胆汁酸盐对囊型肝包虫生长的影响及作用机制

Infection of humans and domestic ruminants with the larval stage (metacestode) of Echinococcus granulosus results in cystic echinococcosis (CE). The metacestode causes a space-occupying lesion in visceral organs, most commonly in the liver.The most effective treatment of hydatid cyst is surgical operation. We found that the hepatic echinococcosis with biliary fistula, hydatid protoscolex have mostly died in..Our preliminary study shows, bile can reduce the activity of protoscolices of Echinococcus granulosus. Bile acids are the main components in the bile. Bile salt signal transduction pathway in Echinococcus granulosus protoscoleces. Bile acid influence andactivity and structure of germinal cells, resulting in loss of activity and growth ability of hydatid disease. The aim of the present work was to determine the efficacy of bile salt against Echinococcus granulosus by using in vitro and in vivo models. Protoscolices were collected aseptically from sheep livers containing hydatid cyst. Viability of protoscolices was determined by 0.1% eosin staining. Freshly isolated E. granulosus protoscoleces were subjected to bile salt treatment, and the effects on parasite viability were monitored by eosin staining and scanning electron microscopy(SEM) and transmission electron microscopy (TEM). A group of 80 female mice were included in the study and randomly divided into four groups of 20 mice each. The scolicidal agents and Echinococcus granulosus were directly injected into the left lobe of the liver. Six months after injection, the mice were killed and autopsied. Developed hydatid cysts were counted,weighed and processed for observation by transmission electron microscopy. This experiments will evaluate the potential of bile salt as a useful anthelmintic for the treatment of human CE.

囊型肝包虫病严重危害人体健康，治疗以手术治疗为主，药物治疗为辅。我们发现囊型肝包虫病合并胆瘘时，包虫内原头蚴大多已经死亡。我们前期研究显示，胆汁可降低细粒棘球蚴原头节活力，胆汁酸是胆汁主要组成成分，在细粒棘球蚴存在胆汁盐信号通路，胆汁酸是否影响原头节和生发细胞的活性及结构，进而导致包虫失去活性和发育能力，目前未见报道。本研究以细粒棘球蚴原头节和生发细胞为研究对象，检测7种胆汁酸对原头节和生发细胞活力的影响，观察7种胆汁酸对原头节和生发细胞形态结构的改变，并通过检测凋亡、内质网应激蛋白表达和MAPK信号通路蛋白表达，探讨7种胆汁酸对细粒棘球蚴原头节和生发细胞的作用及其机制；同时建立肝包虫小鼠模型，进一步探索7种胆汁酸对小鼠肝包虫生长状况的影响，初步探究胆汁导致包虫损伤死亡的机制，旨在进一步了解包虫自然死亡的原因，同时可以为肝包虫手术术中杀灭原头节，预防肝包虫术后复发提供药物治疗新选择。

本研究以体外培养的细粒棘球蚴原头节为研究对象，将不同浓度的胆酸(CA)､石胆酸(LCA)､脱氧胆酸（DCA）､鹅脱氧胆酸(CDCA)和牛磺胆酸(TCA)体外作用于细粒棘球蚴原头节，探讨胆汁中各种胆酸盐对细粒棘球蚴原头节的作用及其机制。不同浓度CA、LCA、DCA、CDCA作用细粒棘球蚴原头节后,倒置显微镜下观察并分别统计存活原头节的个数，根据获得的数据计算出原头节的存活率，接着利用统计学软件分析并描绘出相应的活力曲线，不同浓度CA、LCA、DCA、CDCA均可抑制原头节的生长，随着药物作用时间的延长和药物浓度的增加，原头节活力的降低的越明显。CA、LCA、DCA、CDCA对细粒棘球蚴原头节的生长抑制作用,具有时间依赖性和剂量依赖性。将不同浓度胆酸（CA）、石胆酸（LCA）和鹅脱氧胆酸（CDCA）作用于细粒棘球蚴原头节，在扫描电镜和透射电镜观察，CA、LCA和CDCA可破坏细粒棘球蚴原头节结构。究其机制，DCA、CDCA、LCA、TCA可增加细粒棘球蚴原头节ROS的产生；LCA、TCA降低细粒棘球蚴原头节的SOD、GSH、HO-1和NQO-1活性；DCA、CDCA可增加细粒棘球蚴原头节内钙离子水平；Western blot法检测显示，LCA、TCA增加细粒棘球蚴原头节Ctyc蛋白表达水平；CA､LCA和CDCA激活细粒棘球蚴原头节内Caspase-3酶活性。胆酸､石胆酸､脱氧胆酸､鹅脱氧胆酸和牛磺胆酸在体外可明显抑制细粒棘球蚴原头节的生长，可能为治疗细粒棘球蚴病提供一个新的思路。