The in vitro modulation of intestinal growth and inflammatory indices by fumonisin B1 and hydrolyzed fumonisin B1 through protein network analyses

Abstract

There is an increasing amount of staple feed and food in low- and middle-income countries that can be contaminated by various mycotoxins. Regarding this, tthe intestinal epithelium is constantly being exposed to these ingested harmful contaminants, such as mycotoxins. Fumonisin B1 (FB1) is one of the most toxic and abundant fumonisins found in nature and commonly contaminates maize and maize-based feed and food, causing various adverse effects in many mammalian species. This can often lead to various forms of infection and inflammation, which trigger stress response pathways, promoting delamination of cells and inducing apoptosis. FB1 can be enzymatically converted to form a less potent ceramide synthase inhibitor, hydrolyzed fumonisin B1 (HFB1), which was found to have controversial results in various in vitro and in vivo models. Many studies have focused on in vivo FB1 and HFB1 individually, but have failed to identify the in vitro comparative mechanisms regulating cellular interactions during fumonisin exposure. The identification of various signaling pathways provided after protein analysis, contributes to the increased understanding of the mechanisms behind such pathogenic interactions. In the present study, the modulating effect of FB1, HFB1 and lipopolysaccharides (LPS) on the growth and immune indices of the intestinal porcine enterocyte (IPEC-J2) cell line was investigated. It was then followed by the identification of molecular mechanisms and pathways influenced by FB1 and HFB1, and a comparative proteomic analysis was performed. Initially, IPEC-J2 cells were exposed to various concentrations of FB1, HFB1 and LPS. Thereafter the modulating effect of FB1 and HFB1 on the growth and immune indices was investigated. The cell survival indices (cell viability, apoptosis, and cell proliferation) were measured, while inflammatory responses were monitored by immune-detection of interleukin 8 (IL-8). Co-exposure of FB1 and HFB1 with LPS were also analyzed. It was found that HFB1 elicits a heightened degree of toxicity on intestinal epithelial cells in vitro compared to FB1. IPEC-J2 cells were then exposed to 7.81 μM and 15.63 μM concentrations of both FB1 and HFB1 for 24 hours, respectively. Cells were quantified through proteomic analyses using liquid chromatography- mass spectrometry (LC-MS/ MS). Bioinformatics analyses were conducted, and Differentially Abundant Proteins (DAPs) were identified and visualized with volcano plots. The functional annotation of DAPs was carried out using Gene Ontology (GO), comparing data using the Homo sapiens and porcine databases. A total of 52 significant DAPs were identified between FB1 and HFB1 compared to the control. In the KEGG pathways enrichment analysis, 15.63 μM FB1 exposure elicited a significant enrichment of proteins within multiple cancer pathways and the AGE/ RAGE signaling pathway. During 7.81 μM HFB1 exposure, a significant enrichment of proteins was identified within ribosomal pathways, while exposure to 15.63 μM HFB1 elicited a 3 significant enrichment of proteins within the ECM receptor interaction and proteoglycans in cancer, as well as focal adhesion, and bacterial invasion of epithelial cells. Fibronectin 1 (FN1), an adhesive glycoprotein of the intestine, was the only protein observed amongst all concentrations in cells exposed to FB1 and HFB1. It was found that FB1 up-regulates FN1 and HFB1 down-regulates FN1, which in turn elicited very different cancer promoting pathways using Cytoscape and STRING enrichment analysis. The expression of FN1 is important in cellular integrity maintenance, response to intestinal epithelial injury, and wound healing. Individually, it is noted that HFB1 promotes a greater toxicity on intestinal epithelial cells in vitro compared to FB1. Although HFB1 is known to be less toxic compared to FB1 in vivo, which suggests that HFB1 is metabolized differently in vitro compared to in vivo. This finding was suggested based on the individual and co-exposure data obtained. The potency of FB toxicity on intestinal cells are affected by the complexity of pathways connected. These results further suggest that HFB1 promotes a greater toxicity upon the IPEC-J2 cell line when compared to FB1, due to the abundance of proteins that were affected during exposure and the interconnectedness of pathways that were enriched.