急性肝损伤小鼠肝组织中的自噬现象

The role of hepatocellular autophagy in LPS and D-Gal-induced acute liver injury mice

目的探讨在脂多糖(lipopolysaccharide,LPS)和D-氨基半乳糖(D-galactosamine,D-GalN)诱导的小鼠急性肝损伤模型中,肝脏组织的自噬情况。方法将60只雄性Balb/C小鼠随机分为5组(每组均包括实验亚组和对照亚组,各6只)。实验组小鼠腹腔注射LPS(100μg/kg)和D-GalN(350mg/kg)溶液,对照组小鼠只注射无菌等量生理盐水,分别于注射后2、4、8、12、24h处死小鼠,取其血液及肝脏。另取3只小鼠饥饿处理24h作为自噬阳性对照组。所有小鼠于处死前24h经高压尾静脉注射法注射溶于2ml生理盐水的GFP-LC3质粒100μg。生化法检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平;透射电镜、荧光显微镜下观察小鼠肝脏的自噬情况。结果各实验组血清ALT、AST水平均明显高于相应对照组(P<0.01)。透射电镜显示,实验组和自噬阳性对照组肝组织中可见大量肝细胞发生自噬。荧光显微镜下可见GFP-LC3绿色荧光蛋白。8、12、24h实验组及自噬阳性对照组每高倍镜视野下平均GFP-LC3荧光点数目明显高于对照组(P<0.01)。结论 LPS和D-GalN可诱导小鼠发生急性肝损伤,而自噬是这一过程中的重要形态学特征。

Objective To explore hepatocellular autophagy in LPS and D-GaiN-induced acute liver injury of mice. Methods Sixty male Balb/C mice were randomly divided into 5 groups. Each group included model and control subgroups （n=6）. The model mice were injected intraperitoneally with LPS （100 μg/kg） and D-GaIN （350 mg/kg） in 100 μl physiological saline solution （PSS）, while the control mice were treated with equal volume PSS, and they were sacrificed respectively at 2, 4, 8, 12 or 24 h later. Three mice treated with starvation for 24 h constituted autophagy positive control group. All mice received 2 ml GFP-LC3 plasmid （100 μg） via tail vein hydrodynamic injection at 24 h before sacrifice. Blood and liver tissue samples were collected. The contents of serum ALT, AST were detected. The status of hepatocellular au- tophagy was observed by transmission electron microscopy and fluorescence microscopy. Results The levels of ALT and AST in the model groups were all significantly higher than those of the corresponding control groups （P 〈 0.01）. Transmission electron microscopy showed that autophagy cells were abundant in the hepatocytes of the model groups and starvation-induced group. GFP-LC3 was visualized by fluorescence microscopy. The average GFP-LC3 punctae of the 8,12 and 24 h model groups were significantly higher than those of control groups （P 〈 0.01 ）. Conclusion Acute liver injury can be induced by LPS and D-GaIN in mice, and the autophagy is an important morphological feature in this process.