青蒿琥酯体外抗细粒棘球蚴活性氧对DNA作用机制影响的研究

Study the effect of artesunate anti-echinococcosis on the mechanism about what is ROS acting on DNA

目的了解索青蒿琥酯体外抗细粒棘球蚴氧化损伤机制。方法以二甲基亚砜(DMSO)为溶剂对照组,H2O2为活性氧(reactive oxygen species,ROS)对照组,阿苯达唑(albendazole,ABZ)和硝唑尼特(nitazoxanide,NTZ)为药效对照组,青蒿琥酯低剂量组(65μmol/L),青蒿琥酯中剂量组(130μmol/L)和青蒿琥酯高剂量组(325μmol/L)为实验组,采用1%伊红染色法检测实验组药物干预4d的细粒棘球蚴死亡率,并观察病理组织学和超微结构变化;采用双氢罗丹明123(DHR123)法检测细粒棘球绦虫细粒棘球蚴体内ROS含量动态变化;采用单细胞凝胶电泳法(彗星实验)以Olive尾距(OTM)作为DNA损伤指标对各组细粒棘球蚴的DNA损伤情况进行分析。同时设活性氧清除剂甘露醇(mannitol,man)与药物联合干预细粒棘球绦虫细粒棘球蚴试验组作比较。结果与甘露醇联合药物干预组比较,青蒿琥酯中剂量组与H_2O_2组细粒棘球蚴死亡率降低(χ~2分别=46.371和59.328,P<0.05);病理学观察青蒿琥酯组细粒棘球蚴顶突小钩脱落,角质层、生发层结构均遭到破坏;透射电镜观察细粒棘球蚴生发层出现大量脂滴,并且有异染色质出现;在330min内,青蒿琥酯低、中和高剂量组细粒棘球蚴ROS含量升高程度均明显高于阿苯达唑组、硝唑尼特组,并具有一定的剂量依赖性;青蒿琥酯高剂量组彗星实验图像拖尾明显,证明细粒棘球蚴DNA受损严重;青蒿琥酯低、中、高剂量组与阿苯达唑组、硝唑尼特组相比,OTM值比较均具有显著性差异(t=9.065、13.134、7.845、9.400、12.251和7.877,P<0.01)。结论青蒿琥酯抗细粒棘球蚴的作用机制可能通过产生ROS导致其DNA损伤所致,为青蒿琥酯抗包虫病作用靶点的筛选和抗包虫病新药的开发奠定了基础。

Objective To examine the mechanism of oxidative damage to Echinococcus granulosus by artesunate in vitro. Methods Artesunate was administered at three concentrations（65μmol/L,130μmol/L,and 325μmol/L）.The solvent dimethylsulfoxide（DMSO）was administered to one control group,H_2O_2 was administered to the positive control group（for measurement of reactive oxygen species（ROS））,and albendazole（ABZ）and nitazoxanide（NTZ）were administered as conventional anti-echinococcosis drugs.Mortality,pathological changes,ultrastructural changes,ROS levels,and DNA damage were analyzed to examine the anti-echinococcal mechanism of artesunate in vitro.The ROS scavenger mannitol（man）was also administered and the effects of each treatment were compared. Results Compared to the man group,the group receiving the intermediate dose of artesunate and the H_2O_2 group had a significantly lower rate of worm eradication（χ~2=46.371 and 59.328,P〈0.05）.Pathologic observations indicated that E.granulosus protoscoleces in the artesunate group shed their hooks and that their cuticle and germinal layer were destroyed.Ultrastructural observation indicated that there were a large number of lipid droplets in the germinal layer of protoscoleces,and heterochromatin was evident.Compared to the ABZ and NTZ groups,the three artesunate groups had a marked increase in ROS levels in a dose-dependent manner within 330 minutes of treatment.Results of a comet assay indicated that a high artesunate concentration resulted in substantial DNA damage in E.granulosus.Compared to the ABZ and NTZ group,the three artesunate groups had a markedly different OTM（t=9.065,13.134,7.845,9.400,12.251 and 7.877,P〈0.05）. Conclusion The mechanism of anti-echinococcal action of artesunate may involve DNA damage mediated by ROSes.This finding has laid the foundation for the screening of targets of artesunate and the development of new drugs to combat echinococcosis.