β-glucan Mitigated Toxicological Effects Of Hypoxic Stress And Cu Stress On Large Yellow Croaker Pseudosciaena Crocea

Large yellow croaker（Larimichthys crocea）is one of the most commercially important marine fish species in China.High-density culture is often employed in aquaculture practice to increase profitability,which may result in poor water quality including hypoxia,and white spot disease outbreak caused by the ciliate Cryptocaryon irritans.Copper sulphate（CuSO₄）is one of the most effective agents against Cryptocaryon irritans,which may cause significant amounts of Cu in farming water.Large yellow croak is especially susceptible to environmental variations,and the high mortality of farmed fish partly caused by poor water quality including hypoxia and Cu pollution,resulting in substantial economic losses.Restriction of antibiotics in fish feed has prompted research toward alternative products for health management,such as prebiotics.Therefore,understanding the characteristics ofβ-glucan ameliorating hypoxia-induced oxidative damage and Cu-induced toxicological effect in large yellow croaker may be helpful to the development of strategies and measurements for sustainable development of its culture.In the present study,based on the methodology of aquaculture,environmental stress physiology,biochemistry and molecular biology,the effects ofβ-glucan on energy metabolism,oxidative stress and inflammation in large yellow croaker under hypoxia stress and copper stress were evaluated systematically.β-Glucan decreases intensity of hypoxia-induced oxidative stress in large yellow croaker and its corresponding mechanisms:To evaluate the effects ofβ-glucan on acute hypoxia-induced oxidative stress in the liver of large yellow croaker,lipid peroxidation,activities and mRNA levels of antioxidant enzymes（Cu/Zn-SOD,CAT,GPx,GR and GST）,and gene expression of the Keap1-Nrf2 signaling molecules were determined.Fish were injected withβ-glucan at a dose of 0 or 5 mg kg^-1 body weight on 6,4 and 2 days before exposed to 1.5 and 7.0 DO mg/L for 24 h.The results showed that normoxia withβ-glucan had no effect on lipid peroxidation compared with the normoxic control,hypoxic stress withβ-glucan remarkably reduced lipid peroxidation during the whole experiment compared with hypoxic stress,indicating thatβ-glucan could ameliorate hypoxia-induced lipid peroxidation.Obtained results also showed a coordinated transcriptional regulation of antioxidant genes,suggesting that Nrf2 was required for regulating these genes.Furthermore,a negative relationship between the mRNA levels of Nrf2 and Keap1indicated that Keap1 plays an important role in switching off the Nrf2 response.In conculsion,β-glucan could alleviate cute hypoxia-induced oxidative stress in large yellow croker,emphasizing a central role of transcription factor Nrf2 in the process.Effects ofβ-glucan on ROS production and energy metabolism in yellow croaker under acute hypoxic stress and its corresponding mechanisms:To evaluate the effects ofβ-glucan on acute hypoxia-induced oxidative stress and changes in energy metabolism in the liver of large yellow croaker,ROS production,activities and mRNA levels of energy metabolism enzyme（PK,F-ATPase,SDH and MDH）,and gene expression of HIF-1αat different exposure times（3,6,12,24 and 48h）were determined.The results showed thatβ-glucan enhanced survival rate and reduced ROS during the lethal hypoxic stress,indicating thatβ-glucan could ameliorate hypoxia-induced oxidative stress.Obtained results also showedβ-glucan could upregulate activities and mRNA levels of PK,demonstrating thatβ-glucan increased anaerobic glycolysis capacity.Furthermore,a coordinated transcriptional regulation of energy metabolism enzyme genes,suggesting that HIF-1αis required for regulating these genes.In conculsion,β-glucan could alleviate cute hypoxia-induced oxidative stress in large yellow croker by enhancing anaerobic glycolysis capacity,emphasizing a central role of transcription factor HIF-1αin the process.Differential effects ofβ-glucan on oxidative stress,inflammation and copper transport in two intestinal regions of large yellow croake under acute copper stress:To evaluate investigate the effects ofβ-glucan on oxidative stress,inflammation and copper transport in two intestinal regions of large yellow croaker under acute copper stress,fish were injected withβ-glucan at a dose of 0 or 5 mg kg-1 body weight on 6,4 and 2days before exposed to 0 and 368μg Cu L-1 for 48 h.Biochemical indicators（MDA,Cu content,MTs protein levels,Cu/Zn-SOD,CAT and iNOS activities）,gene expressions of oxidative stresses（Cu/Zn-SOD,CAT,Nrf2,MTs and MTF-1）,inflammatory responses（NF-κB,iNOS,IL-1β,IL-6 and TNF-α）and Cu transporters（ATP7A,ATP7B and CTR1）were determined.In the anterior intestine,β-glucan increased MTs levels,activities of Cu/Zn-SOD,CAT and iNOS,mRNA levels of MTs,CAT,iNOS,ATP7A and ATP7B,and reduced Cu content and CTR1 gene expression to inhibite Cu-induced MDA.Butβ-glucan had no effect on inflammatory gene expressions.In the mid intestine,β-glucan increased activities of Cu/Zn-SOD and iNOS,mRNA levels of Cu/Zn-SOD,CAT and iNOS to maintain MDA content.However,unlike the anterior intestine,β-glucan had no effect on Cu transporter gene expressions.Furthermore,transcription factors（Nrf2,NF-κB and MTF-1）paralleled with their target genes in the mid intestine,but no correlation was observed between NF-κB and IL-1βand TNF-αgene expressions in the anterior intestine.In conclusion,our results unambiguously showed thatβ-glucan induced oxidative stress,inflammation and copper transport were varied between the anterior and mid intestines of fish under Cu stress.