| In intensive aquaculture,fish is constantly affected by various stressors from their living surroundings,which can lead to poor growth performance,disease outbreak and even mortality,causing significant economic losses and deeply affecting food safety.Therefore,improving immunity of fish and reducing disease outbreak has become a key subject for researches in this field.Emodin is one of the most important active anthraquinone constituents extracted from rhubarb.It has many beneficial functions such as anti-bacteria anti-virus,anti-stress,immunity enhancing,feed attractant,and water quality improving.As an immune enhancer,unlike antibiotic and other chemical medicines,herbal medicine causes no insecticide resistance.It can not only control disease,but also promote digestion,absorption and growth as a nutrient.Because of these advantages,herbal medicine now has become a hot research object as an alternative of antibiotics,hormones and chemical medicines,and also has been widely used in human disease prevention and control as well as in acquaculture.Megalobrama amblycephala is one of the major freshwater aquaculture species in China.In light with the frequency of disease outbreak,the present study was aimed to determine the effects of dietary emodin on hepatic differential proteomics,understand the interaction of differentially expressed antioxidant proteins and immunologic mechanism of Megalobrama amblycephala,in order to provide an insight into the usage of emodin in aquaculture of Megalobrama amblycephala.This study was mainly focused on the following aspects:1 Hepatic differential proteomics analysis of Megalobrama amblycephala fed with dietary emodinThis study was conducted to separate and identify the hepatic differentially expressed proteins of Megalobrama amblycephala fed with 30 mg/kg dietary emodin based on 2-DE.Results showed that 39 significant differentially expressed protein spots(up/down-regulated? 2 fold)were separated,of which 38 were successfully identified(8 up-regulated and 30 down-regulated),including 25 unique proteins.Based on the functional annotation of Gene Ontology(GO),the differentially expressed proteins can be divided into 10 categories,including 12 carbohydrate metabolism(8 unique,31%),8 energy metabolism(3 unique,20%),1 immune-related proteins(2%),4 cytoskeleton proteins(2 unique,10%),2 chaperone(2 unique,5%),1 signal transduction(3%),5 antioxidant proteins(2 unique,13%),1 cofactors and vitamins(3%),3 amino acid metabolism(3 unique,5%),and 2 nucleotide metabolism(2 unique,8%).2 Network interaction analysis and functional annotation of differentially expressed proteinsThis research was aimed to build the interaction network and functional annotation of differentially expressed proteins of Megalobrama amblycephma by Cytoscape and DAVID based on the network database of NCBI,STRING,DAVID and GO.The results indicated that 18 unique differentially expressed proteins performed an important role in network interaction,including ApoAl,ALDOB,GAPDH,TP53,GPxl,GST,HSP70,Bhmtl,EEF2b,FTCD,Aldh8al,Aldh9a1a,LDHBB,Eno3,Pgkl,ME1,ETFA and AST.All these proteins were up-regulated except ALDOB and TP53.The GO annotation showed that the differentially expressed proteins were mainly involved in the redox reaction in metabolism.Besides,they also participated in the processes of glycolysis,glucose catabolism,monose catabolism,hexose catabolism,alcohol catabolism,and cellular carbohydrate catabolism.Based on the information retrived,TP53,Aldh9a1a,ME1,Aldh8a1,LDHBB,Sord,ALDOB,HSP70 and GPxl were all closely related to antioxidant reaction,which is same to the result by Cytoscape.All these results indicate that ApoA1,ALDOB,GAPDH,TP53,GPxl,GST and HSP70 play an important role in antioxidation reaction.3 Expression validation on mRNA level by RT-PCR and function analysis of differentially expressed proteinsThis investigation was aimed to use RT-PCR to compare the mRNA expression level of differentially expressed proteins of each group,and then to explore the relation of metabolism,immunity and antioxidant of these proteins.Results showed the 30 mg/kg emodin supplementation group had significantly improved the mRNA expression of ApoA1,ALDOB,GAPDH,HSP70,GPx1 and GST compared with the control group(P<0.05),which was consistent with the protein expression level.The same expression trends were also observed in Aldh8a1,Eno3,Pgk1,ETFA and FTCD,while Sord,FAH and EEF2b showed an opposite trend.In addition,the mRNA expression of ApoAl,GAPDH,HSP70,GPxl and GST first increased and then decreased with the increasing of dietary emodin level,and the highest expression was observed in 30 mg/kg group(P<0.05).Taking all these results into consideration,this research showed that 30 mg/kg supplementation of emodin in feed can enhance the metabolism,immunity and antioxidation of Megalobrama amblycephala. |
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