Department of Intensive Care Medicine, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland.
PLoS One. 2012;7(9):e45195. doi: 10.1371/journal.pone.0045195. Epub 2012 Sep 13.
During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α). Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα) phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB). The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis). Our data suggest that remifentanil increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration.
在脓毒症中，肝功能障碍很常见，已经描述了线粒体无法有效地将氧消耗与能量产生偶联。除了脓毒症之外，用于治疗脓毒症患者的药物也可能导致线粒体功能障碍。这项研究旨在验证这样一个假设，即瑞芬太尼与肝线粒体氧消耗相互作用。用瑞芬太尼处理人肝癌细胞系 HepG2 及其分离的线粒体，或在进一步暴露于肿瘤坏死因子-α（TNF-α）的情况下进行处理。通过高分辨率呼吸测定法测量线粒体氧消耗，通过 Western blot 测定 Caspase-3 蛋白水平，并通过 ELISA 测定细胞因子水平。使用商业 ELISA 试剂盒分析完整细胞中抑制性κBα（IκBα）磷酸化、细胞内 ATP 含量和线粒体膜电位的测量。一小时后孵育瑞芬太尼，细胞呼吸增加，IκBα磷酸化，表明核因子κB（NF-κB）被激活。孵育 2、4、8 或 16 小时后，对细胞呼吸没有影响。瑞芬太尼增加了复杂 I 依赖性呼吸的分离线粒体呼吸控制比，而不干扰最大呼吸。预先用阿片受体拮抗剂纳洛酮孵育可防止瑞芬太尼诱导的细胞呼吸增加。瑞芬太尼的浓度比治疗浓度高 10 倍，可降低线粒体膜电位和 ATP 含量，而不会解偶联氧消耗和基础呼吸水平。TNF-α暴露降低了复合物 I、II 和 IV 的呼吸作用，这种作用可通过预先用瑞芬太尼孵育来预防。此外，预先用瑞芬太尼孵育可防止 TNF-α诱导的 HepG2 细胞释放 IL-6，并减弱前半胱天冬酶 3（细胞凋亡的早期标志物）的切割活性。我们的数据表明，瑞芬太尼增加了人肝细胞的细胞呼吸，并防止了 TNF-α诱导的线粒体功能障碍。这些结果不能用线粒体呼吸的解偶联来解释。
