PIK-75对体外细粒棘球蚴原头节的作用研究

Parasiticidal effects of PIK-75 against Echinococcus granulosus protoscoleces in vitro

目的探讨不同浓度PIK-75体外对细粒棘球蚴原头节生长作用及形态学的影响。方法将体外培养的细粒棘球蚴原头节分为空白对照组及PIK-75处理组(0.4、0.8、1.2、1.6及2.0μmol/L),处理后的原头蚴用经0.1%伊红染色,置于倒置显微镜下观察原头节的形态及活力,试验独立重复3次并绘制活力曲线图。用Caspase-3试剂盒检测作用24、48h后各浓度组原头节体内caspase-3酶活性;使用ELISA试剂盒测定不同浓度PIK-75作用后原头节体内抗氧化酶HO-1及NQO-1的活性变化。结果 PIK处理组原头节的形态结构发生变化,活力减弱,以2.0μmol/L时组原头节变化更明显,且在第6d时原头节均发生死亡。随着药物浓度的增高,PIK-75对细粒棘球蚴原头节生长的抑制作用越强。0.8μmol/L PIK-75处理组作用24h后原头节活性开始下降,在第3d约为67.4%,作用7d后活力为32.1%,空白对照组原头节形态及活力无明显变化。不同浓度PIK-75作用细粒棘球蚴原头节24、48h后caspase-3酶活性显著高于空白对照组(P<0.05)。0.8μmol/L PIK-75作用原头节2d、5d后,HO-1活性分别为(704.265±5.082)pg/ml和(656.05±0.095)pg/ml,NQO-1活性分别为(2.236±0.018)ng/ml和(1.446±0.026ng/ml),与空白对照组比较显著降低(P<0.05),且作用5d后的原头节酶活性显著低于作用2d组(P<0.05)。结论 PIK-75能抑制体外细粒棘球蚴原头节的生长,降低抗氧化酶的活性,具体机制有待进一步研究。

Objective The aim of this study was to examine the parasiticidal effects of different concentrations of PIK-75 a- gainst Echinococcus granulosus protoscoleces in vitro. Methods E. granulosus protoscoleces were collected aseptically from cysts in infected sheep and cultured in vitro with RPMI 1640. The following final concentrations of PIK-75 were used： 0. 4 μmol/L, 0.8 μmol/L, 1.2 μmol/L, 1.6 μmol/L, and 2.0μmol/L）. Evidence of parasite damage and death was revealed by 0. 1% eosin staining. The experiment was independently performed in triplicate and a viability curve was plotted. Protoscoleces were treated with different concentrations of PIK-75 and Caspase-3 activity was detected using an assay kit. The effects of PIK-75 on the levels and activity of HO-1 and NQO1 were characterized using ELISA. Results Morphological damage and reduced viabili- ty were evident in PIK-75-treated protoscoleces. Protoscoleces cultured with 2. 0 μmol/L of PIK-75 were no longer viable after 6 days. PIK-75 increased the mortality of protoscoleces in a dose-dependent manner. In addition, treatment with 0.8 μmol/L of PIK-75 reduced the number of viable protoscoleces within a period of 1 day and it reduced that number 67.4% after 3 days; 7 days after treatment, 32. 1 % of protoscoleces were still viable. In the control group, changes in morphology and viability were not noted during the experimental period. All concentrations induced a significant increase in caspase-3 activity at 24 h and 48 h compared to the control group （P〈0. 05）. ELISA results indicated that treating cultured protoseoleces with 0.8 μmol/L of PIK- 75 for either 2 or 5 days caused a significant decrease in both HO-1 and NQO-1 production in a time- and dose-dependent manner （P〈0.05）. Conclusions PIK-75 had an inhibitory effect on the growth of Echinococcus granulosus protoscoleces and marked- ly decreased the activity of antioxidant enzymes. Nonetheless, the detailed mechanisms by which PIK-75 kills E. granulosus pro- toscoleces still need to be studied further.