靶向Hedgehog信号通路Smo受体的Cyclopamine杀伤日本血吸虫的作用与机制研究

Schistosomiasis is a parasitic zoonosis caused by blood-flukes that remains widely spreading and seriously threatening the people health. So far, the treatment of schistosomiasis worldwide relies exclusively on praziquantel, a drug discovered in the mid-1970s that lacks efficacy against the larval stages of the parasite. Furthermore, the repeated administration of one drug has caused a decreased sensitivity of Schistosoma mansoni to praziquantel in many endemic areas. There are also several schistosomiasis cases caused by Schistosoma haematobium in which repeated standard treatment failed to clear the infection. Praziquantel-resistant field isolates were also found from schistosomiasis serious endemic areas. Under such condition, the novel, effective and safe drugs against schistosomiasis must be developed as soon as possible. Typically a drug target is a key molecule involved in a particular metabolic or signaling pathway that is specific to the infectivity, development or survival of a pathogen. The Hedgehog signalling pathway is a key pathway that has a crucial role in the embryonic development, in the regeneration of tissues in adult organisms and in many other processes of all multicellular eukaryotic animals. Hedgehog signals control multiple aspects of development, including the proliferation, fate specification, polarity, and migration of cells. In our previous work, we found that Cyclopamine, which targeting Smo receptor of Hedgehog pathway, can effectively kill Schistosoma japonicum larvae in vitro, suggesting that inhibition of schistosome Hedgehog pathway may cause worm death. Based on this research result, we intend to comprehensively evaluate the killing effect of Cyclopamine on different developmental stage schistosome in vitro and in vivo. To identify the recognition and combination between Cyclopamine and Smo receptor by using immunofluorescence, and analyze the action mechanism of Cyclopamine by using Hedgehog small molecular agonist, apoptosis analysis and RNAseq analysis. The research outcome will help to evaluate the potential of Smo as a drug target for screening new drugs against Schistosoma japonicum and promote the development of new drugs.

血吸虫病是血吸虫感染引起的危害严重的人畜共患寄生虫病，迄今依赖唯一化疗药吡喹酮防治。吡喹酮对童虫杀伤力弱，且单一反复用药存在产生抗药性的风险，急需研发抗血吸虫新药。对病原的发育或生存至关重要的代谢途径或信号通路的关键分子是常用的药靶。Hedgehog（Hh）信号通路在多细胞真核生物胚胎发育和成体内环境稳定中起关键调节作用，该通路的异常调节常引起发育畸形甚至死亡或肿瘤发生。本研究前期工作发现靶向Hh通路Smo受体的Cyclopamine能有效杀伤体外培养的血吸虫童虫，提示Hh通路抑制可能造成血吸虫死亡。本研究拟全面评价Cyclopamine对体外培养及小鼠体内血吸虫的杀伤作用，利用免疫荧光技术分析其能否识别血吸虫的Smo受体，并采用Hh通路激动剂、凋亡分析和RNAseq技术分析其杀伤血吸虫的作用机制，评价Smo受体可否作为抗血吸虫新药筛选的靶标，为推进抗血吸虫新药的研发提供理论指导。

血吸虫病是血吸虫感染引起的危害严重的人畜共患寄生虫病，迄今依赖唯一化疗药吡喹酮防治。吡喹酮对童虫杀伤力弱，且单一反复用药存在产生抗药性的风险，急需研发抗血吸虫新药。对病原的发育或生存至关重要的代谢途径或信号通路的关键分子是常用的药靶。Hedgehog（Hh）信号通路在多细胞真核生物胚胎发育和成体内环境稳定中起关键调节作用，该通路的异常调节常引起发育畸形甚至死亡或肿瘤发生。本研究前期工作发现靶向Hh通路Smo受体的Cyclopamine能有效杀伤体外培养的血吸虫童虫，提示Hh通路抑制可能造成血吸虫死亡。本研究证实血吸虫体内存在Hh通路，且受配体基因在童虫阶段转录水平较高，Smo受体蛋白在雌雄生殖系统及体被下的实质细胞内均有分布。RNA干扰能降低14天童虫的受配体基因的转录水平，其中Smo受体基因的抑制导致发育不良虫体的比率显著增加，提示Hh通路对维持虫体的正常生长发育有重要的调节作用。体外培养实验发现5µM以上浓度的Cyclopamine能在培养48小时左右导致14天、18天和23天虫体在光镜下可见的明显损伤，组织切片的HE染色显示虫体内部已发生了不可修复的破溃或消融。虫体呈僵硬状态，不能像正常虫体那样自如伸缩移动，各发育阶段的虫体在后续的培养中都最终死亡，而所有对照虫体都健康存活。本项目的研究结果表明，Cyclopamine具有杀伤血吸虫的作用，可作为候选药物，推进抗血吸虫新药的研发。