Creating an Intestinal Epithelial Cell Culture Model to Screen Novel Phytochemical Therapies for Postmenopausal Women (FS09-05-19)

Abstract

ObjectivesThe intestinal epithelium serves as barrier maintained by tight junction (TJ) proteins. TJ integrity is compromised by release of matrix-metalloproteases (MMPs). Degradation of TJ proteins may lead to increased permeability of the small intestine. Previous research has shown that MMPs are up-regulated with estrogen deficiency. In postmenopausal women, there may be a causal relationship between intestinal permeability and inflammation-related disease. The purpose of this project is to create an in vitro cell culture model, representative of the estrogen-deficient small intestine in vivo, to screen phytochemicals that may attenuate increased intestinal permeability observed with a decline in estrogen levels. MethodsHuman colorectal adenocarcinoma (CaCo-2) cells seeded on 12 mm Transwell® inserts were co-cultured with 3T3 Swiss fibroblasts, a major source of MMPs in the intestines, in 12-well plates. Cells were pre-treated with 10 μM universal estrogen receptor inhibitor (ICI 181,780; ABCAM, Boston, MA) for 1 hour, then treated with 10 nM estrogen (17 β-Estradiol; Sigma-Aldrich, St. Louis, MO) with or without MMP-9 Inhibitor (MMP-9 Inhibitor I; Millipore Sigma, Burlington, MA). Transepithelial electrical resistance (TER) was measured at 24, 48, and 72 hours following treatment. Confocal immunocytochemistry was used to determine the expression and distribution of TJ proteins. Differences between treatment groups were analyzed via ANOVA using SAS 9.3. ResultsInhibition of the estrogen receptors resulted in downregulation of TJ proteins in this model, suggesting a decline in intestine epithelial barrier integrity. MMP 9 inhibition concomitant with suppression of estrogen signaling resulted in increased TER values. ConclusionsMMP 9 inhibition when estrogen signaling is blocked enhances the intestinal epithelial barrier integrity. Future work will use this model to identify food components, especially those known to inhibit MMP 9, that may attenuate the decline in intestine epithelial barrier integrity observed with estrogen deficiency. Our long-term goal is to identify nutrition-related therapeutic targets to decrease the various inflammation-related comorbidities associated with estrogen deficiency, thereby improving the health of postmenopausal women. Funding SourcesCollege of Allied Health Student Research and Creativity Grant.