去氢骆驼蓬碱及其衍生物通过抑制TopoⅡ活性抗囊型包虫病机制的研究

Cystic echinococcosis (CE) is one of the neglected and life-threatening zoonoses,which is caused by the larval stage of Echinococcus granulosus (Eg). It’s urgentto develop novel drug targets for the helminth.Studies have shown that harmine (HM) has good curative effect on cystic echinococcosis, but neurotoxicity is greater, so we carried out structural modification, the pharmacodynamics and safety evaluation, screening two High efficiency and low toxicity compounds.TEM was found, HM and its derivatives can be caused the accumulation of heterochromatin in parasites’ nuclear, gene microarray results show, HM may increase parasite genes associated with DNA repair, but the mechanism of action is unknown.Studies have shown that HM can inhibit the activity of topoisomeraseⅡ(TopoⅡ), which can combine with inhibitors to form a complex, resulting in DNA strand breakage damage.Therefore, we propose the scientific hypothesis that "harmine and its derivatives cause DNA damage through the inhibition of TopoⅡactivity, eventually leading to the death of E. granulosus”,We intend to in vitro and in vivo intervention by HM and its derivatives, TopoⅡ inhibitors and DNA damage repair agents through the cloning of related genes and identification function, to verify the hypothesis from the aspects of morphology, gene and protein expression.This study will lay the theoretical foundation for the elucidation of harmine and its derivatives against cystic hydatid disease mechanism and development of new drugs for the treatment of hydatid disease.

细粒棘球蚴（Eg）感染所致的囊型包虫病（CE）危害严重，亟需新药物。有研究表明去氢骆驼蓬碱（HM）对囊型包虫病有较好的疗效，但神经毒性较大，因此我们对其进行了结构修饰，经药效学和安全性评价，筛选出两种高效低毒的化合物。透射电镜实验发现，HM及其衍生物均可造成虫体细胞核异染色质堆积，基因芯片结果显示，HM可以上调与虫体DNA损伤修复相关的基因，但作用机制未知。文献报道HM可以抑制拓扑异构酶Ⅱ（TopoⅡ）的活性，造成DNA链断裂损伤。因此，我们提出“去氢骆驼蓬碱及其衍生物通过抑制TopoⅡ活性，造成DNA损伤，最终导致虫体死亡”的科学假说，拟通过细粒棘球蚴DNA损伤修复相关基因及鉴定功能，采用HM及其衍生物、TopoⅡ抑制剂及DNA损伤修复剂进行体外体内干预，从形态、基因及蛋白表达等层面验证假说。此研究将为阐明去氢骆驼蓬碱及其衍生物抗囊型包虫病的作用机制和研发新型治疗包虫病药物奠定基础。

本项目针对去氢骆驼蓬碱（HM）及其衍生物（HMD）治疗囊型包虫病（CE）作用机制及靶点不清的难题，围绕“HM及HMD通过抑制Topo2a抗囊型包虫病”这一假说，首先根据HM可以上调细粒棘球蚴（Eg）同源重组修复关键基因Rad51、Rad54表达的现象，通过彗星实验、qRT-PCR、Western Blot实验及siRNA干扰等技术，研究HM及HMD（DH-004，DH-330）致细粒棘球蚴DNA损伤的机制，明确了HM、DH-004及DH-330可激活EgATM-P53-Topo2a通路致EgDNA损伤，其中EgATM获得激酶活性后通过激活磷酸化组蛋白EgH2AX及EgTopo2a将损伤信号进一步放大，激活其下游靶蛋白EgBRCA1、EgMRE11、EgKU70等DNA末端修饰因子，从而募集EgRad51发挥修复功能，初步确定EgTopo2a可能为HM和DH-004，DH-330抗CE的作用靶点之一。在成功克隆了细粒棘球蚴DNA损伤修复基因EgRad54后，证明了EgRad54基因在细粒棘球蚴体内能够稳定表达，将EgRad54敲除后联合HM、DH-004和DH-330治疗可上调EgATM和EgH2A的表达、下调EgRad54和EgTopo2a的表达，表明下调EgRad54可进一步增强细粒棘球蚴的DNA损伤，抑制细粒棘球蚴的生长。由此推测EgRad54可能是HM和HMD抗CE潜在的新治疗靶点。其次，针对细粒棘球蚴DNA损伤信号可能激活下游DNA末端修饰因子，募集EgRad51发挥修复功能的现象，开展了HM及DH-330、DH-004联合DNA损伤修复抑制剂RI-1治疗CE的药效学研究，发现HM及DH-004，DH-330可引起DNA损伤修复同源重组基因RAD51、BRCA1、MRE11表达上调，说明HM及HMD可能通过导致基因组不稳定诱发DNA损伤，作用机制可能是诱发细粒棘球蚴虫体DNA双链断裂损伤。DNA损伤修复抑制剂RI-1联合HM及HMD后具有增强HM及DH-004，DH-330体内外治疗CE的作用，表明了DNA损伤修复抑制剂RI-1和HM及DH-004，DH-330联合治疗CE具有可行性和有效性。该研究为寻找HM及HMD抗包虫作用靶点奠定理论基础，对后续开拓包虫病患者预防和新治疗方式具有较好的指导意义。