| Lipids are essential nutrients and energy substances in fish,which are of great significance to maintain the normal growth,development and reproduction.Unsaturated fatty acids(UFA)are the key nutritional elements for the lipid requirement of fish,however,they are prone to be oxidized under normal condition.Dietary oxidized lipid could disorder intestinal secretion,increase intestinal permeability,induce intestinal oxidative stress and disease,which will affect the growth,body health and even cause mortality of aquatic animals.Toxicity effects of oxidized lipid have been widely studied in mammals.However,the impairment mechanism of oxidized lipid on fish still remain unclear,it has become a bottleneck that seriously restricted the sustainable development for aquaculture in some extent.Thus,it has become a scientific hotspot of concern in recent years.Therefore,to explore the effective means that could reduce the impairment of oxidized lipids on fish is of great important for our scientists.Gene is the key determinant for protein expression and phenotype during life processes.However,more and more evidences indicate that individuals with identical genomes intend to have different phenotypes during development,which is closely related to epigenetics.Epigenetics is a kind of stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence,it is a joint effect of environment factors and genome of cells.Epigenetics has become the most exciting frontier in life science,including DNA methylation,histone modification and microRNA(miRNA)regulation.MiRNA is a class of small RNAs that exhibited solid regulatory function,featured as regulating ontogenesis,metabolism,cell proliferation,differentiation,apoptosis,and cell cycle.It is estimated that miRNAs could regulate more than 30%encoding gene expression in mammalian,however,if the regulation is not controlled in tune,it will induce abnormal metabolism and inordinate cell cycle,and even malignant tumors.Megalobrama amblycephala(M.amblycephala)is one of the most widely cultivated freshwater species in China.It is susceptible to be affected by external stress and thus induce a stress response,such as hemorrhage,superficial mucus secretion decreases,and poor feed intake.Meanwhile,M amblycephala is vulnerable to some endogenous factors that could induce intestinal injury,such as dietary lipid oxidation.Therefore,M.amblycephala is an ideal experimental target to study the oxidative impairment mechanism.In this study,we used M.amblycephala to explore the ameliorative mechanism of emodin on oxidative injury stress induced by dietary oxidized fish oil based on approaches of nutriology,high throughput transcriptome and miRNA sequencing,and bioinformatic analysis.With this research,we are expecting to get deep insights into the negative effect of dietary lipid peroxidation on health and growth of M.amblycephala,which may provide a theoretical direction for the application of emodin as an immunopotentiator in aquatic feed.1.Effects of different lipid sources on growth performance and physiological health in M.amblycephalaPolyunsaturated fatty acids(PUFA)contents in soybean oil are different from that in fish oil.Here we conducted a 12-week rearing experiment with diets containing 6%soybean oil(6S)and 6%fish oil(6F)to evaluate the effect of oil rich in different PUFA content on growth performance,immune factors,antioxidant enzymes activity and intestinal histologic structure of M.amblycephala.Results suggested that the weight gain ratio(WGR)and specific growth ratio(SGR)were significantly increased in 6F(P<0.05),while the feed conversion ratio(FCR)was significantly decreased(P<0.05).It has no significant changes in body parameters(P>0.05).Besides,6F significantly increased plasma glucose(Glu)and reduced glutathione(GSH)activity,while decreased plasma alanine aminotransferase(ALT)activity,aspartate aminotransferase(AST)activity,total superoxide dismutase(T-SOD)activity as well as anti-superoxide anion free radicals(ASAFR)activity compared with 6S(P<0.05).Moreover,histological ultrastructure revealed the intestinal cells in 6S group were expanded and emerged with many large electron-dense fat droplets,resulted in displacement of the nucleus to the cell margin as well as a loss cytoplasm.The mitochondria were crowed together and exhibited an irregular arrangement and structural damage to the outer and the inner membrane.However,intestinal tissue in 6F group exhibited a normal ultrastructure,the intestinal cells exhibited dark,slender mitochondria with well-developed integrity,and the intestinal villus(?)were thick and closely arranged,IV length is 6F is significantly longer than that in 6S(P<0.01),suggesting that dietary fish oil improved growth performance,immunity and antioxidant capacity,also protected intestinal structure and villus integrity.2.Effects of dietary oxidized fish oil on growth,plasma biochemistry,intestinal health and antioxidant capacity in M.amblycephalaFish oil was oxidized in the lab until the peroxide value(POV)reach to 375.33 mmol kg-1.Here we conducted a 12-week rearing experiment with diets containing 0,2,4,and 6%oxidized fish oil(6F,4F2OF,2F4OF,and 6OF)to investigate effects of dietary oxidized fish oil on growth,plasma biochemistry,intestinal health and antioxidant capacity of M.amblycephala.Results indicated that 6OF reduced growth performance with increased FCR and reduced FBW,WGR and SGR compare with 6F(P<0.05).Meanwhile,oxidized fish oil treatments also increased antioxidant enzyme activity(P<0.05),suggesting an impaired physiological condition.The plasmatic antioxidant enzyme activity of ALT,AST,T-SOD,GSH-Px,ASAFR,concentration of MDA and cortisol were significantly increased in 6OF,while plasma Glu,albumin(ALB),globulin(GLB),total proteins(TP),total cholesterol(TC),GSH concentration were decreased(P<0.05).Histological ultrastructure revealed the integrity of mid-intestinal cells and villus were destroyed in 6OF.Moreover,the gene expression of PPARs signaling,Nrf2-Keap1 antioxidant signaling and downstream antioxidant factors were activated in response to oxidized fish oil stress under 6%oxidized fish oil treatment.These results indicate that oxidized fish oil may induce growth inhibition,lipid metabolism dysregulation,intestinal integrity damage and antioxidant defense mechanism initiation.3.Oxidative injury stress of M.amblycephala induced by dietary oxidized fish oil based on transcriptome analysisThe transcriptome analysis was carried out on M.amblycephala intestine with treatment of 6%fish oil(6F)and 6%oxidized fish oil(6OF)based on the high-throughput sequencing technology to explore injury stress regulation pathway induced by oxidized fish oil.We found that a total of 60 differentially expression genes(DEGs)in 6OF,among which 26 genes were up-regulated and 34 down-regulated.GO-enriched analysis showed that DEGs were mainly enriched in MHC protein complex,membrane,antigen processing and presentation as well as transporting and binding activity.The KEGG pathway enrichment analysis indicate that DEGs were mainly enriched in the life process of intestinal inflammation and immunity(such as graft-versus-host disease,allograft rejection,autoimmune thyroid disease,intestinal immune network for IgA production,antigen processing,and presentation and Inflammatory bowel disease),organism metabolism(maturity onset diabetes of the young,type I diabetes mellitus),and virus defense(butirosin and neomycin biosynthesis,staphylococcus aureus infection).Furthermore,RT-PCR analysis revealed PI3K-Akt/NF-?B/TCR inflammatory signaling were active under oxidized fish oil administration,indicating its regulatory function in oxidative injury stress induced by dietary oxidized fish oil.4.Ameliorative mechanism of emodin on oxidative injury stress of M.amblycephala induced by dietary oxidized fish oilBased on the above experiments,we conducted a 12-week rearing experiment with 6%fish oil(6F)and 6%oxidized fish oil(6OF)as substrate by adding 30 mg kg-1 emodin(6F+E,6OF+E)to investigate ameliorative mechanism of emodin on oxidative injury stress induced by dietary oxidized fish oil,respectively.Results shown that the FBW,WGR and SGR were significantly increased while the FCR and hepatopancreas somatic indices(HSI)were significantly decreased in 6OF+E(P<0.05).In addition,6OF+E significantly increased plasma antioxidant enzymes activity but decreased immune indexes and MDA content(P<0.05).Moreover,compared with 6OF,6OF+E ameliorated mitochondria and intestinal epithelial cell damage,exhibited fewer fat droplets in the cell and longer intestinal villus(P<0.01).Nevertheless,there were still injuries compared with 6F group.We also found that the expression of PPARs signaling was significantly decreased to 6F level(P<0.01),indicate the protective function of emodin on metabolic and antioxidant in some content.Moreover,gene expression of Nrf2-Keapl antioxidant signaling and downstream antioxidant factors was significantly decreased(P<0.05)in 6OF+E.The results suggested that dietary emodin can ameliorate the growth inhibition induced by oxidized fat oil,enhance immune and antioxidant capacity,improve intestinal morphology as well as increase metabolic capacity.However,the intestinal structure and antioxidant capacity could not recover to the control level.5.Ameliorative mechanism of emodin on oxidative injury stress induced by dietary oxidized fish oil based on miRNA analysisThe miRNA analysis was carried out on M.amblycephala intestine in 6F,6OF,6F+E,and 6OF+E groups based on the high-throughput sequencing technology,aiming to elucidate the epigenetic mechanism of emodin on oxidative injury stress induced by dietary oxidized fish oil.It may provide theoretical basis for ameliorative mechanism of emodin on oxidative injury stress of M.amblycephala.Results indicated that a total of 90,309,315,and 72 differentially expressed miRNAs(DE miRNAs)were identified in 6F+E-6F,6OF-6F,6OF+E-6F,and 6OF+E-6F+E,and annotated to 60362,21725,57200,and 24554 target genes,respectively.GO-enriched analysis showed that target genes were mainly enriched in the terms of biological process(metabolic process,cellular process,single-organism process),cellular component(cell,cell part,membrane,organelle),and molecular function(binding,catalytic activity,transporter activity,structural molecule activity),suggesting that the regulatory mechanism of emodin on injury stress induced by oxidized fish oil mainly focus on the influence on metabolism,membrane integrity,and signal transmission.The KEGG pathway enrichment analysis indicate that target genes of DE miRNAs in 6OF were enriched in antioxidant signaling such as energy metabolism,Ras signaling,carcinogenic miRNA,and MAPK/PI3K-Akt.However,apoptosis,cell cycle and Wnt antioxidant signaling were initiated in 6OF+E,suggesting that emodin relieves injury stress by regulating these signaling.6.Ameliorative mechanism of emodin on oxidative injury stress induced by dietary oxidized fish oil based on miRNA-mRNA analysisThe above results indicate that emodin exhibited ameliorative function on the oxidative injury stress induced by dietary oxidized fish oil.In this section,miRNA-mRNA correlation analysis was conducted to establish miRNA-mRNA regulatory networks,which was important to reveal the transcriptional and epigenetic mechanism of emodin on oxidative injury stress induced by dietary oxidized fish oil.It may provide theoretical basis for reduce the negative effect of lipid peroxidation in feed on health and growth of fish in practical production.MiRNA-mRNA correlation analysis in 6OF indicate that organism metabolism,stress reaction,immunity regulation,cell activation and intercellular communication regulated by Notch/MAPK/PI3K-Akt signaling were involved in injury stress induced by oxidized fish oil.We verified the expression of key target genes and found that miR-144 and miR-200a negatively targeted to MAPK/p53/Notch signaling,miR144 and miR-155 negatively targeted to PTEN/PI3K-Akt signaling,miR-144,miR-153a,miR-200a,miR-155,and miR-196a negatively targeted to Nrf2-Keapl signaling.MAPK/P53/Notch signaling mainly regulates cell cycle and apoptosis,PTEN/PI3K-Akt and MHC/NF-?B signaling mainly involve in immunological recognition regulation,while Nrf2-Keap1 signaling plays a key role in antioxidant regulation.The complex regulatory network of these signaling may plays a vital function in the ameliorative mechanism of emodin on oxidative injury stress induced by dietary oxidized fish oil. |
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