| Gamma-aminobutyric acid(GABA)is a class of non-protein amino acids,which are widely distributed in the central nervous system as the main inhibitory neurotransmitter,and have various physiological functions,such as participate in the regulation of feeding,metabolism,endocrine,and immunity.As a functional feed additive,up to now,most studies of GABA were mainly focused on livestock and poultry animals,whereas the study on aquatic animals,especially shrimp and crab is rare.For benthic crabs,the pond bottom is the living area for most of the time.However,the complex ecological and biological environment at the pond bottom makes crabs more susceptible to pathogens and hypoxia.Therefore,it is crucial to improve the immunity and hypoxia tolerance of Chinese mitten crab by nutrition adjustment strategy.This study employed juvenile Chinese mitten crab(Eriocheir sinensis)as its research object,and a range of research methods and technical approaches were applied in a thorough manner.Firstly,the effects of GABA on the growth and immunity of juvenile crab were investigated from the aspects of regulating food intake,digestion,immunity and antibacterial.Then,the regulatory effect of GABA on hypoxia tolerance was evaluated,and the neural excitotoxicity effect and toxic mechanism induced by hypoxia were explored.Moreover,the molecular mechanism of neuroprotective effect of GABA in hypoxia response were explored.The main results and conclusions are as follows:1.Optimal dietary GABA level and its regulation effect on growth and immunity of juvenile E.sinensisThe objective of this study was to assess the impact of dietary GABA supplementation on food intake,digestive ability,growth performance and immune response of juvenile E.sinensis.Six diets were provided to the crabs(0,40,80,160,320 and 640 mg/kg GABA,respectively)for a period of 8 weeks,and then the lipopolysaccharide(LPS)challenge test was conducted for 24 hours.The results revealed that moderate dietary GABA supplementation could significantly increase the molting frequency(MF),weight gain(WG),feeding rate(FI),and digestion and absorption related enzyme activities of juvenile crabs.The expression of orexigenic neural signal-related genes was significantly up-regulated,and the anorexigenic neural signal-related genes were significantly down-regulated in the cranial ganglia,thoracic ganglia and intestine tissues.Meanwhile,the expressions of the main genes involved in taste modulation and reward regulation were significantly up-regulated,whereas the expression of genes related to punishment regulation was significantly down-regulated in cranial ganglia and thoracic ganglia.The results showed that GABA might act as an orexigenic neural signal or work with other appetite-related nerve signals to promote food intake by participating in regulating the satiety circuit and the reward circuit.A two-slope broken-line regression analysis of specific growth rate and weight gain against the dietary GABA levels revealed that dietary the optimal dietary GABA level was 84-89 mg/kg,and this level can improve growth performance,food intake,digestion and absorption in juvenile E.sinensis.On the other hand,the results showed that moderate dietary GABA supplementation could significantly increase the hemocyanin content,acid phosphatase,and alkaline phosphatase activities,and significantly reduce significantly reduce in serum.Supplementation of moderate dietary GABA could have a significant effect on the superoxide dismutase activity and glutathione content in serum,as well as significantly augmenting the transcriptional expression of antioxidant-related and immune-related genes in hepatopancreas.The m RNA expressions of antilipopolysaccharide factors,inflammatory signaling pathways related genes and proapoptosis-related genes were significantly up-regulated,and anti-apoptosis-related genes were significantly down-regulated in LPS stimulation groups compared with PBS treatment.However,moderate dietary GABA supplementation effectively alleviated LPS-induced immune overresponse and apoptosis.Consequently,this study demonstrates that dietary GABA supplementation could be employed as an immunomodulator to enhance the non-specific immunity and antioxidant capacity,as well as alleviate the immune-mediated immune overresponse of juvenile crabs.In conclusion,dietary GABA supplementation could significantly improve the growth performance and immunomodulatory ability of juvenile E.sinensis.2.The metabolic regulation and neuroprotective effects of GABA on juvenile E.sinensis exposed to acute hypoxia stressIn view of the positive role of GABA in growth and immune regulation,this study further explored the effects of GABA on respiration and energy metabolism of juvenile crabs exposed to acute hypoxia stress,and preliminarily evaluated the nerve damage caused by acute hypoxia and the neuroprotective effect of GABA.Following an 8-week feeding trial,acute hypoxia stress(DO: 1.0 ± 0.1 mg/L)was administered for 24 h.Acute hypoxia stress caused a marked upsurge in m RNA expression level of hypoxiainducible factor 1α(HIF 1α),oxygen consumption rate and anaerobic respiratory metabolism-related enzyme activities,whereas aerobic respiratory metabolism-related enzyme activities were significantly reduced.However,dietary GABA supplementation remodeled the respiratory metabolism pattern of juvenile crabs exposed to hypoxia stress,aerobic respiratory metabolism-related enzyme activities were significantly increased,and anaerobic respiratory metabolism-related enzyme activities were significantly decreased,similarly,the expression level of HIF 1α and oxygen consumption rate were significantly reduced.The expression of crustacean hyperglycemic hormone gene(CHH)was significantly augmented due to acute hypoxia,as well as the contents of hemolymph glucose and lactic acid(LD,in serum and hepatopancreas),whereas the expression of E.sinensis insulin-like peptide gene(Es＿ILP)was significantly down-regulated.The levels of hepatopancreas glycogen and muscle phosphoarginine(PA)were significantly reduced.However,dietary GABA supplementation reversed this trend.In addition,acute hypoxia stress significantly augmented the contents of free glutamate(Glu)and GABA in the nervous tissue.Moreover,the expression levels of N-Methyl-D-aspartate-related receptor(NMDA)and apoptosis-related genes were significantly up-regulated.Dietary GABA could effectively prevent acute hypoxia stress-induced neural damage.Furthermore,dietary GABA could significantly improve the redox status in vivo exposed to hypoxia stress.In conclusion,acute hypoxia could lead to aerobic metabolism inhibition and oxidative stress,and induce nerve tissue damage,whereas dietary GABA supplementation could improve the hypoxia tolerance of juvenile crabs by regulating metabolic patterns,improving antioxidant capacity and exerting neuroprotective effects.3.Neural excitotoxicity and the toxic mechanism induced by acute hypoxia in juvenile E.sinensisPrevious results found that acute hypoxia could induce significant upregulation of apoptosis-related genes in nervous tissue,indicating that acute hypoxia might cause toxic damage to the nervous system.This study investigated the potential toxic effects of acute hypoxia on the nervous system of juvenile crabs,delving into the induction effects and potential mechanism of acute hypoxia stress(DO: 1.0 ± 0.1mg/L)over a period of 24 hours on neural excitotoxicity in juvenile E.sinensis.The results showed that hemolymph glucose and serum LD content were significantly increased,and the m RNA expression of CHH and HIF 1α were significantly up-regulated in the hypoxia group compared with control.RNA-Seq results confirmed that acute hypoxia stress had a more significant impact on carbohydrate metabolism than lipid and protein metabolism.Meanwhile,the hypoxia group exhibited a notably greater rate of nerve cells than the control,and similar patterns were observed in the expression of apoptosis-related genes.RNA-Seq results showed that acute hypoxia stress-induced neuronal apoptosis by regulating multiple apoptosis-related pathways.In comparison to the control group,the hypoxia group exhibited significant greater free Glu and GABA concentrations in the nerve tissue of thoracic ganglia,as well as significantly higher m RNA expression of NMDA related receptors.Similar trends were observed in the expression of calcium-dependent degrading enzymes and endogenous antioxidantrelated proteins or enzymes.Meanwhile,the m RNA expression level of high-affinity neuronal glutamate transporter(n Glt)in the hypoxia group was significantly upregulated compared with the control.Furthermore,NMDA-R antagonists(MK-801 and Ro25-6981)injection showed that NMDA-R served as the bridge and core position of Glu-induced neural neurotoxicity.In conclusion,the inhibition of energy metabolism and redox imbalance caused by acute hypoxia stress affected the reuptake of Glu by glutamate transporters,which led to excessive opening of NMDA-type channels and activation of a series of degrading enzymes,and finally induced neural neurotoxicity and neuronal toxic damage of juvenile E.sinensis.4.Comprehensive physiological and multi-omics analysis explores the neuroprotective mechanism of dietary GABA response to hypoxia stress in juvenile E.sinensisThe above studies showed that acute hypoxia could induce neural neurotoxicity,and GABA exhibited a protective effect.To reveal the neuroprotective pathway and metabolic regulatory mechanism of GABA in E.sinensis exposed to hypoxia stress,an8-week feeding trial and acute hypoxia challenge(DO: 1.0 ± 0.1 mg/L)were performed.Subsequently,this study performed an apoptosis analysis and comprehensive transcriptomic and metabolomic analysis of the thoracic ganglia.The results showed that GABA pretreatment significantly reduced the neuronal apoptosis rates of cranial ganglia and thoracic ganglia compared with the hypoxia group.The results of omics analysis showed that differential genes and differential metabolites were co-annotated to 11 KEGG pathways,and further significant analysis showed that only the sphingolipid signaling pathway and the arachidonic acid metabolism pathway were significantly enriched.In the sphingolipid signaling pathway,GABA treatment significantly increased long-chain ceramide content in thoracic ganglia,which exerted neuroprotective effects by activating downstream signals to inhibit hypoxia-induced apoptosis.In the arachidonic acid metabolism pathway,GABA could increase the content of neuroprotective active substances and reduce the content of harmful metabolites by regulating the metabolism of arachidonic acid for inflammatory regulation and neuroprotection.This study reveals the neuroprotective pathways and possible mechanisms of GABA in juvenile E.sinensis exposed to hypoxia stress and inspires the discovery of new targets for improving hypoxia tolerance in aquatic animals. |
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