The Whole Cell Proteome-based Study on The Mechanism of Apoptosis Induced by Soybean Agglutinin on IPEC-J2
Abstract Background: Soybean agglutinin (SBA), a major anti-nutritional factor in soybean, may induce abnormal health and metabolism of intestinal cells, resulting in the reduction of the production performance of animals. The anti-nutritional mechanisms of SBA are not fully understood, in terms of the cell life activities and metabolism of intestinal cells. This research aims to find the effects of SBA on the cell cycle, apoptosis and proteomic, and furtherly to get more findings for verifying the mechanism of SBA anti-nutritional characters.Methods: The IPEC-J2 cell line was cultured with the medium containing 0.0, 0.5 or 2.0 mg/mL SBA, respectively, for 24 h. The percentage of the cells at different cell cycle phases (G0/G1 phase, S phase and G2 phase) and cell apoptosis rates were measured with flow cytometry. The expressions of Cyclin D1, active p21, Bcl-2, and Bax were determined by western blotting. The activity of caspase-3 (Casp-3) and caspase-9 (Casp-9) were tested with ELISA. The whole-cell quantitative proteome were detected by TMT/iTRAQ Labeling, HPLC fractionation and LC-MS/MS Analysis. The functions and characteristics of the differential expressed proteins in the proteome results were analyzed from the aspects of GO annotation and KEGG pathway. The relationship between the results of proteomics and apoptosis or cell cycle were analyzed and discussed.Results: The percentage of the cells at G0/G1 phase, cell apoptosis rates, expressions of Bax and p21, and the activities of Casp-3 and Casp-9 were increased, cyclin D1 and Bcl-2 expression were declined with the increased of the SBA treatment levels (p<0.05). The proteomic measurements showed that a numbers of differentially expressed proteins, caused by SBA treatment, were mainly enriched in the DNA replication, base excision repair, nucleus excision repair, mismatch repair, ubiquitin-mediated proteolysis pathway, cell structural-proteins, and structures and functions of mitochondria. Moreover, the differential expressed proteins enriched in AMP-activated protein kinase (AMPK) pathway and the process of synthesis and metabolism of proteins were only found in 2.0 mg/mL SBA treatment.Conclusion: The results of this experiment demonstrated that cell cycle arrest and apoptosis induced by SBA may be resulted from the downregulating the expression of the proteins related to DNA replication and repair, protein translation, signal-conducting relation, cell structure, and subcellular structure and function.
