The Regulation Function Of Key Molecules In Glycogen Metabolism In Immune Response Of Chinese Mitten Crab Eriocheir Sinensis

The immune system continues to perceive and respond to the threat of the environment which needs to consume a lot of energy,while lack of energy will lead to immune system suppression and reduced resistance,so the regulation of energy during the immune response is critical.Because immune cells lack the storage of nutrients such as glycogen,only the increased intake of nutrients from the microenvironment can sustain these reactions.In this study,Chinese mitten crab Eriocheir sinensis was used as a research object to study the effects of glycogen synthase kinase 3 and its upstream regulatory kinase,protein kinase B(Akt)and extracellular signal regulating kinase 2(ERK2),to explore the role of PI3 K and ERK signaling in the innate immune response of E.sinensis.Glycogen synthase kinase-3(GSK3)is a protein kinase that catalyzes serine / threonine,which was first identified as a modulator of glycogen synthesis.Recently,it has been considered to be the key regulator of the immune reaction.In the current research,a GSK3 homologous gene(designated as EsGSK3)was cloned from Chinese mitten crab,E.sinensis.The open reading frame(ORF)was 1824 bp,which encoded a predicted polypeptide of 607 amino acids.There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3.Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3-? from oriental river prawn Macrobrachium nipponense in the invertebrate branch,while GSK3 s from vertebrates formed the other distinct branch.EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas,eyestalk,muscle,gonad,haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas,and EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis.The expression levels of Es GSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge(p < 0.05),and then gradually decreased to the initial level at 48 h in comparison with control group(p>0.05).The mRNA expression of Lipopolysaccharide-induced tumor necrosis factor(TNF)-? factor(Es LITAF)was also induced by A.hydrophila challenge.However,the mRNA expression of EsLITAF and production of TNF-? was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium,while the phagocytosis of crab haemocytes was significantly promoted.The catalase,alkaline phosphatase activity and nitric oxide(NO)synthesis levels were significantly enhanced.These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E.sinensis by promoting TNF-? production and inhibiting haemocyte phagocytosisERK(extracellular signal-regulated kinases),the upstream kinase of GSK3 pathway,was considered as a regulator of the metabolism of a series of genes in MAPK signaling pathway.In the present study,a homolog gene of ERK(designated as EsERK)was cloned from Chinese mitten crab E.sinensis.The open reading frame(ORF)of EsERK was of 1746 bp,encoding a protein of 581 amino acids.The EsERK mRNA expression levels in haemocyte increased significantly at 6 h after A.hydrophila challenge(p < 0.05),and then gradually decreased till 48 h(p < 0.05).After injecting dsERK into E.sinensis for 24 h,the mRNA transcripts of I?B(Inhibitor of nuclear factor kappa-B)rised significantly(p< 0.05)and superoxide dismutase(SOD)activity was also significantly increased(p< 0.05).Moreover,after lithium was injected into crabs as the specific inhibitor of EsGSK3,the I?B mRNA expression was significantly up-regulated at 6 h post injection(p < 0.05).A novel Akt gene was identified from the cDNA library of E.sinensis and named as EsAkt.Its open reading frame(ORF)was of1395 bp,encoding a protein of 464 amino acids.The theoretical molecular mass of EsAkt was 53.17 kDa and its theoretical isoelectric point was 5.83.Based on RT-PCR analysis,the mRNA transcripts of EsERK were found expressed in all examined tissues,including haemocytes,gonads,hepatopancreas,gills,muscles and eyestalks.The highest expression level was detected in the muscle.The level of transcription of EsAkt was significantly up-regulated after being A.hydrophila challenge.The above results indicated that EsGSK3 could regulate the innate immune response of E.sinensis by inducing TNF-?,inhibiting the phagocytosis of haemocytes,regulating the activity of immune-related enzymes and the synthesis of NO.EsERK could be involved in immune responses by regulating the expression of I?B and TNF-? transcription factors,influencing the inflammatory reactions mediated by PI3K/Akt and Ras/ERK pathways.In addition,EsAkt also showed the response to the bacteria stimulation.In this study,GSK3,ERK and Akt were identified from E.sinensis,not only as critical regulatory molecules of glycogen metabolism,but also vital regulators in the innate immune response,which provide the basis for in-depth study of the PI3 K and Erk signaling pathway for the control of infection and inflammation in E.sinensis.