In vitro investigation of monoglycerides and zinc glycinate: anti-inflammatory and epithelial barrier function

Abstract

The objectives of this study were to investigate the in vitro immune-modulatory effects of monoglycerides and zinc glycinate with porcine alveolar macrophages (PAM) and their impact on epithelial barrier integrity using the intestinal porcine enterocyte cell line (IPEC-J2). Cell viability was assessed using a Vybrant MTT assay to determine the appropriate dose range of monoglyceride blend (C4, C8, and C10) and zinc glycinate. In Exp. 1, IPEC-J2 cells (5 × 105 cells/mL) were seeded and treated with each compound (monoglycerides: 0, 25, 100, 250, 500, and 1,000 µg/mL; zinc glycinate: 0, 2, 5, 12.5, 25, and 50 µg/mL). Transepithelial electrical resistance (TEER) was measured by Ohm’s law method at 0 h (before treatment) and at 24, 48, and 72 h post-treatment. In Exp. 2, PAM were collected from six clinically healthy piglets (7 wk of age) and seeded at 106 cells/mL. After incubation, the cells were treated with each compound and/or lipopolysaccharide (LPS). The experimental design was a 2 × 6 factorial arrangement with 2 doses of LPS (0 or 1 μg/mL) and 6 doses of each compound (monoglycerides: 0, 50, 100, 250, 500, and 1,000 µg/mL; zinc glycinate: 0, 25, 50, 100, 250, and 500 µg/mL). Cell supernatants were collected to analyze the concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) by enzyme-linked immunosorbent assay kits. Data were analyzed by ANOVA using PROC MIXED of SAS with a randomized complete block design. IPEC-J2 cells treated with 250 or 1,000 μg/mL of monoglycerides, or 5 μg/mL of zinc glycinate had increased (P < 0.05) TEER values at 48 or 72 h post-treatment, compared with control. The LPS challenge increased (P < 0.05) the production of TNF-α and IL-1β from PAM. In the non-challenge group, 50 or 100 μg/mL of monoglycerides stimulated (P < 0.05) TNF-α and IL-1β production from PAMs. Treatment with 25 or 100 μg/mL of zinc glycinate also enhanced (P < 0.05) TNF-α production from PAM. In LPS-treated PAM, 1,000 μg/mL of monoglycerides increased (P < 0.05) IL-1β production, while zinc glycinate suppressed (P < 0.0001) the secretion of TNF-α and IL-1β at the doses of 100, 250, and 500 μg/mL. In conclusion, the results of this in vitro study indicate that monoglycerides positively affect the barrier function of the epithelium, while zinc glycinate may have strong immune regulatory benefits. Future animal studies will be required to verify their impacts on animal gut health, systemic immunity, and growth performance.