Apoptotic insults to human HepG2 cells induced by S-(+)-ketamine occurs through activation of a Bax-mitochondria-caspase protease pathway
Apoptotic insults to human HepG2 cells induced by S-(+)-ketamine occurs through activation of a Bax-mitochondria-caspase protease pathway
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DOI：
10.1093/bja/aen322
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发表时间：
2009-01-01
影响因子：
9.8
通讯作者：
Chen, R. -M.
中科院分区：
文献类型：
--
作者：
Lee, S. -T.;Wu, T. -T.;Chen, R. -M.
背景氯胺酮被广泛用作静脉注射麻醉剂和滥用药物。肝细胞有助于内源性和外源性物质的代谢。本研究以人肝癌HepG 2细胞为实验模型，探讨S-（+）-氯胺酮的毒性作用及其可能机制。HepG 2细胞暴露于S-（+）-氯胺酮。测定细胞活力、乳酸脱氢酶（LDH）和γ-谷氨酰转肽酶（GPT）的释放，以确定S-（+）-氯胺酮对HepG 2细胞的毒性。细胞形态学，DNA片段化和凋亡细胞进行了分析，以评估S-（+）-氯胺酮诱导的细胞死亡的机制。通过免疫印迹法定量细胞质或线粒体中Bax（一种凋亡蛋白）和细胞色素c的量。使用生物发光测定法分析细胞三磷酸腺苷水平。Caspase-3、Caspase-9和Caspase-6的荧光测定。S-（+）-氯胺酮可增加HepG 2细胞LDH和GPT的释放，但降低细胞活力（均P < 0.01）。S-（+）-氯胺酮呈时间依赖性地引起HepG 2细胞皱缩。S-（+）-氯胺酮暴露导致显著的DNA断裂和细胞凋亡（P=0.003和0.002）。S-（+）-氯胺酮可增加Bax向线粒体的转位，降低线粒体膜电位和细胞内腺苷三磷酸水平（P均< 0.01）。随后，S-（+）-氯胺酮给药后，胞浆细胞色素c水平和半胱氨酸天冬氨酸蛋白酶-9，3和6的活性增加（均P < 0.001）。Caspase-6抑制剂Z-VEID-FMK可减轻S-（+）-氯胺酮诱导的Caspase-6活性增强、DNA断裂和细胞凋亡（P均< 0.001）。本研究表明，S-（+）-氯胺酮可通过线粒体-caspase蛋白酶途径诱导人HepG 2细胞凋亡损伤。因此，我们认为临床相关或滥用浓度的S-（+）-氯胺酮可能由于其对肝细胞的毒性而诱导肝功能障碍。
Background. Ketamine is widely used as an i.v. anaesthetic agent and as a drug of abuse. Hepatocytes contribute to the metabolism of endogenous and exogenous substances. This study evaluated the toxic effects of S-(+)-ketamine and possible mechanisms using human hepatoma HepG2 cells as the experimental model.Methods. HepG2 cells were exposed to S-(+)-ketamine. Cell viability and the release of lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GPT) were measured to determine the toxicity of S-(+)-ketamine to HepG2 cells. Cell morphology, DNA fragmentation, and apoptotic cells were analysed to evaluate the mechanism of S-(+)-ketamine-induced cell death. Amounts of Bax, an apoptotic protein, and cytochrome c in the cytoplasm or mitochondria were quantified by immunoblotting. Cellular adenosine triphosphate levels were analysed using a bioluminescence assay. Caspases-3, -9, and -6 were measured fluorometrically.Results. Exposure of HepG2 cells to S-(+)-ketamine increased the release of LDH and GPT, but decreased cell viability (all P < 0.01). S-(+)-Ketamine time-dependently caused shrinkage of HepG2 cells. Exposure to S-(+)-ketamine led to significant DNA fragmentation and cell apoptosis (P=0.003 and 0.002). S-(+)-Ketamine increased translocation of Bax from the cytoplasm to mitochondria, but decreased the mitochondrial membrane potential and cellular adenosine triphosphate levels (all P < 0.01). Sequentially, cytosolic cytochrome c levels and activities of caspases-9, -3, and -6 were augmented after S-(+)-ketamine administration (all P < 0.001). Z-VEID-FMK, an inhibitor of caspase-6, alleviated the S-(+)-ketamine-induced augmentation of caspase-6 activity, DNA fragmentation, and cell apoptosis (all P < 0.001).Conclusions. This study shows that S-(+)-ketamine can induce apoptotic insults to human HepG2 cells via a Bax-mitochondria-caspase protease pathway. Thus, we suggest that S-(+)-ketamine at a clinically relevant or an abused concentration may induce liver dysfunction possibly due to its toxicity to hepatocytes.