School of Basic Medical Sciences, Lanzhou University, Lanzhou, 730000, Gansu Province, People's Republic of China.
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO Collaborating Center for Tropical Diseases, National Center for International Research on Tropical Diseases, Key Laboratory of Parasite and Vector Biology of the Chinese Ministry of Health, Shanghai, 200025, People's Republic of China.
Parasit Vectors. 2021 Feb 15;14(1):108. doi: 10.1186/s13071-021-04618-4.
Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca/CaM-CaMKII) in infected mice.
The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction.
In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm.
Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.
包虫病由带科绦虫幼虫引起，是一种全球性的人畜共患寄生虫病，对人类和动物的健康构成严重威胁。阿苯达唑是治疗包虫病的首选药物，但由于其长期使用后治愈率低且副作用大，难以达到临床治疗目标。因此，迫切需要寻找新的抗寄生虫靶点和有效的治疗替代品。先前的研究表明，维拉帕米（Vepm）可以抑制细粒棘球蚴幼虫的生长，但这种作用的机制尚不清楚。本研究旨在深入了解 Vepm 对包虫的抗包虫作用，特别是 Vepm 对感染小鼠中钙/钙调蛋白依赖性蛋白激酶 II（Ca/CaM-CaMKII）的调节作用。
评估了 Vepm 对体外细粒棘球蚴原头蚴（PSC）和感染小鼠中的泡球蚴后生蚴的抗包虫作用。通过扫描电子显微镜（SEM）观察 Vepm 诱导的包虫形态改变，并通过 SEM-能谱分析、电感耦合等离子体质谱和茜素红染色测量寄生虫和小鼠血清及肝脏中的钙含量变化。此外，通过免疫组织化学和/或实时定量聚合酶链反应评估感染小鼠中 CaM 和 CaMKII 的蛋白和 mRNA 水平以及细粒棘球蚴 PSC 中 CaMKII 的 mRNA 水平在 Vepm 治疗后的变化。
在体外，浓度为 0.5 μg/ml 或更高的 Vepm 可在 8 天内杀死细粒棘球蚴 PSC。在这些条件下，PSC 的超微结构受损，并且伴随着明显的钙损失和 CaMKII mRNA 表达下调。在体内，用 40mg/kg Vepm 治疗后，泡球蚴后生蚴的重量和钙含量减少，并且感染小鼠的血清和肝脏中的钙含量升高。此外，在用 Vepm 治疗后，感染泡球蚴后生蚴的小鼠肝脏中 CaM 和 CaMKII 的蛋白和 mRNA 表达下调。
Vepm 通过下调过度激活的 Ca/CaM-CaMKII 的表达，在体外和体内均对包虫具有杀虫作用。结果表明，Ca/CaM-CaMKII 可能是一个新的药物靶点，维拉帕米可能是未来控制包虫病的一种有潜力的抗包虫药物。
