(P30-009-23) Effect of Branched-Chain Amino Acid In Vitro Supplementation on Cytokines Synthesis in Intestinal Caco-2 Cells

Objectives: To investigate the effects of branched-chain amino acids (BCAA) in vitro supplementation on cytokine synthesis in intestinal Caco-2 cells.

Methods: Caco-2 cells were cultured until they reached 80% confluency. The cell cultures were distributed to six groups, being two control groups — control with Dulbecco's Modified Eagle's Medium without BCAA (CTL0) and with BCAA (CTL) (0.8 mmol/L of leucine, 0.8 mmol/L of valine and 0.8 mmol/L of isoleucine) — and four BCAA-supplemented groups — leucine group (LEU) (2 mmol/L of leucine), isoleucine group (ISO) (2 mmol/L of isoleucine), valine group (VAL) (2 mmol/L of valine) and BCAA association (LIV) (2 mmol/L of leucine, 2 mmol/L of isoleucine and 2 mmol/L of valine). Cells were supplemented for a period of 24 hours and then stimulated with lipopolysaccharides (LPS) for 24 hours. Cell viability was assessed by the MTT reduction assay ([3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide]). Determining the concentration of cytokines interleukin (IL)-1β, IL- 6, IL-8, IL-10 and tumor necrosis factor (TNF)-α in the culture medium was measured using a Multiplex kit for immunoassay. Comparisons between groups were performed by ANOVA. Significant differences were then compared using Tukey’s Honestly Significant Difference test with a significance coefficient of 0.05.

Results: According to the results obtained in the MTT assay, the groups treated with BCAA, in the absence or presence of LPS, had no impairment of cell viability in comparison to the CTL group without LPS. Cells stimulated with LPS showed a significant increase in IL-8 synthesis (p < 0.05), however, this effect was attenuated by the in vitro administration of leucine (p = 0.90) and isoleucine (p = 0.71). BCAA supplementation did not influence the in vitro synthesis capacity of IL-1β, IL-6, IL-10, and TNF-α in Caco-2 cells.

Conclusions: The amino acids leucine and isoleucine have an anti-inflammatory effect in Caco-2 cells by modulating the synthesis of IL-8, which is involved in the inflammatory response in the intestine, as it stimulates the migration of neutrophils.

Funding Sources: The São Paulo Research Foundation (FAPESP)