| Bioactive peptides are molecular polymers between amino acids and proteins,which have many functions such as antioxidant and antibacterial.The protein content in the shell of Procambarus clarkii accounts for about 24%of the mass of the shell,and the active peptide obtained from the hydrolysate of the shell has been verified to have antioxidant activity in vitro.Copper(Cu)is an essential trace element for the life activities of the body,and its lack and excess are easy to cause negative effects on the growth performance of fish.Cu is also a trace element that is easy to be overused in diet,which can easily lead to oxidative stress,lipid metabolism disorder and lipid accumulation.Fish is a kind of organism that is sensitive to environmental changes.Zebrafish is a model animal in fish with short growth cycle and highly similar genomes to humans.In this study,zebrafish were used as the research object to analyze the antioxidant response,lipid metabolism and lipid deposition of shrimp shell active peptide LPLWPY on zebrafish under Cu stimulation,and the primary hepatocytes of zebrafish were treated with nrf2 and pparγspecific inhibitors,respectively,to study the mediating role of Nrf2 and PPARγsignaling pathways in the regulation of antioxidant and lipid metabolism in zebrafish regulated by shrimp shell active peptides.The main research results are as follows:1.Preparation of active peptides from the shell of P.clarkii and screening of antioxidant active peptides.To obtain active peptides from shell of P.clarkii,five common proteases were used to hydrolyze the shell,and the degree of hydrolysis and antioxidant activities of the hydrolysates(DPPH·free radical and ABTS~+·free radical scavenging)were tested.The results showed that in the hydrolysis test of this study,the alkaline protease had the highest hydrolysis degree,and the hydrolysis obtained the enzymatic hydrolyzed liquid had the highest antioxidant capacity.The fraction smaller than 1 k Da in alkaline protease hydrolysate had the strongest antioxidant activity.The active peptide of this fraction was analyzed by mass spectrometry.After the active peptide sequence was determined,the antioxidant score and molecular docking with Keap1 were used to determine the active peptide LPLWPY had a strong antioxidant activity.2.Effects of diet Cu excess and shrimp shell active peptide LPLWPY addition on zebrafish growth,tissue morphology and hepatic oxidative stress,lipid metabolism disorders and lipid deposition.To study the effects of diet Cu excess and shrimp shell active peptide LPLWPY addition on zebrafish growth,tissue morphology,hepatic oxidative stress,lipid metabolism disorders and lipid deposition,a control group,Cu treatment group(500mg/kg Cu added to diet),Cu+low concentration peptide treatment group(500 mg/kg Cu and 100 mg/kg LPLWPY added to diet)and Cu+high concentration peptide treatment group(500 mg/kg Cu and 400 mg/kg LPLWPY added to diet)were set up.Zebrafish were fed with the corresponding diet for each treatment group for 8 weeks.The results showed that the weight gain rate and condition factor of zebrafish in the Cu treatment group were significantly reduced,and there was no significant difference between the weight gain rate and condition factor of zebrafish in the Cu+high concentration peptide treatment group and the control group.The analysis of H&E staining,oil red O staining results and TG content of liver tissue showed that excess diet Cu led to vacuolar denaturation and lipid accumulation of zebrafish liver cells,which was alleviated by the Cu+low concentration peptide treatment group,but the effect was not as good as that of the Cu+high concentration peptide treatment group.Enzyme activity analysis showed that excessive dietary Cu significantly increased the content of MDA,the end product of lipid peroxidation,and the activity of antioxidant enzyme CAT,and decreased the activity of antioxidant enzyme SOD and GPx in zebrafish liver.The analysis of gene expression results showed that the expression levels of antioxidant-related genes sod1,sod2,gpx3,gpx4b,keap1 and nrf2 in the Cu treatment group,as well as genes related to lipolysis,were significantly decline.The addition of shrimp shell active peptide to the Cu excess diet reduced the MDA content in zebrafish liver,and the MDA content of the Cu+high concentration peptide treatment group was lower than that in the control group,showing a stronger antioxidant effect compared with the Cu+low concentration peptide treatment group.The expression levels of lipid metabolism genes in the two treatment groups with shrimp shell active peptide were higher than those in the Cu treatment group.In conclusion,dietary supplementation of shrimp shell active peptide can alleviate the decreased growth performance,abnormal liver lipid accumulation,oxidative stress and lipid metabolism disorder of zebrafish induced by dietary Cu excess.3.Mechanism of shrimp shell active peptide LPLWPY mediating Cu induced oxidative stress and lipid metabolism disorder in zebrafish liver.To further explore the molecular mechanism of shrimp shell active peptide regulating antioxidant and lipid metabolism reactions in zebrafish,zebrafish primary liver cells were isolated and cultured,the safe use concentration of LPLWPY was determined by MTT method.And the primary zebrafish liver cells were incubated with Cu,nrf2inhibitor ML385,pparγinhibitor T0070907 and shrimp shell active peptide LPLWPY.The results showed that the contents of MDA and TG in zebrafish liver cells incubated with 10μM Cu were significantly higher than the control group,and LPLWPY could eliminate the increase of TG content and the up regulation of nrf2 expression decline by Cu.ML385 significantly inhibited the activity of nrf2 and decline the expression level of transcription factor pparγgene associated with lipid deposition,MDA content were increased.In the Cu+ML385+LPLWPY group,the expression level of nrf2 and pparγincreased,the contents of MDA and TG decreased significantly.The expression level of pparγin the Cu+T0070907 treatment group decreased significantly,and the TG content in the Cu+T0070907+LPLWPY treatment group showed a decreasing trend.Therefore,this study suggested that shrimp shell active peptide LPLWPY may regulate Cu mediated cellular antioxidant response and lipid metabolism and alleviate oxidative stress and lipid deposition through Nrf2 and PPARγin primary zebrafish liver cells. |
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