The Effectiveness Of Nrf2 Inhibitor On Echinococcus Granulosus Protoscoleces

Object: The aim of this study was to investigate the in vitro efficacy of the Nrf2 inhibitor DRB against Echinococcus granulosus protoscoleces.Methods: 1. In vitro culture of E. granulosus protoscoleces: Echinococcus granulosus protoscoleces were collected from cysts of infected sheep, and were cultured in RPMI 1640 medium. 2. Drug treatment of E.granulosus protoscoleces: Protoscoleces treatment was initiated within 5 days of in vitro culture. Every holes panel with at least 1500 protoscoleces in 20 ml culture medium were supplemented with 25, 50, 75g/l,DRB [stock solution in 10 mg/ml in dimethyl sulphoxide(DMSO)], and DMSO control was supplemented with 20 ml DMSO alone. The viability of protoscoleces was assessed by observation of inverted microscope with eosin staining. Drawing dynamic curve. Observe the protoscolex ultrastructural by electron microscope. Use the western blot analysis the protoscoleces Nrf2. Detecting the NQO-1 and HO-1changes of protoscoleces treated with DRB by ELISA.Result:Control group > 95% of the parasites were still viable after 6 days. Protoscoleces cultured with75g/l DRB were killed considerably faster than protoscoleces cultured with 25, 50g/l DRB. At 50 and 75g/l DRB had a clearly decreased efficacy, 75 g/l killed all protoscoleces by day 6; 50 g/l left 50% of the protoscoleces viable after 4 days of treatment, and left small but detectable numbers of viable protoscoleces after 6 days. DRB at 25 g/l had no significant effect on protoscoleces. Scanning electron microscopy(SEM)demonstrated the morphological and structural damage occurring in DRB-treated protoscoleces. In the control group, no ultrastructural or morphological damage was seen in six days after culturing One day after 50 g/l DRB treatment, no differences were detected in protoscoleces; however, on day 3, they revealed ultrastructural changes, with multiple pits arising on the outer surfaces of the germinal membrane,disruption of the external plasma membrane, collapse of the sucker region, and contraction. The 75 g/l DRB treatment caused extensive damage to the tegument, including loss of microtriches on the rostellum on day 3, and on day 6 had caused the destruction of the rostellum and the germinal layer. The expression of Nrf2, NQO-1 and HO-1 decreased in the protoscoleces.Conclusion: It revealed that DRB in vitro had obviously killing effect on Echinococcus granulosus protoscoleces, and the damage to the ultrastructure, DRB as an agent downregulating its expression.