| Preterm infants, born before 37 weeks gestation, often need breast milk supplementation in order to achieve growth rates comparable to those in utero. It has been previously shown that iron supplements in human breast milk increase lipid peroxidation, generate reactive oxygen species (ROS) in cultured enterocytes, and induce apoptosis. Several studies have shown that intracellular ROS alter the expression of gap junction proteins (connexins) and influence gap junctional intercellular communication (GJIC). In the present study, I demonstrate the effects of oxidative stress and iron-supplemented breast milk on the expression and phosphorylation of connexin-43 (Cx43) in the human gut. For these purposes, I have used a well-characterized cell line, CaCo-2BBE, as an in vitro model of the intestinal epithelium. Cell cultures were exposed to various dietary factors (including iron) supplemented to human breast milk which have previously been shown to generate intracellular oxidative stress. Cx43 expression was assessed using a number of different molecular biological techniques, including real-time reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and by immunolocalization. GJIC was assessed by dye coupling. Finally, an important function of the intestinal epithelium is to provide a selective permeability barrier. This was assessed by measuring the transepithelial electrical resistance (TEER) across confluent epithelial cell monolayers.;After exposing cells to both peroxide and iron supplemented breast milk treatments, Cx43 gene expression was significantly reduced compared to untreated controls. Interestingly, those same treatments caused a significant increase in overall Cx43 protein expression as well as in the phosphorylated isoform of the protein. Both peroxide and iron caused a decrease in GJIC, with iron showing a dose response. Consistent with other reports in the literature, treatments which decreased GJIC also decreased TEER suggesting a possible role for gap junctions in maintaining epithelial function. |
