Effects Of Emodin And Vitamin C On Growth, Non-specific Immunity And Stress Resistance Of Wuchang Bream (Megalobrama Amblycephala, Yih)

Emodin is an active anthraquinone constituent extracted from the rhizome of rhubarb Rheum officinale Baill. It has many functions such as anti-bacterial and anti-inflammatory, antivirus, antioxidation and scavenging free radicals, reducing blood lipid, protecting the liver, and regulating immunity. Vitamin C (Vc), also known as L-ascorbic acid, is an essential nutrient for normal physiological functions in fish. Vc is an important immunostimulant and antioxidant. but most teleosts are unable to synthesize ascorbic acid due to lack of L-gulonolactone oxidase (EC 1.1.3.8) responsible for the de novo synthesis of Vc. Therefore, exogenous Vc is required in fish diet. In order to study the effects of dietary emodin, high-dose Vc and their combination on growth, non-specific immunity and anti-stress of fish, Wuchang bream (Megalobrama amblycephala, Yih), one of the major freshwater aquaculture fish, was selected as the research subject. Firstly, two full-length cDNA sequences of two HSP70s genes were isolated and cloned from the liver of Wuchang bream, and their gene structure and expression characteristics were analyzed. Then, two HSP70s prokaryotic expression systems were constructed on the basis of above two HSP70s genes, and the expression conditions were optimized. The expressed products were purified and identified. At the same time,1200 healthy fish with initial body weight of (133.44±2.11) g were selected and randomly divided into four groups:a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin,700 mg/kg Vc, and the combination of 60 mg/kg emodin+700 mg/kg Vc, respectively. Each diet was tested in triplicate. After continuous feeding for 60 days, the rearing experiment was over. Then, the experiments of crowding stress, heat stress and infected with Aeromonas hydrophila were carried out.1 Molecular cloning and sequence analysis of two HSP70 genes in Wuchang breamTwo full-length cDNA sequences of two HSP70s genes were isolated from the liver of Wuchang bream using RT-PCR and rapid amplification of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70 genes, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly(A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kD, and theoretical isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule, belonging to the constitutive HSC70 whereas Ma-HSP70 gene did not contain any intron in the coding region, belonging to the inducible HSP70. Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates while Ma-HSP70 shared more than 85.0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region. The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinaseⅡphosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins wereα-helix and random coil, and their spatial structures included N-terminal ATPase domain and C-terminal polypeptide-binding domain.2 Effects of emodin and vitamin C on growth, physiological and biochemical parameters, and two HSP70s mRNA expression of Wuchang breamAt the end of the rearing experiment, the growth performance, muscle composition, biochemical parameters of serum and liver, and the mRNA levels of liver two HSP70s genes were investigated. The results showed that compared with the control, the weight gain and specific growth rate, the levels of total protein (TP), lysozyme (LSZ) and alkaline phosphatase (AKP) in serum, the activity of superoxide dismutase (SOD) and basal expression level of inducible HSP70 mRNA in liver significantly increased (P<0.05) while feed coefficient, mortality, the contents of serum cortisol (COR) and triglycerides (TG), and liver malondialdehyde (MDA) decreased (P<0.05) in emodin and Vc groups. Although serum TP, LSZ levels, and the mRNA level of liver HSP70 gene increased significantly (P <0.05), and liver MDA content also significantly decreased (P<0.05) in combination group, the synergism didn’t appear. In addition, liver catalase (CAT) activity was also significantly improved in emodin group compared with the control (P<0.05) while the CAT activities in Vc and combination groups were not significantly different with the control (P> 0.05). The other indices, such as serum glucose (GLU), glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), cholesterol (CHOL), the mRNA level of liver constitutive HSC70, the fish body indices, and muscle composition were not significantly different among all groups (P> 0.05).3 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream under crowding stressAt the end of the rearing experiment,25 fish per tank were exposed to crowding stress (100 g/L) for 48 h, the biochemical parameters of blood and liver, mRNA levels of liver two HSP70s genes, and cumulative mortalities of fish in each group were investigated at 0 h,12 h,24 h and 48 h, respectively. The results showed that, after crowding stress, compared with the control, serum TP and AKP levels, liver SOD and CAT activities, and the expression levels of liver HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees, and serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent, while the LSZ activity decreased at first and then increased significantly (P<0.05). In combination group, although these parameters had similar changing trend as above ones, most of them were not significant differences with the control(P>0.05), and did not show any synergism either. In addition, serum CHOL levels among all groups before and after crowding stress were not significantly different (P> 0.05). The statistics showed that, the cumulative mortalities of the emodin and Vc groups were significantly lower than that of the control at 24 h and 48 h (P<0.05), but there was no significant difference between the combination and control groups (P> 0.05).4 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream under high temperature stress At the end of the rearing experiment,25 fish per tank were exposed to high temperature stress of 34℃. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that, after heat stress, compared with the control, serum TP, LSZ, and AKP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. In addition, serum cholesterol levels among all groups before and after heat stress were not significantly different (P>0.05). Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P<0.05) while the difference between the combination and control groups was not significant (P>0.05).5 Effects of emodin and vitamin C on biochemical parameters and two HSP70s mRNA expression of Wuchang bream infected with Aeromonas hydrophilaAt the end of the rearing experiment,15 fish per tank were intraperitoneally infected with A. hydrophila (BSK-10), and the biochemical parameters of serum and liver, the mRNA expression levels of liver two HSP70s genes before and after infection were determined. Another ten fish taken from each tank were challenged with A. hydrophila as the same method as above, the cumulative mortality of each group was counted at different time point of post-infection. The results showed that after infection of fish, compared with the control, serum TP, LSZ and AKP levels, liver SOD, CAT activities, and the expression levels of HSC70 and HSP70 mRNA increased in emodin and Vc groups in varying degrees, while serum COR, GLU, GPT, GOT, TG and liver MDA levels decreased to some extent. In combination group, although these parameters had similar changing trend as above ones, most of them were not significant differences with the control(P>0.05), and did not show any synergism either. In addition, serum cholesterol levels among all groups before and after infection were not significantly different (P>0.05). Statistics also showed that after infected with A. hydrophila, the cumulative mortalities of emodin and Vc groups were significantly lower than that of the control in 24 h and 48 h (P<0.05), but the difference of the cumulative mortality between the combination and control groups was not significant (P>0.05).6 Prokaryotic expression, purification and identification of two HSP70s in Wuchang breamThe fragments of constitutive HSC70 and inducible HSP70 ORFs of Wuchang bream were amplified by PCR, and then cloned into prokaryotic expression vectors of pET-22b(+), respectively. The two recombinant plasmids, determined by double-endonuclease digestion and sequencing, were transformed into competent E. coli BL21 (DE3), respectively. The two recombinant strains were induced by 1 mmol/L IPTG at different temperatures and times, establishing optimal conditions for inducible expression. The expression products were purified by Ni-NTA His Bind Resins affinity chromatography and DEAE-Sepharose FF anion-exchange chromatography, and detected by SDS-PAGE and Western-blotting, respectively. The results show that two recombinant expression plasmids of pET-22b(+)/Ma-HSC70 and pET-22b(+)/Ma-HSP70, each expressing a fusion protein of about 72 kD identified by rabbit source anti-HSP70 polyclonal antibody, were successfully constructed, and the purity of two purified fusion proteins was more than 95%. A higher temperature (37℃) is conducive to the rapid expression of fusion protein, but it is easy to form inclusion body. A lower temperature (25℃) is in favor of the soluble expression of fusion protein, but it could also reduce the expression rate of fusion protein. All things considered, the optimal conditions for soluble expression of the two fusion proteins of Ma-HSC70 and Ma-HSP70 are induced for 7 h at 25℃and 30℃, respectively.In conclusion, two full-length HSP70 cDNAs were cloned and characterized from Wuchang bream liver for the first time. There are seven introns in the coding region of Ma-HSC70 gene while there is no intron in the Ma-HSP70 gene, suggesting constitutive and inducible expression, respectively. Through optimizing the expression conditions, two purified fusion proteins of Ma-HSC70 and Ma-HSP70, which purities were more than 95%, have been obtained by prokaryotic expression systems of two HSP70s genes of Wuchang bream. Thus it laid a foundation for further study on molecular structure and function of the two proteins and associated antibodies. The rearing and stress experiments indicate that, the basal diet supplemented with either 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, lower FCR, increase non-specific immunity, antioxidant capacity, and stress resistance, reduce stress mortality. However, the combination of emodin and high-dose Vc showed no better effects. The mechanism of their interaction needs to be further studied.