Protective Effects of Butyric Acid and Zinc on Equine Intestinal CellFunction Under Hypoxic Conditions

Abstract

Spheroids were isolated from small intestinal crypts to evaluate the effect of butyric acid and zinc on dye leakage under hypoxic conditions. Spheroids were luminally injected with butyric acid and zinc-FD4 or FD4 only and cultured in normoxic or hypoxic conditions for 17 hours. Images were taken at the start and end of the experiment and corrected total cell fluorescence (CTCF) was

calculated to determine FD4 dye loss. Treatment with butyric acid and zinc decreased dye loss in both normoxic (p=0.09) and hypoxic conditions (p=0.039) compared to control spheroids. Results support that supplementing equine intestinal spheroids prior to cellular injury with butyric acid and zinc may benefit the intestinal epithelium in horses undergoing gastrointestinal stressors.

Introduction

Intestinal epithelial cells are essential in absorbing nutrients needed by the host while simultaneously creating a barrier that excludes harmful toxins and pathogens from entering the host. Environmental and disease stressors can play a major role in compromising these functions which can lead to intestinal and systemic inflammatory challenges. Butyric acid1-3 and zinc4,5 have been shown to improve intestinal barrier function via tight junction expression, a key component in maintaining intestinal integrity. Previous in vitro studies in swine and poultry have demonstrated a protective effect with the combination of butyric acid and zinc on barrier function in heat stress and inflammatory conditions. 6,7

The current study evaluated the protective effects of butyric acid and zinc on intestinal barrier function under normoxic and hypoxic conditions in primary equine spheroids.

Materials and Methods

Equine spheroids were isolated from small intestinal crypts and cultured in a three-dimensional extracellular matrix (Matrigel® Matrix, Corning Inc., Corning, NY) supplemented with growth factors and media. FITC-dextran 4 kDa (FD4), a fluorescent marker, was used to quantify permeability of the spheroids. Spheroids were luminally injected with butyric acid and zinc-FD4 or FD4 only (3-5 spheroids/

treatment/group). Successful injection criteria included: the entire lumen needed to be filled with dye, no dye could be seen outside of the spheroids and initial exposure time on the microscope needed to be low. After injection, spheroids were placed in normoxic (1% O2) or hypoxic (1% O2) conditions for 17 hours. Fluorescence images were taken at the start of the experiment (0 hour) and 17 hours after and corrected total cell fluorescence (CTCF) was calculated to determine FD4 dye loss. Multiple t-tests were performed with corrections for multiple comparisons using the Holm-Sidak method to compare percentage of dye loss of the treatment (butyric acid and zinc) to control in normoxic and hypoxic conditions.

Results

In normoxic conditions, the spheroids injected with butyric acid and zinc showed a reduction (15% difference) in dye loss compared to the control. There was a significant reduction (13% difference) in dye loss with butyric acid and zinc spheroids compared to control in hypoxic conditions.

At 0 hours: Dye loss for control was 70%; butyric acid and zinc was 44%

At 17 hours: Dye loss for control was 92%; butyric acid and zinc was 55%

Conclusions

Results of this study demonstrate that supplementing equine intestinal spheroids with butyric acid and zinc may have a protective effect on intestinal barrier function under hypoxic conditions. Further research using this novel model will provide an avenue to develop and investigate additional solutions to protect against intestinal injury.

References

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