Proteomic analysis of hemocyte reveals the immune regulatory mechanisms after the injection of corticosteroid-releasing hormone in mud crab Scylla Paramamosain

Abstract

Corticosteroid-releasing hormone (CRH) is a crucial neuroendocrine-immune factor regulating the immune response of Scylla paramamosain. To understand the regulatory mechanisms of CRH in S. paramamosain, the hemolymph of S. paramamosain with injection of CRH (1.5 ng/crab) at 24 h were chosen to perform proteomic analysis in this study. Furthermore, quantitative real-time PCR (RT-PCR) method was used to validate the accuracy of proteomic data at 24 h after CRH injection. The proteomic data showed that 255 DEPs were identified, in which 231 and 24 were up- or down-regulated, respectively. Besides, the results of enriched pathways showed that the DEPs were involved in signaling pathways, cellular immunity, humoral immunity and the response of immune related processes. These results revealed that CRH promoted the activation of signal transduction, regulated immune systems and antioxidation, and enhanced the immune related processes (such as protein synthesis, protein transport, carbohydrate mobilization and energy redistribution). These findings will benefit to foster the understanding on the effects of glucocorticoids on neuroendocrine-immune (NEI) networks of crustacean, and supply a substantial material and foundation for further researching of the NEI response. SignificanceCorticotrophin-releasing hormone (CRH) is a 41-amino acid neuropeptide and has been preliminarily studied in aquatic animals. CRH can regulate many important physiological activities comprising protein synthesis, energy metabolism, growth, breeding and behavior in fish, which play an important roles in neuroendocrine-immune (NEI) regulatory network of fish. The neuroendocrine system of crustacean has a primary research, that inspired by fish NEI network. Despite the research on the neuroendocrine system in crustacean has rapidly increased in recent years, our understanding of the regulation between neuroendocrine system and immune system in crustacean is still limited. The research on the strategy of NEI network in crustaceans becomes a significant issue.In the present study, the isobaric tags for relative and absolute quantification (iTRAQ) technology approach were applied to examine the NEI network of Scylla Paramamosain. control group and treatment group (CRH: 1.5 ng/crab) were settled for the iTRAQ experiment, and sampled at 24 h after CRH injection. The study aimed to gain knowledge on the immune response in Scylla Paramamosain after CRH injection and identify related differentially expressed proteins (DEPs) of the crab. The results of this study provide a preliminary resource for analysis the immune mechanism for crustaceans.In general, our work represents the first report of the utilization of the iTRAQ proteomics method for the study of NEI regulatory network in Scylla Paramamosain after CRH injection. We identified a number of DEPs involved in diverse pathways including immune signaling pathways, cellular immunity, humoral immunity, immune related process. These results demonstrated a very complex network involving immune and multiple related metabolic pathways in hemocytes of Scylla Paramamosain and will be of great value in understanding the crab neuroendocrine-immune immune mechanism.