In vitro protective effects of a standardized extract of Opuntia ficus indica L. and Olea europaea L. against indomethacin-induced intestinal epithelial cells injury

The pathogenesis of enteropathy induced by non-steroidal anti-inflammatory drugs (NSAIDs) is still unclear, and there are no established treatments. NSAIDs can cause increased intestinal permeability and the reduction of intestinal mucus granting luminal substances, such as bile acid and intestinal bacteria, to access to the mucosa. These in turn lead to inflammation through neutrophils infiltration, upregulation of proinflammatory mediators, and the production of reactive oxygen species. Strategies of therapy are often represented by physical protection of mucosa with mucoadhesive materials and by treatment of correlated inflammatory and cytotoxic events. Nowadays, research is focused on natural substances as protective agents for treatment and prevention of NSAID-induced small intestinal damage. In this study, we used a model of intestinal epithelial cells injury induced by NSAID indomethacin, to investigate the in vitro protective effects of a standardized extract (OFI+OE) containing polysaccharides obtained from cladodes of Opuntia ficus indica L. with mucoadhesive properties, and polyphenols from leaves of Olea europaea L. known for multiple biological activities. Caco-2 human intestinal epithelial cells were cultured for 18 days post confluence to obtain fully differentiated cells. Then, cells were pre-treated with OFI+OE (200 and 400 μg/mL) for 24 hours and subsequently exposed to indomethacin 1 mM for 24 hours to simulate in vitro pro-inflammatory and cytotoxic events correlated to mucosal diseases. Monolayer integrity and function were evaluated by analysis of fluorescein permeability. Western blot analysis and qPCR techniques were used to determinate proteins and genes expression involved in oxidative stress, in inflammatory NF-κB pathway and in apoptotic process. In addition, reactive oxygen species (ROS) and Total Antioxidant Activity (TAA) levels evaluation allowed to clarify OFI+OE protective effects on redox imbalance involved in mucosal diseases. Pre-treatment with OFI+OE was able to prevent intestinal epithelial barrier damage induced by indomethacin, as demonstrated by the decrease in fluorescein permeability. Furthermore, OFI+OE enhanced the redox status, reducing ROS intracellular production and increasing TAA values, and inhibited NF-κB pathway and pro-inflammatory cascade. High ROS levels, stimulated by indomethacin, were able to induce endoplasmic reticulum (ER) stress and apoptotic cell death, but OFI+OE prevented indomethacin-induced apoptosis as demonstrated by its effects on Bcl-2/Bax ratio and Caspase-3 protein levels. Finally, herein we demonstrate the protective effects of Opuntia ficus indica L. and Olea europaea L. combined extract against indomethacin-induced intestinal epithelial cells injury, via modulation of oxidative, inflammatory, and apoptotic signaling pathways. In conclusion, this study provides a basis to the possibility of using this natural combined extract as a strategy for treatment and prevention of gastrointestinal mucosal damage.