Study On Mechanisms And Protective Effects Of L-carnitine On Oxidative Stress In Juvenile Rhynchocypris Lagowski Dybowski

Due to its unique physiological condition and complicated water environment,Fish is susceptible to oxidative stress.To solve the stress problem in aquaculture,researchers have been focused on the nutritional antioxidants.Meanwhile,L-Carnitine（LC）,because of its unique physiological function,plays an important role in the treatment of human diseases,especially related to oxidative stress.But the effect of LC on fish is still very shallow,and the application of LC has not been widely used in aquaculture.Furthermore,the study of LC in antioxidant function is also less,and the antioxidant mechanism is still unclear.In the present study,Nrf2 gene was firstly cloned in fathead minnow muscle cell line（FHM）,then the bioinformatics of the sequences was analyzed.Based on the sequence,the siRNA was designed,and the Nrf2 siRNA silenced FHM cells was establised.Meanwhile,H₂O₂ as oxidative stress induced factor,the effect of L-carnitine on anti-oxidation function in normal and Nrf2siRNA silenced FHM cell was investagate.Second,the effects of dietary fish oil oxidation on growth and anti-oxidation function of Rhynchocypris lagowski Dybowski in vivo were investagted,and on the basis,effects of dietary oxidized fish oil with LC supplementation on growth performance and anti-oxidation function of Rhynchocypris lagowski Dybowski were evalued.Therefore,the antioxidation function of LC was explored in vivo and in vitro to confirm the regulation in Keap1-Nrf2-ARE signaling pathways,as well as the antioxidation mechanism for the basic theory of LC as an antioxidant used in aquaculture.This study includes four parts test,the results and conclusions were presented as follows:（1）Cloning and bioinformatics analysis of nuclear factor erythroid 2-related factor 2 gene in FHM cellThe full-length Nrf2 cDNA was composed of 2578 bp,containing 124 bp in the5’-untranslated region（UTR）,1770 bp in the open reading frame（ORF）and 684 bp in 3’-UTR with a poly（A）tail.The ORF encodes a polypeptide of 364 amino acids without a signal peptide.The predicted molecular mass of the mature protein（364 aa）was 66.3 kDa with an estimated pI of 4.47.SMART and CDD predictions show that the protein contains a BZIP domain.The main function is to bind to the specific DNA sequence and predict the activity of the transcription factor.Regarding similarity with other vertebrates,the deduced Nrf2 showed identity（more than 70%）to those fishes.A phylogenetic tree was constructed by the NJ method based on the amino acid sequences of Nrf2.Phylogenetic analysis revealed that Nrf2 gene of FHM most closely clustered together with Cyprinus carpio.（2）L-carnitine prevents H₂O₂-induced oxidative stress by activating the Keap1-Nrf2 antioxidative defense system in FHM cellThe present study aimed to clarify how LC modulate Nrf2/Keap1 system to exert its antioxidant function.Based on the siciencing effect,predesigned siRNA-242against FHM Nrf2 were choosed.Then,normal and silencing Nrf2 FHM cell were stimulated with 1 mM H2O2 for 1 h after 6 h LC pre-treatment,to investagete the protective effect of LC against H2O2-induced oxidative stress by activating the Keap1-Nrf2 antioxidative defense system.Cell cycle and apoptosis analysis were detected by using flow cytometry instrument,and the activity of antioxidant enzyme and the mRNA relative expression of antioxidant related gene were measured.The results indicated that H₂O₂ declined FHM cell proliferation by arresting the cells at G1phase of cell cycle（P<0.05）,and induced cell apoptosis（P<0.05）.Moreover,MDA level significantly increased（P<0.05）,the antioxidant enzymes（T-SOD,CAT,GSH-PX）activities and GSH content were significantly decreased（P<0.05）.However,L-carnitine pretreatment can callback the bad influence by down-regulating Keap1mRNA relative expression and up-regulating the mRNA relative expressions of Nrf2,Maf,HO-1.Therefore,FHM cell proliferation improved and the cell apoptosis reduced,and the antioxidative defense system was stimulated.But Nrf2 siRNA reduced the cell protection of LC against H₂O₂-induced oxidative stress.（3）Establishment of oxidative stress model induced by oxidized fish oil in juvenile Rhynchocypris lagowski DybowskiFive isonitrogenous and isolipidic diets containing fresh fish oil and four degrees of oxidized fish oil（POV:100;200;300;400）were formulated to investigate the effects of dietary oxidized fish oil on growth performance,anti-oxidation function of Rhynchocypris lagowski Dybowski.After a 8-week feeding trial,fish fed oxidized oil diets obtained significantly lower weight gain and specific growth rate due to their lower feed efficiency and protein efficiency.The analysis of parameters related to antioxidant levels indicated significant differences in various test diets.With the fish oil oxidation degree deepen,MDA levels significantly increased,the antioxidant enzymes（T-SOD,CAT,GSH-PX）activities and GSH content were significantly decreased（P<0.05）.Further analysis,Keap1 mRNA relative expression was increased（P<0.05）,and the mRNA relative expressions of Nrf2,Maf,HO-1 were significantly decreased（P<0.05）.However,some parameters showed reverse trend,the possible reason is the different degree of oxidation.Under the conditions of this experiment,by checking indicators of growth,activities of main antioxidant enzymes,the related gene expressions,feeding the oxidative fish oil（POV:100 and400）for 8w can successfully induce oxidative damage of mild and severe in Rhynchocypris lagowski Dybowski.（4）The protective effects of L-carnitine on oxidative stress in juvenile Rhynchocypris lagowski DybowskiAn 8-week feeding trial Rhynchocypris lagowski Dybowski was conducted in triplicate groups with test diets containing two degrees of OFO（100 and 400 meq/kg）supplement with two levels of LC（500 and 1000 mg/kg）,respectively.Fresh fish oil without LC supplementation was employed as control group.Fish fed oxidative fish oil indicated significantly lower growth,significantly higher in MDA levels,significantly lower in the antioxidant than control group（P<0.05）.Increased dietary LC levels led to reduce the damage,while the highest value was observed in fish fed high OFO100 with 1000mg/kg LC supplemented diets.Further analysis,Keap1mRNA relative expression was increased（P<0.05）,and the mRNA relative expressions of Nrf2,Maf,HO-1 were significantly decreased（P<0.05）.LC pretreatment can callback the bad influence by down-regulating Keap1 mRNA relative expression and up-regulating the mRNA relative expressions of Nrf2,Maf,HO-1.Finally,LC supplementation led a normal growth of Rhynchocypris lagowski Dybowski fed oxidized fish oil.In conclusion,these results showed that cytotoxicity of H₂O₂ exposure in FHM cells and the detrimental effects of dietary oxidized fish oil on growth in Rhynchocypris lagowski Dybowski,as well as LC prevents oxidative stress by activating the Keap1-Nrf2 antioxidative defense system in vivo and in vitro.