The Effects On Echinococcus Granulosus Protoscoleces By Down-regulated The Expression Of GRP78

Objective: The most confident way for treatment of hydatid cyst is surgical operation.Spillage of the cyst contents during the operation is the major cause of recurrence afterhydatid cyst surgery. Instillation of scolicidal agent into hydatid cyst is the most commonlyemployed measure to prevent this complication. The aim of this study was to thedown-regulated the expression of GRP78via small interfer RNA on Echinococcus granulosusprotoscoleces to explore the role of GRP78in Echinococcus granulosus protoscoleces.Methods: Echinococcus granulosus protoscoleces were collected from cysts of infectedsheep under the sterile conditions. Protoscoleces of Echinococcus granulosus were incubatedin vitro with siRNA-GRP78via electroporation which was based on exist GRP78onEchinococcus granulosus protoscoleces. At the same time, set a control group. While, theexpression level of GRP78was analyzed by Western-Blot and RT-PCR. A0.1%eosin solution(eosin powder in distilled water) was added to the samples in a ratio of1:1. After15min, theviability of protoscolices was determined by observing the change of color under a lightmicroscope. The live protoscolices did not change in color but the dead ones were stained red.The experiment was repeated three times, and draw vitality curve of protoscolices. Of eachculture, a small sample was processed for scanning electron microscopy (SEM). Eachexperiment was repeated three times. At the same time, the expression level of GRP78andCaspase-12was analyzed by Western-Blot, caspase-3activity was detected with Caspase-3ActivityAssay kit. The apoptosis of protoscoleces was detected with TUNEL.Result: Western bolt showed that GRP protein levels decreased in protoscoleces of theexperimental groups. RT-PCR showed that GRP mRNA levels decreased in protoscoleces ofthe experimental groups. Control Echinococcus granulosus protoscoleces incubated inNon-specific siRNA were not altered and remained viable after1days of incubation. Incontrast, a loss of viability in siRNA-GRP78cultures was observed, with a50.19%reductionin the number of viable protoscoleces. The number of dead E. granulosus protoscolecesincreased with siRNA-GRP78exposure time decreased. The maximal protoscolicidal effect ofsiRNA-GRP78was observed after days of incubation. After9days about87.65%ofProtoscoleces were dead. In the early, the results were confirmed on the ultrastructural levelby SEM. SEM analysis demonstrated the drug-induced morphological and structural damagein siRNA-treated protoscoleces. The primary site of drug damage was the parasite tegument.At1day post-incubation, signs of ultrastructural alteration were evident: the soma region wascontracted. The activity of caspase-3was found to be higher in protoscoleces which wereincubated with higer concentions of siRNA-GRP78compared to the controlprotoscoleces.TUNEL assay was found that the siRNA-GRP78can promote the apoptosis of protoscoleces.Conclusion:1. The expression of GRP78protein and GRP78mRNA can be down-regulatedby the RNAinterference.2. It revealed that down-regulated the expression of GRP78in Echinococcusgranulosus protoscoleces had obviously killing effect on Echinococcusgranulosus protoscoleces, the caspase-3and Caspase-12were activated too.