18β-Glycyrrhetinic acid altered the intestinal permeability in the human Caco-2 monolayer cell model

Purpose Glycyrrhizin (GL) and its metabolites 18 alpha-glycyrrhetinic acid (18 alpha-GA) and 18 beta-glycyrrhetinic acid (18 beta-GA) are used as traditional medicine and food sweeteners. As the major rout of their administration is oral way, therefore their impact on intestinal epithelial cells are investigated. Methods The effects of GL and its metabolites on cell viability using MTT assay, on cytotoxicity using LDH release, on integrity of intestinal epithelial cells by measuring the transepithelial electrical resistance (TEER) and Luciferase permeability tests, on the expression of tight junction proteins at mRNA and protein level by qPCR and western blot techniques, and ultimately on the rate of test compounds absorption via Caco-2 cells monolayer were investigated. Results MTT assay showed a concentration- and time-dependent decrease in metabolic activity of Caco-2 cells induced by GL, 18 alpha-GA, and 18 beta-GA, while only 18 beta-GA increased the LDH leakage. The monolayer integrity of Caco-2 cells in TEER assay only was affected by 18 beta-GA. The permeability of paracellular transport marker was increased by 18 alpha-GA and 18 beta-GA and not GL. In transport studies, only metabolites were able to cross from Caco-2 cells monolayer. qPCR analyses revealed that 18 beta-GA upregulated the expression of claudin-1 and -4, occludin, junctional adhesion molecules and zonula occludens-1, while 18 alpha-GA upregulated only claudin-4. The expression of claudin-4 at protein level was downregulated non-significantly at 50 mu M concentration of 18 beta-GA. Conclusion Our results suggest that 18 beta-GA may cause cellular damages at higher concentrations on gastrointestinal cells and requires a remarkable attention of the nutraceutical and pharmaceutical industries.