Introduction
Potential therapeutic effects of natural compounds, such as grape polyphenols, have been suggested in the prevention and treatment of mucositis induced by radiotherapy or bacterial infections. Lipopolysaccharide (LPS) significantly increases the paracellular permeability of gut epithelium downregulating the expression of tight junctions. In this study, the protective effect of proanthocyanidin-rich grape seed extract (GSE) was investigated on epithelial barrier damage and ROS production induced by LPS and ionizing radiations in an in vitro model.
Aims & Methods
GSE chemical analysis was performed by high-performance liquid chromatography (HPLC-DAD) and nuclear magnetic resonance (NMR).
Human intestinal epithelial cell line Caco-2, previously treated with LPS [10 µg/m] (pathogenic strain of Escherichia Coli 0111:B4) [1], GSE [6,25 mg/ml] and LPS + GSE, were irradiated with 10 Gy subdivided in 5 daily fractions using TrueBeam™ radiotherapy system by Varian Medical System. Epithelial barrier integrity was investigated measuring the paracellular flux of fluorescein isothiocyanate-dextran (FD-4) [1] and ROS production was assessed by adding 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCF-DA) [3] using a multiplate reader (Tecan). Each experiment was performed twice in triplicates with independent controls among the three conditions.
Results
HPLC-DAD and NMR revealed a 70% content of total procyanidins in both the extract obtained from the virgin hydroalcoholic mixture and quantify 24 metabolites classified as amino acids, organic acids, classified as amino acids, organic acids, carbohydrates and miscellaneous molecules. Among them, it is interesting the presence of ascorbate, procyanidin B1 and polymeric procyanidins.
Our in vitro experiments showed a significant increased permeability was observed in LPS-treated cells over time compared to control (5.215 vs 0.3418; p<0.05), which is prevented by co-treatment with GSE (5.215 vs 0.3407; p<0.05). Irradiation significantly increased intestinal permeability over time and in a dose dependent manner compared to control both in Caco2 cells without LPS treatment (at day 5 with maximal dose of 10 Gy: 3.64 vs 0.441; p<0.05) and in LPS-treated cells (25.5 vs 2.42; p<0.05). GSE treatment significantly reduced this damage in both conditions (25.5 vs 2.04; p<0.05 and 38.87 vs 2.675; p<0.05 respectively). Moreover, LPS significantly increased ROS production in Caco2 cells (12,9 vs 104; p<0.0001) and GSE treatment significantly prevented this damage (104 vs 11.3; p<0.0001). Irradiation increased ROS production in LPS-treated cells (3056 vs 362; p<0.0001) and also in this case GSE treatment was able to reduce ROS production due to irradiation (502 vs 357; p<0.0001). this beneficial effect was observed also in LPS treated cells and then exposed to irradiation (3056 vs 681; p<0.0001).
Conclusion
In an in vitro model, GSE is able to prevent the intestinal epithelium permeability damage and ROS production induced by LPS and by ionizing radiations suggesting a potential therapeutic effect. Further studies are needed to better understand the mechanism underlying radiation-induced mucositis and the antioxidant effect of GSE in its prevention and management.
References
[1] Gori M, Altomare A, Cocca S, Solida E, Ribolsi M, Carotti S, Rainer A, Francesconi M, Morini S, Cicala M, Guarino MPL. Palmitic Acid Affects Intestinal Epithelial Barrier Integrity and Permeability In Vitro. Antioxidants (Basel). 2020 May 13;9(5):417
[2] Calabriso N, Massaro M, Scoditti E, Verri T, Barca A, Gerardi C, Giovinazzo G, Carluccio MA. Grape Pomace Extract Attenuates Inflammatory Response in Intestinal Epithelial and Endothelial Cells: Potential Health-Promoting Properties in Bowel Inflammation. Nutrients. 2022 Mar 11;14(6):1175