谷氨酰胺对白细胞介素-1β刺激下大鼠肝细胞一氧化氮的产生及线粒体膜电位的影响

Glutamine attenuates the production of nitric oxide production and the reduction of mitochondrial membrane potential of cultured rat hepatocytes induced by IL-1β

目的利用原代培养的大鼠肝细胞，观察不同浓度的谷氨酰胺（Glutamine，Gln）在体外对白细胞介素-1β（Interleukin-1β，IL-1β）刺激下肝细胞一氧化氮（Nitricoxide，NO）过度产生和线粒体膜电位降低的影响。方法选用6周龄sD大鼠，采用原位胶原酶循环灌注消化法获取高纯度原代大鼠肝细胞，培养48h后分别用生理盐水、IL-1β（1nmol／L）以及IL-1β（1nmoL／L）联合不同浓度的Gln（2mmol／L、5mmol／L、10mmol／L）刺激肝细胞24h，留取细胞和上清液，采用生化法测定上清液丙氨酸转移酶（alanine aminotransferase，ALT）和NO的水平，采用荧光探针JC-1结合流式细胞仪检测肝细胞线粒体膜电位的变化。对各组数据之间进行方差分析，明确是否具有统计学意义。结果IL-1β刺激后肝细胞培养液ALT平均浓度为38．2U／L、NO的平均浓度为72．7μmol／L，均显著高于生理盐水组（分别为7．4U／L和41．7μmol／L，P〈0．01），而肝细胞线粒体膜电位水平却明显低于生理盐水组（强红色荧光细胞比例分别为30．0％和62.8％，P〈0．01）；同时给与Gln能够抑制IL-1β刺激下肝细胞NO的产生，减轻ALT的升高以及线粒体膜电位的降低，并且这些作用随着Gln浓度的增加而增强。结论谷氨酰胺对炎症应激导致的肝细胞损伤具有保护作用，这种保护作用可能与抑制肝细胞NO的过度产生改善线粒体呼吸功能有关。

Objective To investigate the effects of different concentrations of glutamine （Gln） on the production of nitric oxide （NO） and reduction in mitochondrial membrane potential in rat heputocytes activated in vitro by interleukin （IL）-1β, Method The primary cultured rat hepatocytes with high-purity were isolated from 6- week-old male Sprague-Dawley rats by an in situ circulatory eollagenase perfusion method. After incubation for 48 hours, hepatocytes were stimulated by saline, or IL-1β（ 1 nmol/L） or IL-1β（ 1 nmol/L）combined with glutamine in concentrations ranging from 2 to 10 mmol/L. The culture medium and hepatocytes were collected at 24 hours after stimulation. The concentrations of alanine aminotransferase （ALT） and NO in the medium were detected by bio- chemical methods. The mitoehondrial membrane potential of the hepatocytes was detected with flow cytometry after incubated with fluorescent probe JC-1. Statistic package of SPSS 11.5 was used for the data analysis and significant differences between means were evaluated by ANOVA analysis. Results The average concentration of ALT and NO in the cttlture medium after IL-1βstimulation was 38.2 U/L and 72.7/anol/L, respectively, which were significantly higher than those of control group （7.4 U/L and 41.7 μmol/L, respectively, P 〈 O. 01 ）. The mitochondrial membrane potential of hepatocytes in IL-1βgroup was much lower than that in control group （30.0 % vs. 62.8 %, P 〈 0.01 ）. Gin inhibited NO production induced by IL-1β, ALT releasing and reduction in mitochondrial membrane potential of cultured rat hepatocytes in a dose-dependent manner. Conclusions Glutamine, the most abundant free amino acid in the body, can attenuate hepatocye injury mediateel in vitro by pro-inflammatory cytokine mediated. This protective effect may be associated with the inhibition of NO production and thereby amelioration of mitochondrial function.