| Eomecon chionantha Hance is a perennial wild herbaceous plants of Chelidonium Eomecon in Papaveraceae family.It is a endemic species of China and abundant in resources, and also having many pharmacological effects. It has been proved that the Eomecon chionantha alkaloids (ECA) extracted from the roots and the rhizomes of Eomecon chionantha Hance was a good molluscicide and well worth developing. Study of the molluscicide mechanism of ECA on Oncomelania hupensis will provide scientific evidences for the application of Eomecon chionantha Hance as a plant molluscicide.This research was supported by National Natural Science Fund and Changsha Technology Project. The molluscicide mechanism of ECA on Oncomelania hupensis was studied by using the snail livers as samples. According to the immerse method recommended by WHO for molluscicide screening, Oncomelania hupensis were immersed in 10mg/L ECA or clean water for 36 hours and livers were isolated:①The total protein and glycogen content of the livers, ALT, AST, ACP,AKP, LDH and MDA level were determined.②Differentially expressed proteins between ECA group and control group were separated by two-dimensional gel electrophoresis and analyzed by mass spectrometry. ③The DNA of liver cells were extracted and analyzed by agarose gel electrophoresis.④Comminute the liver tissues to obtain the liver cell suspension, and the apoptosis of liver cells induced by ECA were analyzed by flow cytometry. The main results attained were as follows:(1) The total glycogen and protein content, the activity of AKP, ALT, AST, LDH were evidently decreased in ECA processed snails, there were significant differences between ECA treated group and control group(P<0.05). The differences of ACP and MDA between ECA treated group and control group were not significant(P>0.05).(2) The conditions of 2-DE for liver proteins from Oncomelania hupensis were established, a high resolution and reproducible 2-DE image was successfully obtained.There were 354±10 and 311±12 spots observed in ECA group and in water group respectively(n=3). Eleven differential expressed proteins were identified and all were up-regulated in ECA group. The eleven identified protein were hypothetical protein, ENSANGP00000022175,class I aldehyde dehydrogenase, unknown protein, ENSANGP00000027067, protein similar to ankyrin 3 isoform 1 isoform 4, protein similar to hepatitis A virus cellular receptor 1 short form, mitochondrial isocitrate dehydrogenase 2-like, protein similar to keratin, keratin-10, keratin-1 respectively.(3) The typical DNA Ladder map was not observed in agarose gel electrophoresis of Oncomelania hupensis liver cells treated by ECA. Flow cytometry displayed that the early apoptotic cells percentage of ECA treated group and control group were 13.77% and 13.70% respectively,late stage of apoptotic cells were 10.74% and 10.62% respectively,the total apoptosis rate of liver cells 24.51% and 24.32% respectively, the difference were not significant (P>0.05).From above results we concluded:(1) Through inhibiting the AKP, ALT, AST, LDH enzyme activity, decreasing glycogen and protein content, the drug ECA destroy the liver metabolic balance, lead to liver disfuction, then eventually kill the snail.(2) The conditions of 2-DE for liver proteins from Oncomelania hupensis were established.(3) Expression of liver proteins of Oncomelania hupensis were changed after treated with ECA and eleven differential expressed proteins were successfully identified in this study. Identify of the differential expressed proteins may provided new information for elucidating the underlying mechanism of ECA-induced liver injury.(4) Tentative confirmed that cell apoptosis was not the way of ECA to kill the snails.
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