内源性过氧亚硝基阴离子介导脂多糖致肺动脉内皮细胞损伤

谷振勇1,2,*, 凌亦凌2, 徐小虎1, 朱铁年2, 丛斌2

1汕头大学医学院, 汕头515031;2河北医科大学病理生理教研室, 石家庄 050017

摘要: 在培养的牛肺动脉内皮细胞(BPAEC)水平上, 观察脂多糖(LPS)对BPAEC诱生过氧亚硝基阴离子(peroxynitrite, ONOO-)能力及内皮源性ONOO-在LPS致BPAEC损伤中的作用。结果显示, (1) LPS剂量依赖性地引起BPAEC诱生ONOO-生成标志物硝基酪氨酸(nitrotyrosine, NT)的荧光强度(即ONOO-)明显增多, NT阳性细胞数和百分率也明显增多或增高(P<0.05); iNOS选择性抑制剂氨基胍(AG)明显抑制LPS诱生ONOO-增多(P<0.05), 而NT阳性细胞数和百分率分别减少或降低, 但无明显差异。(2)在LPS作用下BPAEC培养上清中的MDA含量和LDH活性明显增多和增高, 呈现剂量依赖性效应。加AG后MDA含量明显降低(P<0.001)、 LDH活性呈降低趋势。(3) LPS可诱导BPAEC凋亡明显增多, 用EB荧光染色后可见细胞染色质浓集、 核变小等凋亡征象。AG可致LPS引起的BPAEC凋亡明显减少, 但仍明显高于溶剂组。LPS可导致BPAEC线粒体呼吸抑制及膜电位下降。上述结果表明, LPS可引起BPAEC生成ONOO-增多, ONOO-参与介导LPS所致BPAEC过氧化损伤与细胞凋亡。

关键词: 过氧亚硝基阴离子, 脂多糖, 肺动脉, 内皮细胞

中图分类号: Q463; R363.2

Endogenous peroxynitrite mediates lipopolysaccharide-induced injury of cultured pulmonary artery endothelial cells

GU Zhen-Yong1,2,*, LING Yi-Ling2, XU Xiao-Hu1, ZHU Tie-Nian2, CONG Bin2

1Shantou University Medical College, Shantou 515031;

2Department of Pathophysiology, Hebei Medical University, Shijiazhuang 050017

Abstract: This study, using cultured bovine pulmonary artery endothelial cells (BPAEC), was undertaken to investigate the roles of endogenous ONOO- in LPS-caused injury to endothelial cells. The fluorescent intensity of nitrotyrosine (NT), a specific marker of ONOO- generation, in BPAEC represented the content of endogenous ONOO- generation. The fluorescent intensity of NT and the number of NT positive cells were detected with flow cytometry (FCM), the percentage of NT positive cells was calculated. (1) LPS (1, 5 and 10 μg/ml) caused a marked increase in fluorescent intensity of NT in a dose-dependent manner, which was significantly increased compared to vehicle group (P<0.01).The number and percentage of NT positive cells were markedly increased (both P<0.05 vs vehicle group). Aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase (iNOS), inhibited LPS- induced increase in fluorescent intensity of NT in BPAEC. However, the number and percentage of NT positive cells had a tendency to reduce. (2) LPS caused the enhancement of MDA content and activity of LDH in cultured supernatant. AG reversed the enhancement of MDA content induced by LPS (P<0.01). In contrast, AG had marginal effect on the activity of LDH. (3) LPS induced an increase in apoptotic rate in BPAEC in a dose-dependent manner.The number of apoptotic cells markedly increased as well. Some BPAEC stained with fluorescent probe ethidium bromide showed morphological features of apoptosis with chromatin condensation and nuclear fragmentation. AG reduced the apoptotic rate and the number of apoptotic cells, both of which were still higher than those of vehicle group (P<0.05). LPS caused inhibition of mitochondrial respiration and membrane potential in an accumulation manner. In conclusion, LPS caused injury to cultured BPAEC and increased the production of ONOO-.The cytotoxicity of LPS may be mediated by the endogenous ONOO- .

Key words: peroxynitrite; lipopolysaccharide; pulmonary artery; endothelial cells