Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
J Antimicrob Chemother. 2013 Apr;68(4):789-99. doi: 10.1093/jac/dks498. Epub 2013 Jan 3.
The pterocarpanquinone LQB-118, previously demonstrated to be effective in vivo via oral delivery, was investigated for its mechanism in selective parasite killing.
Oxidative stress in Leishmania amazonensis was analysed by evaluating reactive oxygen species (ROS) production (2',7'-dichlorodihydrofluorescein diacetate) and the loss of mitochondrial membrane potential (ΔΨm) using rhodamine, JC-1 and MitoCapture. Ultrastructural analysis was performed using transmission electron microscopy (TEM). DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL).
Treatment with LQB-118 induced ROS production in the promastigotes of L. amazonensis in a concentration-dependent manner for the first 4 h and was sustained for 24 h. TEM analysis revealed several alterations typical of apoptosis. Promastigotes presented a reduction of ΔΨm after 24 h of incubation with 2.5 μM (18.7%), 5 μM (63.7%) or 10 μM (70.7%) LQB-118. A sub-G0/G1 cell cycle phenotype was observed in 21%-83% of the promastigotes incubated with 1.25-10 μM LQB-118. Concentration-dependent DNA fragmentation was observed in promastigotes treated with 2.5-10 μM LQB-118, and selective DNA fragmentation was observed in intracellular amastigotes after 72 h with 2.5 μM treatment.
Our results suggest that LQB-118 selectively induces ROS-triggered and mitochondria-dependent apoptosis in this parasite.
先前经口服给药在体内证明有效的紫檀烷醌 LQB-118，其选择性杀伤寄生虫的机制已被研究。
通过评估活性氧物种（ROS）产生（2'，7'-二氯二氢荧光素二乙酸酯）和使用罗丹明、JC-1 和 MitoCapture 检测线粒体膜电位（ΔΨm）的丧失来分析利什曼原虫中的氧化应激。使用透射电子显微镜（TEM）进行超微结构分析。使用末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）评估 DNA 片段化。
LQB-118 以浓度依赖的方式诱导 L. amazonensis 前鞭毛体中的 ROS 产生，在最初的 4 小时内呈递增趋势，并持续 24 小时。TEM 分析显示了几种典型凋亡的改变。在孵育 24 小时后，用 2.5 μM（18.7%）、5 μM（63.7%）或 10 μM（70.7%）LQB-118 孵育后，前鞭毛体的 ΔΨm 减少。在孵育 1.25-10 μM LQB-118 的前鞭毛体中观察到亚 G0/G1 细胞周期表型，在 21%-83%的前鞭毛体中观察到。在孵育 2.5-10 μM LQB-118 的前鞭毛体中观察到浓度依赖性的 DNA 片段化，在用 2.5 μM 处理 72 小时后，在细胞内无鞭毛体中观察到选择性 DNA 片段化。
我们的结果表明，LQB-118 选择性地诱导这种寄生虫中 ROS 触发和线粒体依赖性凋亡。
