sepsis have significantly lower plasma LPC than patients who survive a septic episode. These clinical findings support our hypothesis that supplementation with LPC may be beneficial for patients with sepsis. LPC is one of the metabolites derived from the oxidation of low-density lipoprotein, and these metabolites are thought to be involved in the pathogenesis of atherosclerosis. However, the beneficial effects of treating sepsis with LPC in the short term (possibly within a week) could far exceed the potential atherogenic effects of this lipid, as LPC could prevent the devastating consequences of sepsis. Appropriate caution should be used in patients with cardiac ischemia, however, because 16:0 LPC may cause electrophysiological alterations in ischemic myocardium. At the doses used in this study, LPC did not induce any apparent toxic effects in mice (data not shown). In addition to sepsis, the enhancing effect of LPC on neutrophil bactericidal activity should be useful in cases of microbial infections that have not yet progressed to sepsis. This new approach for combating microbial infections would be complementary to the approach of directly attacking microbial pathogens with antimicrobial agents. Such an approach could be important, considering the continuous appearance of new pathogenic microbes that are resistant to the currently available antimicrobial agents. In conclusion, we have identified a new therapeutic application of LPC for use in sepsis and microbial infections. These findings suggest that a clinical evaluation of these effects of LPC will be useful. [4]

C24H50NO7P

495.6301

299.113

9008-30-4

5311264

White to off-white solid powder

-1.8

1

7

10

19

320

1

CC(=O)OC[C@H](COP(=O)([O-])OCC[N+](C)(C)C)O

RYCNUMLMNKHWPZ-SNVBAGLBSA-N

InChI=1S/C10H22NO7P/c1-9(12)16-7-10(13)8-18-19(14,15)17-6-5-11(2,3)4/h10,13H,5-8H2,1-4H3/t10-/m1/s1

[(2R)-3-acetyloxy-2-hydroxypropyl] 2-(trimethylazaniumyl)ethyl phosphate

Lysophosphatidylcholine; Lysophosphatidylcholines; 9008-30-4; [(2R)-3-acetyloxy-2-hydroxypropyl] 2-(trimethylazaniumyl)ethyl phosphate; 1-acetyl-sn-glycero-3-phosphocholine; UNII-CQD833204Z; Lysophosphatidylcholine, soybean; CQD833204Z;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

MEthanol : ~25 mg/mL
H2O : < 0.1 mg/mL
DMSO :< 1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)