Department of Pharmacology, Institute of Post Graduate Medical Education and Research (IPGME&R), Kolkata, WB 700 020, India.
Department of Biomedical Sciences, Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria.
Phytomedicine. 2022 Aug;103:154221. doi: 10.1016/j.phymed.2022.154221. Epub 2022 Jun 3.
The global burden of leishmaniasis is exacerbated by the limited repertoire of drugs, resulting in an urgent need to develop new therapeutic alternatives. Endoperoxides like ascaridole have emerged as promising anti-parasitic candidates, and its effectiveness was established in an animal model of cutaneous leishmaniasis (CL). However, its impact on Leishmania donovani parasites, causative of visceral leishmaniasis (VL) remains to be established.
This study aimed to delineate the underlying mechanisms contributing towards the leishmanicidal effect of ascaridole in terms of its impact on the cellular redox status and metabolic bioenergetics of L. donovani parasites.
The anti-promastigote activity of ascaridole was established by a cell viability assay in L. donovani [MHOM/IN/1983/AG83] and anti-amastigote activity by microscopy and ddPCR (droplet digital polymerase chain reaction). The cellular redox status, mitochondrial membrane potential (MMP), annexin V positivity and cell cycle arrest was evaluated by flow cytometry, while cellular and mitochondrial bioenergetics was assessed using Agilent XFp Analyzer, and the levels of ATP was measured by chemiluminescence.
Ascaridole demonstrated strong anti-promastigote and anti-amastigote activities in l. donovani, IC (half maximal Inhibitory concentration) being 2.47 ± 0.18 µM and 2.00±0.34 µM respectively, while in J774.A1 and murine peritoneal macrophages, the CC (half maximal cytotoxic concentration) was 41.47 ± 4.89 µM and 37.58 ± 5.75 µM respectively. Ascaridole disrupted the redox homeostasis via an enhanced generation of reactive oxygen species (ROS), lipid peroxidation and concomitant depletion of thiols. However, it failed to increase the generation of mitochondrial superoxide, which minimally impacted on mitochondrial respiration and was corroborated by energy metabolism studies. Instead, ascaridole inhibited glycolysis of promastigotes, caused a loss in MMP, which translated into ATP depletion. In promastigotes, ascaridole enhanced annexin-V positivity and caused a cell cycle arrest at sub- G/G phase.
In summary, ascaridole displays its leishmanicidal activity possibly due to its ability to auto-generate free radicals following cleavage of its endoperoxide bridge that led to disruption of the redox homeostasis, inhibition of glycolysis and culminated in an apoptotic like cell death.
由于药物种类有限，导致利什曼病的全球负担加重，因此迫切需要开发新的治疗方法。过氧化物如ascaridole 已成为有前途的抗寄生虫候选药物，其在皮肤利什曼病（CL）动物模型中的有效性已得到证实。然而，其对内脏利什曼病（VL）的致病原利什曼原虫的影响仍有待确定。
本研究旨在描述ascaridole 发挥杀利什曼原虫作用的潜在机制，具体涉及对利什曼原虫细胞氧化还原状态和代谢生物能量的影响。
通过细胞活力测定法在 L. donovani [MHOM/IN/1983/AG83]中确定ascaridole 的抗前鞭毛体活性，通过显微镜和 ddPCR（液滴数字聚合酶链反应）确定抗无鞭毛体活性。通过流式细胞术评估细胞氧化还原状态、线粒体膜电位（MMP）、膜联蛋白 V 阳性和细胞周期停滞，同时使用 Agilent XFp 分析仪评估细胞和线粒体生物能量，通过化学发光法测量 ATP 水平。
ascaridole 在 l. donovani 中表现出很强的抗前鞭毛体和抗无鞭毛体活性，IC（半最大抑制浓度）分别为 2.47 ± 0.18 µM 和 2.00 ± 0.34 µM，而在 J774.A1 和小鼠腹腔巨噬细胞中，CC（半最大细胞毒性浓度）分别为 41.47 ± 4.89 µM 和 37.58 ± 5.75 µM。ascaridole 通过增强活性氧（ROS）的产生、脂质过氧化和巯基同时耗竭来破坏氧化还原稳态。然而，它未能增加线粒体超氧化物的产生，这对线粒体呼吸的影响很小，这也得到了能量代谢研究的证实。相反，ascaridole 抑制前鞭毛体的糖酵解，导致 MMP 丧失，从而导致 ATP 耗竭。在前鞭毛体中，ascaridole 增加膜联蛋白-V 阳性并导致细胞周期停滞在 sub-G/G 期。
总之，ascaridole 发挥杀利什曼原虫活性可能是由于其过氧化物桥断裂后能够自动生成自由基，导致氧化还原稳态破坏、糖酵解抑制，并最终导致类似凋亡的细胞死亡。
