STUDIES ON THE SIGNALING PATHWAY OF CpG ODNs ACTIVATING proPO SYSTEM IN CHINESE MITTEN CRAB ERIOCHEIR SINENSIS

Prophenoloxidase system(proPO system) plays a key role in the natural immune response of Crustacean animals,but the signal transduction pathway of proPO system is rarely reported.CpG-oligodeoxynucleotides(CpG-ODN) is synthetic oligodeoxynucleotide containing CpG motif that could simulate the bacterial DNA with immunostimulatory activity.Many studies has proved that it is a kind of good non-specificity immunostimulants and specific immunology adjuvant.CpG ODNs could stimulate the proPO system in haemocytes of Macrobrachium rosenbergii,but so far,it has not yet seen a similar study reported in the Chinese mitten crab(Eriocheir sinensis) at home and abroad.The experiment were processed in order to de-termine the signal transduction pathway involved in the CpG ODNs activating proPO system in Chinese mitten crab(Eriocheir sinensis).The crab haemocytes cultured in M-199 medium was treated in vitro with different concentrations of CpG ODN-1670 and ODN-R,as following,0,5,10,15,20 and 25 μg/mL.Then we detected the effects on the proPO system of several activators or inhibitors for specific signaling components,including Sodium fluoride(NaF,G protein activator),Phor-bol-12-myristate-13-acetate(PMA,PKC activator),Chelerythrine(PKC inhibitor),Genistein(tyrosine protein kinase inhibi-tor),worked together with ODN-1670 and ODN-R in this study.Both intra-cellular and extra-cellular relative phenoloxidase(PO) activity including stimulated activity POs and total activity POT were measured to study the signaling way.The results showed that both ODN-1670 and ODN-R could activate the proPO system.The experimental dose of ODN-1670 could induce proPO transforming into PO,while inhibit the releasing of proPO to outside to certain extent.ODN-R could not only induce transforming proPO into PO,but also promote granules releasing.In addition,the signaling pathway of the proPO system activation stimulated by the two ODNs appeared to be some similar to each other,both the signaling pathways may include G-protein mediated PKC way,and ODN-1670 induced proPO activation was negatively regulated via the tyrosine kinase pathway.And some Toll-Like Receptor might exist in the cell membrane,and the first step of ODNs active proPO system was to combine with this Toll-Like Receptor protein then active G-protein mediated pathway.These results have provided basis to the immune defense mechanism study of crab.