The Effect Of Nrf2 Signaling Pathway Inhibition On Echinococcus Granulosus Protoscoleces

Objective: The aim of the present work was to observe the inhibitory impact of retinoic acid(RA) on Nrf2 signaling pathway in Echinococcus granulosus protoscoleces, and to further explore the in vitro effect of Nrf2 signaling pathway inhibition on Echinococcus granulosus protoscoleces.Methods: Freshly E. granulosus protoscoleces collected by aseptically puncturing the liver hydatid cysts of an naturally infected sheep were incubated in vitro in RPMI 1640 medium. The protoscoleces were incubated in vitro with RA at concentrations of 2, 4, 6, 8, and 10 μmol/L, and set up the blank control group.Immunofluorescence technology was performed to detect the localization and expression of the Nrf2 protien in protoseoleces. WST or ELISA were used to detect the activities of GST and HO-1 in protoscoleces cultured with RA. Viability and changes in morphology were investigated by 0.1% eosin staining test. The test was repeated for three times. The ultrastructural alterations were visualized by scanning electron microscopy(SEM). Additionally, after 24 h and 48 h post-incubation, caspase-3 activity was measured with an caspase-3 activity assay kit.Results: Immunolocalization revealed that Nrf2 protein was mainly distributed in the cytoplasm of E.granulosus protoscoleces. After incubation with 8 μmol/L RA, the localization and expression of the Nrf2 in protoscoleces did not change significantly. RA efficiently decreased the activities of GST and HO-1 in protoscoleces in a time- and dose-dependent manner(P<0.05). The scolicidal action of different concentrations of RA was observed. Obvious protoscolicidal effect was seen with RA at concentrations of 8μmol/L and 10 μmol/L. After 5 days post-incubation, the vability was only 9.58% in 8 μmol/L RA, and there were not survival protoscoleces in 10 μmol/L RA. With the prolonging of exposure time, the lethal effect of RA against protoscoleces was more apparent. SEM showed that the primary site of drug damage was the tegument of the protoscoleces. The ultrastructural alterations included contraction of the soma region, rostellar deformation, and shedding of microtriches. The presence of irregular cavities and digitiform tegumental extensions in the tegument of the soma region were observed in protoscoleces cultured with a higher concentration of RA. Besides, the caspase-3 activity significantly increased in protoscoleces after 24 h and 48 h of RA incubation compared to the untreated controls(P < 0.05).Conclusion: 1. RA efficiently reduced the activities of Nrf2-dependent GST and HO-1 in E. granulosus protoscoleces, but may have no effect on the expression of Nrf2.2. RA demonstrated clearly scolicidal activity against E. granulosus protoscoleces and could induce protoscoleces apoptosis.