Lysophosphatidylcholines   中文名称: 溶血磷脂酰胆碱

ERK1/2; IL-1β

溶血磷脂酰胆碱（LPC）以浓度依赖的方式诱导HUVEC损伤。LPC诱导HUVEC中NO和ROS的过量产生，eNOS抑制剂（L-NAME）和抗氧化剂显著抑制了LPC诱导的HUVEC损伤（p<0.05）。 结论：这些发现表明，LPC诱导NO的过量产生，这可能会增加内皮细胞的氧化应激，导致内皮细胞损伤。[1]

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有证据表明，在氧化修饰的低密度脂蛋白、人血浆和动脉粥样硬化病变中存在溶血磷脂酰胆碱（lysoPC）。我们研究了lysoPC对人单核细胞产生细胞因子的影响。在所有测试的细胞因子（IL-8、TNF-α、MCP-1和IL-1β）中，我们发现lysoPC以剂量和时间依赖的方式最一致地刺激人单核细胞产生IL-1β。将粘附的单核细胞暴露于含有0.5%牛血清白蛋白的细胞培养基中的lysoPC。当暴露于12.5至75微摩尔的lysoPC时，IL-1β的细胞含量增加了2-4倍。在高达50微M的浓度下，没有观察到细胞毒性作用。超过50微摩尔，有证据表明存在毒性。IL-1β水平在24小时达到最高水平，然后下降。48小时后，细胞相关的IL-1β水平较低，但lysoPC刺激的细胞产生的IL-1β仍然是对照组的4.1倍。此外，lysoPC增强了IL-1βmRNA，与IL-1β蛋白水平平行。lysoPC的刺激作用取决于其链长。当酰基链短于16时，对IL-1β的产生没有影响。我们还发现，饱和的lysoPC 18:0比单不饱和lysoPC 18-1更能刺激IL-1β的产生。因此，氧化修饰的LDL中的lysoPC可能刺激巨噬细胞中IL-1β的产生，这可能有助于动脉粥样硬化组织的炎症反应。[2]

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溶血磷脂酰胆碱（lysoPC）是氧化低密度脂蛋白（LDL）的一种成分，参与动脉粥样硬化和炎症的发病机制。先前的研究表明，lysoPC可以诱导血管内皮细胞中的各种蛋白激酶，包括酪氨酸激酶、蛋白激酶C（PKC）和丝裂原活化蛋白激酶（MAPK）。然而，lysoPC活化激酶的作用仍未明确。在这项研究中，我们研究了lysoPC对细胞凋亡的影响，并研究了lysuPC激活的蛋白激酶在人脐静脉内皮细胞（HUVEC）中的作用。通过形态学标准、MTT法、显示特征性凋亡阶梯的DNA片段电泳、TUNEL分析评估凋亡的存在，并通过流式细胞术定量为亚二倍体细胞的比例。lysoPC以时间和剂量依赖的方式诱导细胞凋亡。它刺激HUVEC中细胞外信号调节激酶1/2（ERK1/2）和p38 MAPK的磷酸化。特定药物抑制剂的使用表明，p38 MAPK信号通路（SB203580）是lysoPC诱导的凋亡信号所必需的。此外，DEVD-FMK（一种caspas-3/CP32抑制剂）抑制了lysoPC诱导的细胞凋亡，表明凋亡中有一个重要片段参与其中。这些结果表明，lysoPC通过p38 MAPK依赖途径诱导人内皮细胞凋亡[3]。

败血症是重症监护室的主要死亡原因。在这里，我们表明，施用溶血磷脂酰胆碱（LPC），一种内源性溶血磷脂，可以保护小鼠在盲肠结扎和穿刺（CLP）或腹腔注射大肠杆菌后免于死亡。LPC的体内治疗显著提高了腹腔细菌的清除率，并阻断了CLP诱导的中性粒细胞失活。在体外，LPC通过增强摄入大肠杆菌的中性粒细胞中H（2）O（2）的产生，增加了中性粒细胞的杀菌活性，但没有增加巨噬细胞的杀菌活性。与LPC受体G2A抗体一起孵育，抑制了LPC诱导的CLP致死保护作用，并抑制了LPC在中性粒细胞中的作用。G2A特异性抗体还阻断了LPC对脂多糖（LPS）某些作用的抑制作用，包括致死性和中性粒细胞释放肿瘤坏死因子α（TNF-α）。这些结果表明，LPC可以有效预防和治疗败血症和微生物感染[4]。

中性粒细胞杀菌活性。[4]
中性粒细胞在37°C下在60 mm塑料培养皿中的13 mm塑料盖玻片上孵育1小时（每张盖玻片106个中性粒细胞；每张培养皿6-8个盖玻片），并去除非粘附细胞。然后将中性粒细胞与106个调理的大肠杆菌细胞一起孵育1小时。在冲洗掉未融合的大肠杆菌后，在用30μM 18:0溶血磷脂酰胆碱（LPC）或载体进一步孵育1 h前后，测定中性粒细胞中活菌的数量。杀死的细菌百分比计算为100×（LPC暴露后的1个CFU/LPC暴露前的CFU）46。收集上清液以测量H2O2的产生（图4c）。对于G2A特异性抗体的实验，在暴露于大肠杆菌1小时期间，以及随后暴露于LPC 1小时期间（在线补充方法），将血液中性粒细胞与G2A特异性抗体（1μg/ml）或正常山羊IgG（1μg/ml）一起孵育。

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吞噬细胞在体外释放细胞因子。[4]
在存在或不存在不同浓度的18:0溶血磷脂酰胆碱（LPC）的情况下，将血液中性粒细胞和腹腔巨噬细胞分别与LPS（100ng/ml）孵育3小时和6小时。在一些实验中，在向培养基中加入30μM 18:0 LPC之前，将血液中性粒细胞与G2A特异性抗体（1μg/ml）或正常山羊IgG（1μg/ml）预孵育30分钟。30分钟后向细胞中加入LPS（100ng/ml），并在加入LPS后3小时测量培养基中的TNF-α。

目的：确定溶血磷脂酰胆碱（LPC）/Lysophosphatidylcholine是否通过改变一氧化氮（NO）的产生从而增加活性氧（ROS）来诱导内皮细胞损伤。 方法：培养人脐静脉内皮细胞（HUVECs），并将其暴露于LPC、含N（G）-硝基-1-精氨酸甲酯（l-NAME）的LPC、含抗氧化剂的LPC中。使用LDH和刃天青测定LPC诱导的细胞损伤和存活率。Mann-Whitney U检验用于统计分析[1]。

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透射电子显微镜[3]
用75μM溶血磷脂酰胆碱/Lysophosphatidylcholine (lysoPC)处理HUVEC 24小时后，通过收集分离的细胞和贴壁细胞收获细胞，然后用于电子显微镜研究。在用磷酸缓冲盐水（PBS）中的200mM戊二醛固定之前，用PBS洗涤细胞。使用标准包埋和切片程序对细胞进行进一步处理以进行电子显微镜检查。

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TUNEL分析[3]
用75μM溶血磷脂酰胆碱/Lysophosphatidylcholine (lysoPC)处理HUVEC 24小时后，通过收集分离的细胞和贴壁细胞来收获细胞。根据制造商的方案，使用末端脱氧核糖核苷酸转移酶标记凋亡细胞。

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

105

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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