Hammond R A, Hannon R, Frean S P, Armstrong S J, Flower R J, Bryant C E
Department of Biochemical Pharmacology, St Bartholomew's and the Royal London School of Medicine and Dentistry, United Kingdom.
Am J Vet Res. 1999 Apr;60(4):426-31.
To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses.
Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively.
Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent. Conclusion-Lipopolysaccharide induces COX-2 and iNOS in equine macrophages.
The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses.
测定体外培养的马肺泡巨噬细胞在脂多糖（LPS）刺激下诱导产生的环氧化酶-2（COX-2）和诱导型一氧化氮合酶（iNOS）的量。样本群体——从12匹马获取的肺泡巨噬细胞。
通过支气管肺泡灌洗从12匹马收集肺泡巨噬细胞，并用LPS（0.001至10微克/毫升）或赋形剂孵育6小时。提取并纯化总RNA。在合成第一链cDNA后，使用聚合酶链反应（PCR）技术检测COX-2、iNOS和甘油醛-3-磷酸脱氢酶的mRNA诱导情况。在第二项研究中，细胞用LPS或赋形剂孵育24小时。分别通过测定前列腺素E2（PGE2）和总亚硝酸盐浓度来检测培养基中的COX-2和iNOS活性。
脂多糖可诱导马肺泡巨噬细胞产生COX-2和iNOS mRNA。测序显示，COX-2和iNOS的PCR产物与人类序列具有高度的核苷酸同源性（COX-2为91%，iNOS为93%）。COX-2和iNOS mRNA的产生伴随着酶活性的诱导。比较PCR片段产生情况，iNOS mRNA的表达似乎低于COX-2。COX-2的诱导呈LPS浓度依赖性，而iNOS则不然。结论——脂多糖可诱导马巨噬细胞产生COX-2和iNOS。
脂多糖在马巨噬细胞中诱导iNOS和COX-2表明这些酶可能在败血症的病理生理学中起重要作用。对iNOS和COX-2活性进行药理调节可能是治疗马内毒素血症的新靶点。
