Brooks A C, Menzies-Gow N J, Wheeler-Jones C, Bailey S R, Cunningham F M, Elliott J
Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hertfordshire, AL9 7TA, UK.
Inflamm Res. 2007 Apr;56(4):154-61. doi: 10.1007/s00011-006-6151-6.
The aim of this study was to determine the effects of endotoxin on p38 MAPK activation in equine platelets and leukocytes in vivo and in vitro and its role in thromboxane (Tx) production with reference to equine endotoxaemia.
Six adult Thoroughbred horses were used for in vivo infusion studies and separate in vitro studies. For in vivo studies, following collection of a pre-infusion sample, horses were infused with E. Coli O55:B5 LPS (30 ng/kg; 30 min) during and after which platelets were harvested. For in vitro studies isolated platelets and leukocytes were exposed to LPS (10 pg/ml-1 microg/ml). p38 MAPK activity was assessed by SDS-PAGE followed by immunoblotting. TxA2 release was measured by radioimmunoassay.
LPS infusion caused increased phospho-p38 MAPK in equine platelets and leukocytes (1492 +/- 486 % and 83 +/- 45 above basal, respectively) from 10 min after the start of the infusion, which returned to basal by 60 min. In vitro, platelets were 1,000 times more sensitive to LPS than leukocytes in terms of both TxA2 production (EC50 66 pg/ml versus 110 ng/ml, respectively) and p38 MAPK phosphorylation (EC50 11.1 +/- 2 pg/ml versus 14.8 +/- 4 ng/ml, respectively). p38 MAPK inhibitors SB203580 and PD169316 attenuated LPS-induced TxA2 release in platelets, but not leukocytes.
In vivo, LPS stimulates TxA2 production and p38 MAPK phosphorylation in equine platelets and leukocytes at a concentration within a similar range to those reported in clinical endotoxaemia. These data suggest that LPS-induced eicosanoid production in the early phase of clinical endotoxaemia may involve direct effects of LPS upon platelets, mediated via activation of p38 MAPK.
本研究旨在确定内毒素对马体内外血小板和白细胞中p38丝裂原活化蛋白激酶（MAPK）激活的影响，以及其在马内毒素血症中血栓素（Tx）产生中的作用。
六匹成年纯种马用于体内输注研究和单独的体外研究。对于体内研究，在采集输注前样本后，给马输注大肠杆菌O55:B5脂多糖（LPS，30 ng/kg；30分钟），在此期间及之后采集血小板。对于体外研究，将分离的血小板和白细胞暴露于LPS（10 pg/ml - 1 μg/ml）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）随后进行免疫印迹评估p38 MAPK活性。通过放射免疫测定法测量血栓素A2（TxA2）释放。
LPS输注导致马血小板和白细胞中磷酸化p38 MAPK增加（分别比基础水平高1492±486%和83±45%），从输注开始后10分钟开始，到60分钟时恢复到基础水平。在体外，就TxA2产生（EC50分别为66 pg/ml和110 ng/ml）和p38 MAPK磷酸化（EC50分别为11.1±2 pg/ml和14.8±4 ng/ml）而言，血小板对LPS的敏感性比白细胞高1000倍。p38 MAPK抑制剂SB203580和PD169316减弱了LPS诱导的血小板中TxA2释放，但对白细胞没有作用。
在体内，LPS在与临床内毒素血症中报道的浓度相似的范围内刺激马血小板和白细胞中TxA2产生和p38 MAPK磷酸化。这些数据表明，临床内毒素血症早期LPS诱导的类花生酸产生可能涉及LPS对血小板的直接作用，通过p38 MAPK的激活介导。
