Comparative proteomic analysis of liver antioxidant mechanisms in Megalobrama amblycephala stimulated with dietary emodin

Abstract

Oxidative stress is a toxicological endpoint that correlates with the nutrition status of fish through cellular damage, inflammation, and apoptosis. In order to understand the antioxidant mechanism induced by dietary emodin in Megalobrama amblycephala liver, a comparative proteomic analysis was performed to investigate the proteome alteration under emodin administration. 27 altered protein spots were separated under 30 mg kg^-1 emodin stimulation based on 2-DE, and were all successfully identified using MALDI-TOF/TOF, representing 17 unique proteins. These proteins were functionally classified into antioxidant, metabolism, cytoskeleton, chaperone, signal transduction and cofactor groups. Network interaction and Gene Ontology annotation indicated 10 unique proteins were closely related to antioxidation and directly regulated by each other. Compared with the control group, administration of 30 mg kg^-1 emodin significantly increased the antioxidant-related mRNA expressions of GPx1, GSTm and HSP70, but decreased the mRNA expressions of GAPDH and Sord, which was consistent with the protein expression. Nevertheless, Pgk1 and Aldh8a1 were up- and down-regulated, and ALDOB was down- and up-regulated at the mRNA and protein levels, respectively. These results revealed that the altered proteins enhanced antioxidation via complex regulatory mechanisms, and 30 mg kg^-1 emodin was a suitable immunostimulant for M. amblycephala.

Figure 1. The 2-DE reference maps and functional classification of altered proteins derived from the hepatic cells of the control and 30 mg kg⁻¹ emodin groups (pI 4-7).

(a) Significantly increased or decreased proteins are denoted by a circle, arrow and spot number. Molecular masses of marker proteins and pI are mentioned on the left side and at the top, respectively. (b) The identified M. amblycephala liver proteins were classified based on their GO annotations for biological processes and molecular functions.

Figure 2. Network interaction of altered proteins identified in the 30 mg kg⁻¹ emodin group.

In the network, each node represents a protein; nodes in red and green color represent up- and down-regulated proteins, respectively. The size of a node represents an average of the protein abundance. The width of a line connecting proteins represents the intensity of the protein interaction, as extracted from STRING database.

Figure 3. Relative expression of antioxidant-related proteins at protein and mRNA levels.

Diverse little letters above histogram bars show significant differences (P < 0.05) in different dosage groups in Duncan’s multiple range tests. Significant differences (P < 0.05 or P < 0.01) between values obtained the control 30 mg kg⁻¹ emodin group were marked by asterisks above histogram bars in Independent-Samples t-tests.

Figure 4. Emodin-responsive changes in the abundance of antioxidant-related proteins.

Differentially expressed proteins with increased or decreased abundance are marked in the control and 30 mg kg⁻¹ groups. Fold changes of protein spots are mentioned on the right side.

Figure 5. Heatmap of antioxidant-related altered proteins based on mRNA and protein expressions.

In this figure, weak and strong correlations between variables are displayed in green and red, respectively. The dendrogram for strain clustering is shown on the left-hand side of the heatmap. The width of the cluster merged from the left side represents the distance of the two clusters.

Figure 6. Antioxidant-related mechanism of altered proteins under emodin stimulation in M. amblycephala.

Abbreviations in red represent the altered proteins in present study. Dashed arrows are used for mRNA expression, and solid arrows are used for protein expression. The up-arrow in red represents up-regulation, and down-arrow in green represents down-regulated. G6P, glucose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP, glyceraldaldehyde-3-phosphate; 1,3-BPG, 1,3-bisphosphoglycerate; 3-PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; SOD, superoxide dismutase; MPO; CAT, catalase; NF-κB, nuclear factor κappa Β; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor-α; IL-1β, Interleukins-1β; IL-6, Interleukins-6; IL-12, Interleukins-12.