Preliminary Study On Enkephalinergic Neuroendocrine-immune Regulatory Network In Zhikong Scallop Chlamys Farreri

Neuropeptide is one of the most important components in the neuroendocrine-immuneregulatory network, where enkephalins stand out for its roles both as aneurotransmitter as well as a cytokine in the two-way adjustment of this network.Currently, the research about enkephalins in the neuroendocrine-immune regulatorysystem mainly concentrates on vertebrates. In our study, the molecular features ofmethionine-enkephalin (Met-ENK) and its receptor, and their response patterns aswell as immune regulation in immune response were investigated by using molecularimmunology, biochemistry, cytobiology, etc. technical means to obtain a preliminaryinsight into the pathways of enkephalins and their receptors in scallop Chlamysfarreri.Apparent enkephalin-containing sections were visualized in gill, hemocytes,hepatopancreas, mantle, kidney and gonad via immunofluorescence technique, whilelittle immunoreactivity was observed in adductor muscle. The content of Met-ENK inscallop serum was significantly up-regulated at24and48h post Vibrio anguillarumstimulation,3.5-fold and3.0-fold compared to blank group (P <0.05), respectively.The bacteriostasis and phagocytosis of scallop serum were both enhanced withincreasing concentrations of Met-ENK (0.1-10μM), while the phenoloxidase (PO)activity was only enhanced by0.1μM Met-ENK. It has demonstrated that thelysozyme (LYZ) activity and superoxide dismutases (SOD) activity in scallop serumwere both significantly down-regulated by10μM Met-ENK (P <0.05). Met-ENKinduced higher levels of TNF-activity at the dose of0.1μM,1μM and10μM (P <0.05), respectively, whereas there was no significant difference between them. Opioid growth factor receptor (OGFR) was a receptor for Met-ENK and its gene wascloned and characterized from C. farreri (designated as CfOGFR). The completecDNA sequence of CfOGFR was2381bp, whose deduced protein contained a lowcomplexity domain and a conserved OGFR_N domain at the N-terminal. The aminoacid sequence of CfOGFR shared33-64%similarity with other OGFRs. The mRNAtranscripts of CfOGFR were constitutively expressed in all the tested tissues includingkidney, gonad, hemocytes, mantle, gill, adductor muscle and hepatopancreas, with thehighest expression level observed in hepatopancreas. During the early embryonicdevelopment, the mRNA transcripts of CfOGFR could be detected in all the stages,where the expression level presented a downward trend as a whole. The stimulation ofLPS, Glu or poly (I: C) significantly induced the expression of CfOGFR mRNA inhemocytes (P <0.05). Co-IP and Western blot results revealed that the proteinCfOGFR in hemocytes displayed high affinity and specificity to Met-ENK. Met-ENKwas observed to inhibit the proliferation of HEK293T cells transfected withpcDNA3.1(+)-CfOGFR in a time and dosage dependent manner.It colud be implicated from all the above results that enkephalins and their receptorswere present in C. farreri, and enkephalins could be induced by some bacteria or thepathogen associated molecular patterns (PAMPs). And then, enkephalins functionedto modulate the immune response of scallop through the opioid receptors.Characterization of enkephalins and their receptors in scallops would provide a betterunderstanding of the origin and evolution process of endogenous opioids and theirreceptors, as well as the relationship between the neuroendocrine system and immuneresponse, and the findings on its immunological activity in scallop might contribute tothe health management and disease control in scallop aquaculture.