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

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,Gin)在体外对白细胞介素-1β(Interleukin-1β,IL-1β)刺激下肝细胞一氧化氮(Nitric oxide,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48 h后分别用生理盐水、IL-1β(1 nmol/L)以及IL-1β(1 nmol/L)联合不同浓度的Gln(2 mmol/L、5 mmol/L、10 mmol/L)刺激肝细胞24 h,留取细胞和上清液,采用生化法测定上清液丙氨酸转移酶(lanine aminotransfemse 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38.2 U/L、N0的平均浓度为72.7 umol/L,均显著高于生理盐水组(分别为7.4U/L和41.7 mnol/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N0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.

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

Objective To investigate the effects of different concentrations of glutnine(Gln) on the procfion of nitric oxide (NO)and reduction in mitochondrial membrane potential in rat heputocytes activated in by interleukin(IL)-1β.Method The primary cultured rat hepatocytes with high-purity Wfffe isolated from 6-week-old male Sprague-Dawley raL, by flirt in situ circulatory collagenase peffusion method.After incubation for hours.hepatocytes were stimulated by saline,or IL-l(1 nmol/L)or IL-I(1 mnol/L)combined with gIne in concentrations rangiIfrom 2 to 10 mmol/L.The culture muln and hepatocytes were collected at 24 hours after stimulation.The concentrations of alanine aminotransferase(ALT)and NO in the lnllln were detected by biochemical methods.The mitochondrial membrane potential of the hepatocytes was detected with flow cytometry after incubated with fluorescem probe JC-1.Statistic package ofSPSS 11.5 was used for the data analysand significant differences between mean8 were evaluated byQ、4k analysis.Results The average concentration ofand NOinthe culturemedium afterIL-Istimulationwas 38.2U/L and 72.7tmaol/L,respectively,whichwere sis,cantly big,herthanthose ofcontrol group(7.4 U/L and41.7nol/L,respectively,P<0.01).1hemitochondrial membrane potential ofhepatocytes in IL-lgroup was much lowerthan that in control group(30.O%vs.62.8%.P<0.01).Gin inhibited NO production induced byⅡ,lp,Jeleasing and reduction in mitochondrial II brahetential ofcuhured rat hepatocytes in a dose-dependent HmIll.Conclusions Ghtamine,the most abundant free amino acid in the body,call attenuate hepatocye injury mechateel in vitro by pro-inflammatory cytokine mediated.nlis protective effectmay be associatedwiththeinhibition of NO production and thereby amelioration of mitochondrialfunction.