依托咪酯对鼠肝线粒体能量代谢的影响

Effect of Etomidate on Mitochondrial Energy Metabolism of Rat Livers in vitro

目的探讨依托咪酯对鼠肝线粒体能量代谢的影响。方法雄性SD大鼠9只,每只大鼠直接断头处死,提取线粒体后,随机分为:空白对照组、0.4μg/mL和4μg/mL依托咪酯组,按分组加依托咪酯入线粒体后37℃温育7min。流式细胞仪测定线粒体内Rh123的平均荧光强度和前向光散射(FSC),高效液相色谱仪测定线粒体的腺苷酸含量(AMP、ADP、ATP),并计算能荷(EC),定磷法测定H+-ATPase水解活性及寡霉素的影响。结果0.4μg/mL依托咪酯组和4μg/mL依托咪酯组的线粒体内Rh123的平均荧光强度均较空白对照组升高,4μg/mL依托咪酯组的升高更明显(P<0.05)。4μg/mL依托咪酯组的FSC值、AMP含量、H+-ATPase水解活性高于空白对照组和0.4μg/mL依托咪酯组,ADP含量、EC低于后两者(P<0.05或0.01);0.4μg/mL依托咪酯组与空白对照组比较差异均无统计学意义(P>0.05)。寡霉素并不改变依托咪酯对H+-ATPase水解活性的影响(P>0.05)。结论0.4μg/mL的依托咪酯对鼠肝线粒体的氧化磷酸化没有明显的抑制作用,而4μg/mL的依托咪酯则对线粒体的能量代谢有一定的损害作用。

Objective To investigate the change of energy metabolism in isolated rat liver mitochondria by the general anesthetic etomidate. Methods Nine healthy male SD rats weighted 230- 280 g were used to isolate liver mitochondria. According to randomized block design,the isolated liver mitochondria wered divided into three groups： control group, 0.4μg/mL etomidate group, and 4 μg/mL etomidate group. Then the samples were incubated at 37 ℃ for 7 min. The fluorescence intensities of lipophilic cation Rhodamine-123 and forward light scatter（FSC）were analyzed by flow cytometry. The size of the adenine nucleotides（AMP, ADP, ATP）in mitochondria was measured by high performance liquid chromatography （HPLC）. The energy charge （EC） = （ ATP ＋ 0.5ADP） / （ ATP ＋ ADP ＋ AMP）. The inorganic phosphoruss method was used to measure the H^＋-ATPase hydrolytic activity and the oligomycin-sensitive H^＋-ATPase activity. The enzyme activity was express as nmol Pi/（mg pro · min）. Results The fluorescence intensities level of Rhodamine-123 in the 0. 4 μg/mL etomidate group was higher than in the control group,but still lower than in the 4 μg/mL etomidate group（P〈0.05）. The size of FSC had no significant change in the 0.4 μg/mL etomidate group,whereas the 4 μg/mL etomidate group had increased size of FSC（P〈 0.05）. Compared to the control group, no significant changes of content of adenine nucleotides, energy charge and hydrolytic activity of H^＋-ATPase were found in the 0.4 μg/mL etomidate group. However,the intra-mitochondrial ADP,ATP and energy charge levels decreased and the AMP increased in the 0. 4 μg/mL etomidate group （P〈 0.05）. The hydrolytic activity of H^＋-ATPase increased in the 4 μg/mL etomidate group （P〈0.01） ,but it was not influenced by oligomycin（P〉0.05）. Conclusions Etomidate at 0.4 μg/mL has no significant effect on isolated rat liver mitochonddia,but at 4 μg/mL it can lead to distruption of mitochondrial energy metabolism.