Veliparib联合青蒿琥酯体外抗细粒棘球蚴作用机制的研究

Study on the mechanism of the effect of veliparib combined with artesunate against echinococcosis in vitro

目的观察DNA损伤修复抑制剂Veliparib联合青蒿琥酯体外对细粒棘球蚴活性的影响,分析Veliparib联合青蒿琥酯抗囊型包虫病的作用机制。方法将细粒棘球蚴分为空白组,DMSO组,Veliparib(10μmol/L)组,硝唑尼特(nitazoxanide,NTZ)组,H2O2组,青蒿琥酯低(65μmol/L)、中(130μmol/L)、高(325μmol/L)剂量组,H2O2+Veliparib组,青蒿琥酯低剂量+Veliparib组,青蒿琥酯中剂量+Veliparib组、青蒿琥酯高剂量+Veliparib组,共12组。采用1%伊红染色法检测药物干预2、3、4d细粒棘球蚴的活性,计算虫体死亡率。给药组干预细粒棘球蚴4d后,观察虫体组织病理学和超微结构变化;通过彗星试验评价DNA损伤情况;采用免疫荧光法观察虫体内DNA损伤标志物8-羟基脱氧鸟苷(8-oxo-dG)的变化。结果与DMSO组和青蒿琥酯各剂量组相比,青蒿琥酯低、中、高剂量联合Veliparib组细粒棘球蚴死亡率均显著升高(P<0.01);组织病理学观察青蒿琥酯各剂量联合Veliparib组细粒棘球蚴虫体损坏情况较青蒿琥酯各剂量组严重且明显固缩,着色加深,并有空泡形成;超微结构观察青蒿琥酯各剂量联合Veliparib组的细粒棘球蚴合胞体带混沌,细胞结构破坏,异染色质边际化,出现脂滴、空泡样结构,微绒毛明显破坏或减少;彗星试验显示青蒿琥酯各剂量联合Veliparib组细粒棘球蚴彗星拖尾较低、中、高青蒿琥酯组明显加长,Olive尾矩(OTM)值差异均有统计学意义(t值分别为5.358,2.482和4.224,P<0.05)。免疫荧光显示青蒿琥酯高剂量+Veliparib组8-oxo-dG的阳性核数较青蒿琥酯高剂量组增多。结论 DNA损伤修复抑制剂Veliparib增强青蒿琥酯抗囊型包虫病作用,其作用机制是使虫体的DNA损伤更严重,从而导致棘球蚴死亡,为开发新的抗包虫病药物奠定了理论基础。

Objectives To observe the effect of the DNA repair inhibitor veliparib combined with artesunate（AS）on the activity of Echinococcosis granulosus in vitro and to analyze the mechanism of veliparib combined with AS against cystic echinococcosis. Method Twelve groups were infected with protoscoleces（PSCs）of E.granulosus：a control group,agroup treated with DMSO,agroup treated with veliparib（10μmol/L）,agroup treated with nitazoxanide（NTZ）,agroup treated with H2 O2,3 groups treated with a low（65μmol/L）,medium（130μmol/L）,or high concentration（325μmol/L）of AS,agroup treated with H2 O2＋veliparib,agroup treated with a low concentration of AS（65μmol/L）＋ veliparib,agroup treated with a medium concentration of AS（130μmol/L）＋ veliparib,and a group treated with a high concentration of AS（325μmol/L）＋ veliparib.The activity of PSCs was detected with 1% eosin staining 2,3,and 4 dafter treatment,and mortality was calculated.Four d after treatment,histopathological and ultrastructural changes in PSCs were observed in different groups.DNA damage was evaluated with a comet assay,and changes in 8-oxo-dG in PSCs were observed using immunofluorescence. Results Compared to the groups treated with DMSO andAS,groups treated with AS combined with veliparib had significantly increased PSC mortality（P〈0.01）.Compared to groups treated with AS,groups treated with AS combined with veliparib had PSCs that were seriously damaged,markedly contracted,and darker in color and that contained cavities according to pathological observations.Ultrastructural observation indicated that the PSCs in groups treated with AS combined with veliparib had a disrupted tegumental syncytium,a damaged cellular structure,marginalized heterochromatin,evidence of lipid droplet-like and cavity-like structures,and marked damage to or a reduction in microvilli. Results of the comet assay indicated that PSCs in groups treated with AS combined with veliparib produced a significantly longer trailing tail and a significantly greater Olive tail moment（OTM）（t=5.358,2.482,4.224,P〈0.05）.Immunofluorescence results indicated that the group treated with a high concentration of AS combined with veliparib had a greater number of 8-oxo-dG-positive cells compared to the group treated with a high concentration of AS. Conclusion The DNA repair inhibitor veliparib markedly enhanced the anti-echinococcal effect of AS by increasing the severity of DNA damage in PSCs,resulting in their death.This study has laid the foundation for the development of new anti-echinococcosis drugs.