达克罗宁对细粒棘球蚴原头节体外作用及其ROS水平的影响

In vitro effects of dyclonine on the Echinococcus granulosus protoscoleces and its ROS level

目的初步探究达克罗宁对细粒棘球蚴原头节体外生长的影响以及原头节内ROS的表达情况。方法用达克罗宁与细粒棘球蚴原头节体外共培养,采用0.1%曙红排斥试验观察原头节的形态,统计存活率并绘制原头节的生存曲线;将DCFH-DA探针加入孵育原头节的培养基以检测其ROS活性;用SOD和Caspase-3试剂盒测定SOD和Caspase-3的活性。结果40μg/ml达克罗宁可使体外培养的原头节在第4d全部死亡;达克罗宁以剂量依赖的方式使原头节的ROS和Caspase-3表达量增加,同时降低SOD的活性。结论达克罗宁具有体外抗细粒棘球蚴原头节作用,通过激活ROS诱导氧化应激导致Caspase-3被激活从而引起细粒棘球蚴原头节的凋亡。

Objective To explore the in vitro effect of dyclonine on the growth of Echinococcus granulosus proto-scoleces and the expression of ROS in the protoscoleces.Methods Freshly isolated Echinococcus granulosus proto-scoleces were incubated with dyclonine at concentrations of 10,20and 40μg/ml.Morphology of protoscoleces was moni-tored with 0.1%eosin rejection test.Every experiment was repeated three times,and survival rate was recorded daily.The DCFH-DA probe was added to the culture medium to detect ROS released from the Echinococcus granulosus proto-scoleces.SOD activity was measured using a SOD activity assay kit,and Caspase-3activity was measured using a caspase-3activity assay kit.Results Control protoscoleces incubated in RPMI 1640or in RPMI 1640+DMSO remained viable after 8days of incubation.In contrast,incubation in 40μg/mL dyclonine could cause all of the protoscoleces cultured in vitro to die on the fourth day.Morphological of protoscoleces changed included reduction in volume,deformed adhesive discs and destroyed internal organization.Dyclonine increased the expression of ROS in protoscoleces at early time.SOD activity in protoscoleces decreased in a dose-dependent.And Caspase-3activity was significantly increased in protoscoleces treated with Dyclonine for 3days,when compared with that in controls(P<0.05).Conclusion Dyclonine may induces oxidative stress by activating ROS,which leads to the activation of Caspase-3,and resulting in apoptosis of the Echino-coccus granulosus protoscoleces at last.