In Vitro Effects Of CA、LCA And CDCA On Echinococcus Granulosus Protoscoleces

Objective: The aim of this study was to investigate the in vitro efficacy of the cholic acid(CA)、lithochalic acid(LCA) and chenodeoxycholic acid(CDCA) against Echinococcus granulosus protoscoleces.Methods: Echinococcus granulosus protoscoleces isolated from cysts of infected sheep were cultured in RPMI 1640 medium for 5 days. Protoscoleces of Echinococcus granulosus were incubated in vitro with CA、LCA and CDCA at concentrations of 500, 1000, 2000, 3000 μmol/L. The viability of protoscoleces was assessed on a daily basis by microscopic observation of movements, flame cell activity, and 0.1% eosin staining. The corresponding numbers of viable/non-viable protoscoleces were determined in 10 randomly chosen fields by phase contrast microscopy using an inverted microscope.The experiment was repeated three times and then draw the vitality curve. Of each culture, a small sample was processed for transmission electron microscopy(TEM). Each experiment was repeated three times. At the same time, the expression level of GRP78 was analyzed by Western-Blot and caspase-3 activity was detected with Caspase-3 Activity Assay kit.Result:Protoscoleces incubated in the culture medium containing dimethyl sulfoxide(DMSO) showed no changes during the entire experimental per iod. Protoscoleces of Echinococcus granulosus were exposed to CA、LCA and CDCA at concentrations of 500, 1000, 2000, 3000 μmol/L. The viability of CA were 94.12%、90.56%、81.43%、59.15% after 6 days of incubation; continuous observation, after 12 days, the viability were only 85.86%、74.53%、56.84%、26.45%, the maximal protoscolicidal effect of 3000μmol/L CA was observed after 18 days of incubation, when viable protoscoleces could no longer be observed in cultures treated with 3000μmol/L CA. The viability of LCA were 93.63%、87.59%、75.00%、42.70% after 1 day of incubation; continuous observation, after 3 days, the viability were only 59.26%、43.97%、32.56%、7.45%, the maximal protoscolicidal effect of 3000μmol/L LCA was observed after 5 days of incubation, when viable protoscoleces could no longer be observed in cultures treated with 3000μmol/L LCA. The viability of CDCA were 95.97%、87.95%、83.17%、23.70% after 3 days of incubation; continuous observation, after 5 days, the viability were only 95.23%、81.48%、49.04%、1.54%, the maximal protoscolicidal effect of 3000μmol/L CDCA was observed after 6 days of incubation, when viable protoscoleces could no longer be observed in cultures treated with 3000μmol/L CDCA. For protoscoleces incubated in DMSO, no changes in ultrastructure were observed throughout the experimental period. In comparison with control protoscoleces, the protoscoleces treated with CA、LCA and CDCA showed marked structural changes: contraction of the soma region, formation of blebs, rostellar disorganization, loss of hooks, destruction of microtriches, and formation of vesicles, lipid droplets, and lamellar bodies. Additionally, loss of morphology was evident in some protoscoleces. Western bolt showed that GRP78 protein levels decreased in protoscoleces of the experimental groups. The activity of caspase-3 was found to be higher in protoscoleces which were incubated with higer concentions of CA、LCA and CDCA compared to the control protosc oleces. Thus confirmed that the inhibition of growth were more obvious when with the increase of CA、LCA and CDCA treatment time and concentration.Conclusion: It revealed that CA、LCA and CDCA in vitro had obvious ly killing effect on Echinococcus granulosus protoscoleces, which may represent a new strategy in treating hydatid cyst echinococcosis.