摘要
目的利用原代培养的大鼠肝细胞，观察谷氨酰胺（Gln）在体外对白细胞介素-1β（IL-1β）刺激下一氧化氮合酶（iNOS）过度表达的影响。方法原位胶原酶灌注法获取原代大鼠肝细胞，分别用生理盐水、IL-1β（1nmol／L）以及IL-1β（1nmol／L）联合不同浓度的Gin（2、5、10mmol／L）刺激24h，留取细胞和培养液，采用生化法测定培养液-氧化氮（NO）的水平，采用实时定量逆转录多聚酶链反应（RT—PCR）法及蛋白质印迹法检测肝细胞内iNOS信使RNA和蛋白质水平，采用电泳迁移率转变试验检测肝细胞核内核因子KB（NF—KB）的活性。结果IL-1β刺激后培养液NO的平均浓度为72．7μmol／L明显高于生理盐水组41．7μmol／L，肝细胞iNOS蛋白和mRNA水平分别增加了35％和90％，同时给予Gln能够抑制iNOS的过度活化减少NO的产生，并且这种抑制作用随着Gln浓度的增加而增强；IL-1β刺激后肝细胞核内NF—KB的结合活性增强约50％，Gin对NF．KB的活化没有抑制作用，在5、10mmol／L浓度时反而对NF—KB有进-步的激活。结论谷氨酰胺能够下调肝细胞在炎症应激诱导下iNOS基因的过度表达，减少NO的产生，这-作用不是通过NF—KB信号通路实现的。
Objective To investigate the influences of glutamine （Gin） on nitric oxide （NO） production and inducible nitric oxide synthase （iNOS） expression in hepatocytes activated by interleukin （IL）-1β, a pro-inflammatory eytokine. Methods Primary hepatocytes were isolated from SD rats by in situ collagenase perfusion, and cultured with normal saline （NS）, IL-1β（ 1 nmol/L） alone, IL-1β（ 1 nmol/L） with Gin of 2, 5, or 10 mmol/L for 24 hours. The concentration of NO in the supernatant was detected by biochemical method. Western blotting and real-time quantitative reverse transcription PCR （RT-PCR） were used to detect the protein and mRNA expression of iNOS in the hepatocytes. The binding activity nuclear factor kappa B （NF-KB） in hepatocytes cellular localization of nuclear factor kappa B （NF-KB） in the nuclei of hapatocytes was investigated using electrophoretic mobility shift assay （EMSA）. Results The average NO concentration of the IL-1 [~ group was 72.7 p, mol/L, significantly higher than that of the NS group [ （41.7）μmol/L, P〈0.01 ]. The NO concentrations in the supernatant of the IL-1β ＋ Gin 5 and 10 mmol/L groups were 52.9 and 44.4 μmol/L respectively, both significantly higher than that of the NS group （both P 〈 0.05）. The iNOS mRNA and protein expression levels of the IL-1β group were 11 and 2.5 times as those of the NS group （ both P 〈 0.05 ）. Gin dose-dependently decreased the iNOS mRNA and protein expression levels in the hepatocytes. The NO concentration and levels of iNOS mRNA and protein expression in the hepatocytes of the Gln 10 mmol/L group were not significantly different from those of the NS group. The NF-~B levels in the nuclei of hepatocytes of the IL-113 group, 3 IL-1β ＋ Gin group, and only Gin group were all higher than that of the NS group. Conclusion Glutamine down-regulates the iNOS gene over- expression mediated by IL-1β in hepatocytes through an NF-kB independent pathway.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2009年第10期695-698,共4页
National Medical Journal of China
基金
基金项目：国家自然科学基金（30500404）
