Functional Study Of G Protein-coupled Receptor (GPCR89) Of Eriocheir Sinensis On Regulating Cerebral Antimicrobial Peptides Expression

G protein-coupled receptors(GPCRs)are the most diverse family of receptors on cell membrane surface receptors.They participate in a variety of physiological processes by transmitting extracellular signals to intracellular effectors,including cells Metabolism,secretion,neurotransmission,cell differentiation and growth,and inflammation and immune response.G protein-coupled receptors have become important drug targets due to their involvement in a large number of human pathological processes,but there function in invertebrate innate immunity is unclear.In view of the important role of GPCRs in a variety of physiological functions and their research status in invertebrates,we took Chinese mitten crab as the research object and explore the way in which GPCRs regulate innate immunity and its molecular mechanism.In this study,a complete GPCR gene cDNA was first screened and cloned from Eriocheir sinensis and identified as GPCR89(EsGPCR89)after sequence comparison.After analysis,the full length of cDNA was 2277 bp and contained an open reading frame(ORF)consisting of 465 amino acid residues.Bioinformatics analysis showed that this gene conformed to the structural characteristics of the GPCRs family,and has highly conserved among species in the amino acid sequence corresponding to the domain.Tissue distribution test proved that EsGPCR89 was widely distributed in Chinese mitten crab,and the expression was higher in hepatopancreas,brain and gill.Staphylococcus aureus in vivo challenge experiments revealed the involvement of Es GPCR89 in the antibacterial immune response in brain.Therefore,we tested the expression of antibacterial peptides in the brain after baterciral simulation,and found that antibacterial peptides could respond to pathogenic stress and show changes in different strengths and different trends,suggesting a potential connection between geneGPCR89 and antibacterial peptides.After Es GPCR89 interference in vivo,the expression of Es GPCR89 in the brain remained normal due to the presence of the bloodbrain barrier,but the expression of antibacterial peptides in the brain after bacterial stimulation was significantly down-regulated compared to untreated crabs.The results further indicate that there is a correlation between EsGPCR89 and antibacterial peptides in the brain,and that the expression of cerebral antibacterial peptides may depend on signals sent by other organs or tissues after pathogen infection.In order to study the pathways of Es GPCR89 involved in the regulation of antibacterial peptides in the brain,we finally found through RNA interference and SDS-PAGE and other methods that Es GPCR89 in hemocytes after bacteria infects the body will cooperate with brain tissue to produce antibacterial peptides to achieve the antibacterial immune process in the brain.In summary,this study found that the expression of antimicrobial peptides in the brain of Eriocheir sinensis was regulated by hemocytes.It is speculated that it may release secreted proteins through hemocytes and activate its antibacterial peptides expression signal transduction through potential brain receptors,thereby achieving antibacterial immune response in brain tissue.