PSVI-9 - In vitro effects of monoglycerides and zinc glycinate: anti-inflammatory and epithelial barrier function

Abstract: The objectives of this study were to investigate the in vitro anti-inflammatory effects of monoglycerides and zinc glycinate with porcine alveolar macrophages (PAMs) and their impacts on epithelial barrier function using the IPEC-J2 cell line. MTT assays were performed for each compound to determine cell viability and appropriate treatment doses. PAMs were isolated from 6 piglets at 7 weeks of age, and then seeded into 24-well plates with 10⁶ cells/mL. After 24 h incubation, cells were treated with each compound or lipopolysaccharide (LPS). The experimental design was 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 1000 µg/mL; zinc glycinate: 0, 25, 50, 100, 250, and 500 µg/mL). The supernatants were collected after another 24 h incubation to analyze concentration of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) by ELISA. IPEC-J2 cells (5 × 10⁵ cells/mL) were seeded into 12-well plates on transwell cell culture inserts and cultured until confluence, and then were treated with each compound (monoglycerides: 0, 25, 100, 250, 500, and 1000 µg/mL; zinc glycinate: 0, 2, 5, 12.5, 25, and 50 µg/mL). Transepithelial electrical resistance (TEER) was measured in Ωcm² using a Millicell ERS-2 voltohmmeter at 0 h (before treatment) and at 24, 48, and 72 h post-treatment. Data were analyzed by ANOVA using PROC MIXED of SAS with a randomized complete block design. LPS challenge increased (P < 0.05) the production of TNF-α and IL-1β from PAMs. In the non-challenge group, supplementing 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 PAMs. In LPS-treated PAMs, 1000 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. IPEC-J2 cells treated with 250 or 1000 μg/mL of monoglycerides, or 5 μg/mL of zinc glycinate had increased (P < 0.0001) TEER values at 48 and 72 h post-treatment, compared with control. However, cells treated with 50 μg/mL of zinc glycinate reduced (P < 0.0001) TEER values at 24, 48, and 72 h post-treatment compared with other treatments. In conclusion, results of this in vitro study indicate that monoglycerides have a positive effect on the barrier function of epithelium, while zinc glycinate may have strong immune regulatory benefits. Future in vivo animal studies will be needed to verify their impacts on animal gut health, immune responses, and growth performance.