Evaluation of the Effect of Diet Viscosity on Mucus Production of the Intestinal Epithelium Using Swine Enteroids
Background: Majority of digestion occurs in the small intestine and intestinal goblet cells produce mucins, which are proteins that produce the mucus barrier. The mucus barrier is very important for the small intestine as it aids in the protection of the intestinal wall from bacteria, friction from intestinal contents, and aids in the digestive process. The main mucin in the small intestine is Mucin 2, encoded by the gene MUC2. High amounts of soluble fiber in the diet change the viscosity of the chyme (food in the process of digestion in the intestine) and high viscous chyme reduces the digestibility of nutrients and increases the secretion of mucin in the small intestine. It is known that increased viscosity is associated with decreased nutrient digestibility in swine, however, the underlying mechanism of said association is not clearly understood. Objective: The present study aims to evaluate the effect that solutions with different viscosity have on goblet cell numbers and mucin expression of swine enteroids using a Microfluidic System.
Methods: Enteroids, also known as mini-guts, are spherical, three dimensional structures formed of intestinal epithelial cells grown in the laboratory that are able to reproduce functions of the intestinal cells in a live organism. Enteroids were grown in special enteroid culture media (Intesticult) for 7-10 days and embedded in extracellular matrix before being inverted (by removing extracellular matrix) and placed in media with a composition that promotes cell differentiation. Enteroids were then plated on a 96 well plate and wells assigned to one of four treatments: Dulbecco's Modified Eagle Medium (DMEM) or DMEM with 0.1g, 0.4g, or 0.7g of citrus pectin in 50 mL. Citrus pectin was used to alter the viscosity of the solution, with the lowest pectin concentration being the least viscous. The viscosity of these solutions was quantified using a glass viscometer and the kinematic viscosities were 0.997, 0.999, 1.09, and 1.22 m2s-1, respectively. The enteroids were subjected to no-flow (N) or flow (F) solutions using a Microfluidic System (Elveflow OBII) for two hours. The flow rate was set to 60 μL/minute. Once the experiment was complete, enteroids were either placed in 500 mL of QIAzol Lysis Reagent for RNA extraction and gene expression analysis or embedded in OCT compound for cryosectioning and histological analysis. Periodic acid-Schiff (PAS)/ Alcian blue stain was completed to determine the area of total cells (mucosa) occupied by mucin producing Goblet cells. RNA extraction was performed and qPCR was run for evaluation of relative expression of MUC2 to the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Two independent experiments were performed and because of the sample size, we did not perform statistical analysis and only results are described.
Results: The proportion of Goblet cells in the enteroids was numerically greater in all the samples exposed to pectin solutions in the N conditions. Only the F with 0.7g of pectin in 50 mL showed more area when considering the samples with F. No clear differences were observed in presence of Goblet cells when compared samples with N to F conditions. For the relative gene expression of MUC2, pectin seems to increase the expression compared with DMEM when no flow (N). However, because of the high variation between the samples under flow (F), we cannot make a conclusion on the effect of flow and viscosity on expression of mucin 2.
Conclusion: Although we need to have more experimental replicates to make a conclusion, our results suggest that pectin increases the presence of Goblet cells and the expression of mucin 2 in swine enteroids. However, the effect is not dependent on flow.