N-乙酰半胱氨酸对新生小鼠肝细胞内毒素损伤的保护作用  

Protective effect of N-acetylcysteine against lipopolysaccharide injury in hepatocytes of neonatal mice

作　　者：王琳[1] 徐建波[2] 田元[2] 吴河水[2] 刘亚兰[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院儿科,武汉430022 [2]华中科技大学同济医学院附属协和医院普外科,武汉430022

出　　处：《中华儿科杂志》2007年第1期30-33,共4页Chinese Journal of Pediatrics

摘　　要：目的探讨 N-乙酰半胱氨酸对新生小鼠肝细胞内毒素损伤时的保护作用,为新生小鼠内毒素肝损伤的防治研究提供理论依据。方法采用胶原酶消化组织块法分离新生小鼠的肝细胞,将细胞分内毒素处理组(LPS 组)及 N-乙酰半胱氨酸预处理组(NAC 组)。LPS 组培养基中加入 LPS10μg/ml;NAC 组先在培养基中加入 N-乙酰半胱氨酸5 mmol/L,孵育1 h 后再加入 LPS 10μg/ml。分别于加入 LPS 0、6和12 h 收集各组肝细胞上清和肝细胞,每组12例,以生化法检测培养上清的丙氨酸氨基转移酶(alanine aminotransferasc,ALT)和一氧化氮(nitric oxide,NO)的水平,并以逆转录聚合酶链反应(RT-PCR)法检测各组细胞诱导型一氧化氮合成酶(induced nitric oxide synthase,iNOS)mRNA 的表达。结果 LPS 组在0、6、12 h 的 ALT 值分别为(21.1±4.78)U/L、(59.8±8.59)U/L、(89.6±15.30)U/L,NO 水平分别为(1.6±0.31)μmol/L、(6.6±0.81)μmol/L、(7.8±1.01)μmol/L,iNOS mRNA 水平分别为0.17±0.023、0.71±0.091、0.71±0.097。LPS 组的 ALT 和 NO、iNOSmRNA 水平在6、12 h较0 h 明显升高,差异有极显著统计学意义(P<0.01);NAC 组上清中 ALT 和NO 水平在0、6、12 h 分别为(20.6±5.98)U/L、(40.8±7.30)U/L、(55.4±5.48)U/L 和(1.7±0.26)μmol/L、(3.2±0.71)μmol/L、(4.0±0.71)μmol/L,肝细胞 iNOSmRNA 的表达水平在0、6、12h 分别为0.18±0.026、0.41±0.060、0.40±0.067。经 NAC 预处理后,在6、12 h 上清中 ALT 的水平与 LPS 组比较明显降低,而且 NO 水平以及肝细胞 iNOS mRNA 的表达与 LPS 组比较也明显降低,差异均有极显著统计学意义(P<0.01)。结论 NAC 对新生小鼠肝细胞内毒素损伤有保护作用,NAC可能通过抑制 LPS 激活 iNOS 而发挥保护作用。Objective N-Acetylcysteine （NAC） is a sulthydryl donor molecule with antioxidant and antiinflammatory effects. A major role has been described for inducible nitric oxide （NO） synthase in several inflammatory liver diseases. NAC attenuates NO generation following lipopolysaccharide injection in rats. The purpose of this study was to investigate the effect of NAC against lipopolysaccharide injury in hepatocytes of neonatal mice and the molecular mechanisms by which NAC influences inflammatory responses of the hepatocytes. Methods The liver of neonatal mouse was digested by collagenase to dissociate the hepatocytes. The hepatocytes were cultured and isolated. After 7 days of culture the normal hepatocytes were divided into two groups： LPS group and NAC group. In LPS group, 10 μg/ml LPS was added into the culture medium. In NAC group, 5 mmol/L NAG was added into the culture medium firstly, 10 μg/ml LPS was added after 1 h of culture. There were 12 mice in each group. The cell supernatants and the hepatocytes were collected at 0, 6 and 12 hours after adding LPS. The cell supernatants were taken to measure the alanine aminotransferase （ALT） level and nitric oxide （NO） production by the biochemical methods. The cells were taken to analyze the gene expression of induced nitric oxide synthase （iNOS） by the RT-PCR. Results In LPS group, the levels of ALT, NO and iNOS mRNA increased significantly at the time points 6 h and 12 h compared with the time point 0, （P 〈 0.01）. Compared with the LPS group, the levels of ALT, NO and iNOS mRNA of NAC group were lower at the time points 6 h and 12 h （P 〈 0.01）. Conclusions NAC may play a protective role in the hepatocytes injury caused by LPS in the neonatal mice. The protective mechanism works partially through the inhibition of iNOS activation by LPS.

关 键 词：N-乙酰半胱氨酸 内毒素 肝细胞 一氧化氮 诱导型一氧化氮合成酶 

分 类 号：R722.1[医药卫生—儿科]