Hepatotoxic mechanism of diclofenac sodium on broiler chicken revealed by iTRAQ-based proteomics analysis

Abstract

Background: At the therapeutic doses, diclofenac sodium (DFS) has few toxic side effects on mammals. On the other hand, DFS exhibits potent toxicity against birds and the mechanisms remain ambiguous.

Objectives: This paper was designed to probe the toxicity of DFS exposure on the hepatic proteome of broiler chickens.

Methods: Twenty 30-day-old broiler chickens were randomized evenly into two groups (n = 10). DFS was administered orally at 10 mg/kg body weight in group A, while the chickens in group B were perfused with saline as a control. Histopathological observations, serum biochemical examinations, and quantitative real-time polymerase chain reaction were performed to assess the liver injury induced by DFS. Proteomics analysis of the liver samples was conducted using isobaric tags for relative and absolute quantification (iTRAQ) technology.

Results: Ultimately, 201 differentially expressed proteins (DEPs) were obtained, of which 47 were up regulated, and 154 were down regulated. The Gene Ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway analysis were conducted to screen target DEPs associated with DFS hepatotoxicity. The regulatory relationships between DEPs and signaling pathways were embodied via a protein-protein interaction network. The results showed that the DEPs enriched in multiple pathways, which might be related to the hepatotoxicity of DFS, were "protein processing in endoplasmic reticulum," "retinol metabolism," and "glycine, serine, and threonine metabolism."

Conclusions: The hepatotoxicity of DFS on broiler chickens might be achieved by inducing the apoptosis of hepatocytes and affecting the metabolism of retinol and purine. The present study could provide molecular insights into the hepatotoxicity of DFS on broiler chickens.

Keywords: Diclofenac sodium; chicken; liver; proteomics; toxicity.

Fig. 1. Results of the serum biochemical examination presented via bar plots.

(A) Activity of ALT for different periods after DFS administration; (B) Activity of AST for different periods after DFS administration; (C) The content of uric acid in serum for different periods. The data are presented as mean ± SEM. AST, aspartate aminotransferase; DFS, diclofenac sodium; ALT, alanine aminotransferase; SEM, standard error of the mean. ^*p < 0.05, ^**p < 0.01.

Fig. 2. H&E stained histopathology sections of the liver. (A) Normal liver section from the control group, H&E ×400. (B) Liver section from dosed chickens that succumbed, H&E ×400. Focal hepatic necrosis (a), inflammatory cell infiltration (b), and dilatation of hepatic sinusoids (c).

H&E, hematoxylin-eosin

Fig. 3. Effects of DFS on the apoptosis-related genes.

(A) Relative mRNA expression level of Bax; (B) Relative mRNA expression level of Bcl-2; (C) Relative mRNA expression level of caspase 3. Data are presented as mean ± SEM. DFS, diclofenac sodium; SEM, standard error of the mean; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ^*p < 0.05, ^**p < 0.01.

Fig. 4. Identification results of the proteins. (A) Peptide length distribution; (B) Peptide number distribution; (C) Peptide mass distribution; (D) Protein sequence coverage. The identification results of the proteins suggested the high quality of data.

Fig. 5. Volcano plot generated with the fold change (log₂ transformed) as a horizontal coordinate and p value (−log₁₀ transformed) as the vertical coordinate. Based on the threshold of significance (p < 0.05), the data were classified into three categories: the blue dots indicate down-regulated DEPs, red dots correspond to up-regulated DEPs, and grey dots mean no significant differential expression.

DEP, differentially expressed protein; LT, livers from treated group; LC, livers from control group.

Fig. 6. GO classification results. These pie charts demonstrate the proportion of the number of DEPs in each term under their category.

GO, Gene Ontology; DEP, differentially expressed protein.

Fig. 7. KEGG pathway enrichment results. The horizontal coordinate indicates different pathways, and the vertical coordinate denotes the number of DEPs in each pathway as a percentage of total DEPs. Deeper color means smaller corrected p value.

KEGG, Kyoto Encyclopedia of Genes and Genomes; DEP, differentially expressed protein; LT, livers from treated group; LC, livers from control group; ECM, extracellular matrix. ^*p < 0.05, ^**p < 0.01.

Fig. 8. PPI network of the DEPs. These signaling pathways are distinguished by their distinct colors, and the various nodes indicate the individual DEPs.

PPI, protein-protein interaction; DEP, differentially expressed protein; ECM, extracellular matrix.

Fig. 9. mRNA transcript levels of screened DEPs in the liver after DFS administration. Data are presented as mean ± SEM.

DEP, differentially expressed protein; SEM, standard error of the mean; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ^*p < 0.05, ^**p < 0.01.