In Vitro Effectiveness Of P38Inhibitor On Echinococcus Granulosus Protoscoleces

Objective: The aim of this study was to investigate the in vitro efficacy of the P38inhibitor ML3403against Echinococcus granulosus protoscoleces.Methods: Echinococcus granulosus protoscoleces were collected from cysts of infected sheep, and thenwere cultured in RPMI1640medium for5days. Protoscoleces of Echinococcus granulosus were incubatedin vitro with ML3403at concentrations of12.5,25,50,80,100μmol/L. The viability of protoscoleces wasassessed on a daily basis by microscopic observation of movements, flame cell activity, and0.1%eosinstaining. The corresponding numbers of viable/non-viable protoscoleces were determined in10randomlychosen fields by phase contrast microscopy using an inverted microscope at10×magnification. Of eachculture, a small sample was processed for scanning electron microscopy (SEM) and transmission electronmicroscopy (TEM). Each experiment was repeated three times. At the same time, the expression level ofp38was analyzed by Western-Blot and caspase-3activity was detected with Caspase-3Activity Assay kit.Result：Control Echinococcus granulosus protoscoleces incubated in DMSO were not altered and remainedviable after5days of incubation. In contrast, a loss of viability in50μmol/L ML3403-treated cultures wasobserved, with a50.5%reduction in the number of viable protoscoleces. The number of dead E. granulosusprotoscoleces increased with ML3403exposure time, and the viability decreased. The maximalprotoscolicidal effect of100μmol/L ML3403was observed after5days of incubation, when viableprotoscoleces could no longer be observed in cultures treated with100μmol/L of ML3403. The treatmentwith either50μmol/L or80μmol/L also showed a protoscolicidal effect, which was reached later than thatobserved for100μmol/L. The results of viability tests coincide with the tissue damage observed at thestructural level. Protoscoleces incubated with DMSO (control group) revealed no changes in structurethroughout the experimental period. After1day of incubation, the presence of numerous blebs in thetegument of protoscoleces treated with100μmol/L was observed by inverted microscope. These resultswere confirmed on the ultrastructural level by SEM. SEM analysis demonstrated the drug-inducedmorphological and structural damage in ML3403-treated protoscoleces. The primary site of drug damagewas the parasite tegument. At1day post-incubation, signs of ultrastructural alteration were evident: thesoma region was contracted. Numerous blebs were observed on the tegument surface of the scolex regionof some protoscoleces treated with ML3403. At100μmol/L, rostellar disorganization, loss of hooks andshedding of microtriches of the scolex region were observed. In some protoscoleces, the presence ofdigitiform tegumental extensions appeared on the tegument of the soma region. Additionally, loss ofmorphology was evident in some protoscoleces. Western bolt showed that p38protein levels decreased inprotoscoleces of the experimental groups. The activity of caspase-3was found to be higher in protoscoleceswhich were incubated with higer concentions of ML3403compared to the control protoscoleces.Conclusion: It revealed that ML3403in vitro had obviously killing effect on Echinococcus granulosus protoscoleces, the P38inhibitor ML3403may represent a new strategy in treating hydatid cystechinococcosis.