| Objective: The aim of this study was to investigate the in vitro efficacy of the ERK inhibitor PD98059 against Echinococcus multilocularis protoscoleces.Methods: E.multilocularis metacestodes, originally isolated from humans, were maintained in Mongolian gerbils by intraperitoneal injection of minced metacestode tissue. Obtaining Metacestodes by dissect the gerbils after injecting 4-6 months. After washing three times,metacestodes tissue were minced and passed through a sieve(100-μm mesh size),and then washed repeatedly with PBS(PH=7.2) until host materials were drastically removed. Echinococcus multilocularis protoscoleces were collected and maintained in Mongolian gerbils by intraperitoneal injection of minced metacestode tissue to make mouse models. Then the mouse models were dissect when cultured for 4 months, Echinococcus multilocularis protoscoleces were collected and cultured in Dulbecco’s minimal essential Medium(DMEM) for 10 days.Protoscoleces of Echinococcus multilocularis were incubated in vitro with PD98059 at concentrations of 0, 6.25, 12.5, 25, 50, 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 p-ERK was analyzed by Western-Blot; caspase-3activity was detected with Caspase-3 Activity Assay kit; Apoptosis was detected with TUNEL Detection Kit.Result:Throughout the experiment, the viability of Echinococcus multilocularis protoscoleces incubated in DMSO control group was 85±0.124%, while after 6d when Echinococcus multilocularis protoscolecesthe exposed to 6.25μmol / L, 12.5μmol / L, 25μmol / L,50μmol / L of PD98059,the viability were 53.93±0.082%, 51.69±0.297%, 38.18±0.254%, 28.21±0.241%;continuous observation, in the 14 d, the viability of Echinococcus multilocularis protoscoleces exposed to 6.25μmol / L, 12.5μmol / L, 25μmol / L PD98059 group were only 25.48±0.271%,20.1±0.092%, 12.17±0.197%, 50μmol / LPD98059 group was 0.53±0.012%. Thus confirmed that the inhibition of growth were more obvious when with the increase of PD98059 treatment time and concentration. For protoscoleces incubated in DMSO, no changes in ultrastructure were observed throughout the experimental period: microtriches the structure surfaces were complete and internal structures were intact. In comparison with control protoscolices, the protoscolices treated with PD98059 showed marked structural changes: microtriches on surfaces become incomplete and increase in the number of vacuoles and lipid droplets of the soma region.Additionally, loss of morphology was evident in some protoscoleces. Western bolt showed that p-ERK 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 PD98059 compared to the control protoscoleces.TUNEL assay was found that the PD98059 can promote the apoptosis of protoscoleces.Conclusion: It revealed that PD98059 in vitro had obviously killing effect on Echinococcus multilocularis protoscoleces, the ERK inhibitor PD98059 may represent a new strategy in treating hydatid cyst echinococcosis. |
