摘要目的 观察急性肝衰竭(ALF)中决定肝细胞能量代谢特点的线粒体内关键酶:柠檬酸合成酶(CS)、肉毒碱棕榈酰转移酶1(CPT-1)、细胞色素C氧化酶(COX)的活性变化.方法 40只雄性SD大鼠分为对照组、造模4h组、造模8h组、造模12 h组及造模24 h组.模型组给予D-氨基半乳糖800 mg/kg和脂多糖100μg/kg联合腹腔注射构建大鼠ALF模型,对照组给予0.9％的等渗盐水1ml腹腔注射.检测各组大鼠ALT、AST、总胆红素、血清白蛋白、凝血酶原时间和凝血酶原活动度;观察肝脏病理和线粒体超微结构变化;检测各组大鼠肝脏线粒体中的CS、CPT-1和COX的活性及mRNA水平,并分析其与肝细胞凋亡及线粒体超微结构之间的关系.方差分析进行各组间总体差异的比较,独立样本t检验进行两组之间差异的比较.结果 通过生物化学检查和肝脏病理检查证实本实验的动物模型构建成功.大鼠ALF造模4h时可见肝细胞凋L的出现,8h时凋亡非常明显,12h时可见肝细胞的坏死,24 h时肝脏组织内肝细胞坏死更为显著,并有炎症细胞的浸润.电镜观察线粒体的超微结构发现,模型4h组就已经发生了变化,到24 h时线粒体的外膜结构几乎完全破坏,线粒体崩解.CS、CPT-1、COX的活性在4h均开始升高,8h达到高峰,12h开始下降,24 h下降更为明显.造模8h组与正常组相比,CS[(0.030 8±0.008 2)μmol/min对比(0.004 0±0.001 6)μmol/min]、CPT 1[(0.130 3±0.075 8)μmol/min对比(0.033 4±0.008 3)μmol/min]和COX[(0.022 9±0.002 9)μmol/min对比(0.004 8±0.0007)μmol/min]差异均有统计学意义(t值分别为1.481、2.619和1.014,P＜0.05或P＜ 0.01).结论 大鼠ALF过程中,CS、CPT-1和COX活性在肝细胞凋亡时升高,其参与了ALF的发生和发展,尤其是促进了肝细胞凋亡的发生.

abstractsObjective To determine the roles of mitochondrial apoptosis and energy metabolism in hepatocytes during the pathogenic process of acute renal failure (ALF) by assessing disease-related differential activities of several key mitochondrial enzymes,including citrate synthase (CS),carnitine palmitoyltransferase-1 (CPT-1) and cytochrome c oxidase (COX).Methods Thirty-two male SpragueDawley rats were given D-galactosamine followed by and lipopolysaccharide (LPS) to induce acute liver failure and sacrificed after 4 (4 h group),8 (8 h group),12 (12 h group) and 24 hours (24 h group) of treatment.Eight unmodeled rats served as controls.Effects related to apoptosis were evaluated by pathological analysis of hepatic tissues and TUNEL staining.Ultrastructural changes in mitochondria were assessed by electron microscopy.The activity and expression of CS,CPT-1 and COX were measured.Results Hepatocyte apoptosis was present in the 4 h treatment group and was increased obviously in the 8 h treatment group.Hepatocyte necrosis was first observed in the 12 h treatment group and was significantly higher in the 24 h treatment group,with inflammatory cell invasion.Ultrastructural changes in mitochondria were present in the 4 h treatment group,and the 24 h treatment group showed mitochondria with completely destroyed outer membranes,which resulted in mitochondrial collapse.Activity and protein expression ofCS,CPT-1 and COX were increased in the 4 h group (vs.controls),were at their peak in the 8 h group (CS:t =1.481,P＜ 0.01; CPT-1:t =2.619,P＜ 0.05; COX:t =1.014,P＜ 0.01) and showed a decreasing trend in the 12 h group.In addition,the activities of CS,CPT-1 and COX were enhanced at the stage of hepatocyte apoptosis,suggesting that these enzymes were involved in the initiation and development of ALF.Conclusion Energy metabolism plays an important role in hepatocyte apoptosis during ALF.